The Parathyroids, Second Edition: Basic and Clinical Concepts

Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

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The Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

Editor-in-Chief

John P. Bilezikian, M.D. Professor of Medicine and Pharmacology Chief, Division of Endocrinology Director, Metabolic Bone Diseases Program Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York Associate Editors R o b e r t Marcus, M.D. Professor of Medicine Department of Medicine Stanford University School of Medicine Stanford, California and Director, Aging Study Unit VeteransAffairs Medical Center Palo Alto, California

M i c h a e l A. L e v i n e , M.D. Professor of Pediatrics, Medicine, and Pathology Direct~ PediatricEndocrinology TheJohns Hopkins University School of Medicine Baltimore, Maryland

ACADEMIC PRESS A Harcourt Science and Technology Company

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This book is printed on acid-free paper. 0 Copyright © 2001, 1994 by John E Bilezikian, Robert Marcus, and Michael Levine M1 Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without the permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 A c a d e m i c Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, US http://www, academicpress, com A c a d e m i c Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 00-111700 International Standard Book Number: 0-12-098651-5 PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 MM 9 8 7 6 5 4

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2

1

Contents Contributors

.................................................

Preface to the Second Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface to the First Edition

......................................

ix xv

xvii

Section I: Basic Elements of the Parathyroid System 1.

Parathyroids: Morphology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . .

Virginia A. LiVolsi 2.

Parathyroid H o r m o n e Biosynthesis a n d Metabolism . . . . . . . . . . . . . . . . . . .

17

Henry M. Kronenberg, E Richard Bringhurst, Gino V. Segre, and John T. Potts, Jr. 3.

Parathyroid H o r m o n e - R e l a t e d Protein: Gene Structure, Biosynthesis, Metabolism, and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

William M. Philbrick 4.

Interactions of Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein with Their Receptors . . . . . . . . . . . . . . . . . . . . . .

53

Michael Chorev, Joseph M. Alexander, and Michael Rosenblatt 5.

Receptors for Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein: Signaling a n d Regulation . . . . . . . . . . . . . . . . . .

93

Robert A. Nissenson 6.

Nuclear Actions of P T H r P

.......................................

105

Andrew C. Karaplis and M. T. Audrey Nguyen 7.

............................

117

Receptors a n d Signaling for Calcium Ions . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

Signal Transduction of P T H a n d P T H r P

Lee S. Weinstein and Michael A. Levine 8.

Edward M. Brown, Arthur Conigrave, and Naibedya Chattopadhyay 9.

Immunoassays for P T H a n d PTHrP: Clinical Applications . . . . . . . . . . . . . .

143

L. J. Deftos

Section II: Physiological Aspects of the Parathyroid 10. 11.

Physiology of Calcium Homeostasis

................................

167

Edward M. Brown Parathyroid H o r m o n e : Anabolic a n d Catabolic Effects on Bone a n d Interactions with Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Janet M. Hock, Lawrence G. Raisz, and Ernesto Canalis

183

vi

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12.

Cellular Actions of Parathyroid H o r m o n e on Osteoblast and Osteoclast Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

Physiologic Actions of PTH and PTHrP: I. Skeletal Actions . . . . . . . . . . . . . .

213

Physiologic Actions of PTH and PTHrP: II. Renal Actions

227

Jane E. A ubin and Johan N. M. Heersche

13. 14.

15.

GordonJ. Strewler

E Richard Bringhurst

..............

E n d o c h o n d r a l Bone Formation: Regulation by Parathyroid Hormone-Related Peptide, Indian Hedgehog, and Parathyroid H o r m o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

245

Physiologic Actions of PTH and PTHrP: IV. Vascular, Cardiovascular, and Neurologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

261

Physiologic Actions of PTH and PTHrP: V. Epidermal, Mammary, Reproductive, and Pancreatic Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

Gino V. Segre and Kaechoong Lee

16.

Thomas L. Clemens and Arthur E. Broadus

17.

JohnJ. Wysolmerski, Andrew E Stewart, and John T. Martin

Section III: Clinical Aspects of Primary Hyperparathyroidism 18.

Parathyroid Growth: Normal and Abnormal . . . . . . . . . . . . . . . . . . . . . . . . .

19.

Molecular Basis of Primary Hyperparathyroidism

2O.

293

A. Michael Parfitt Andrew Arnold

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331

Clinical Presentation of Primary Hyperparathyroidism in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

Clinical Presentation of Primary Hyperparathyroidism: Europe . . . . . . . . . .

361

Clinical Presentation of Primary Hyperparathyroidism: India, Brazil, and China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375

23.

Clinical Course of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

387

24.

Molecular Markers of Bone Metabolism in Parathyroid Disease . . . . . . . . . .

399

ShonniJ. Silverberg and John P. Bilezikian

21. 22.

Jonas Rastad, Ewa Lundgren, and Sverker LjunghaU

Ambrish Mithal, Francisco Bandeira, Xunwu Meng, ShonniJ. Silverberg, Yifan Shi, Saroj K. Mishra, Luiz Griz, Geisa Macedo, Gustav Celdas, Cristina Bandeira, John P. Bilezikian, and D. Sudhaker Rao

25.

ShonniJ. Silverberg and John P. Bilezikian

MarkusJ. Seibel Cytokines in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . .

411

26.

H i s t o m o r p h o m e t r i c Analysis of Bone in Primary Hyperparathyroidism . . . .

423

27.

Nephrolithiasis in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

437

Inaam A. Nakchbandi, Andrew Grey, Urszula Masiukiewicz, Maryann Mitnick, and Karl Insogna May Parisien, David W. Dempsteg, Elizabeth Shane, and John P. Bilezikian Vanessa A. Klugman, Murray J. Favus, and Charles Y. C. Pak

Contents

28.

Guidelines for the Medical and Surgical M a n a g e m e n t of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael Kleerekopeg, Robert Udelsman, and Michael A. Levine

29.

Medical M a n a g e m e n t of Primary Hyperparathyroidism John L. Stock and Robert Marcus

30.

Preoperative Localization of Parathyroid Tissue in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John L. Doppman

................

459

475 487

31.

The Surgical M a n a g e m e n t of Hyperparathyroidism Samuel A. Wells,Jr. and Gerard M. Doherty

32.

Ectopic Locations of Parathyroid Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norman W. Thompson and Paul G. Gauger

499

33.

Parathyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elizabeth Shane

515

34.

Acute Primary Hyperparathyroidism Lorraine A. Fitzpatrick

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527

Multiple Endocrine Neoplasia Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . StephenJ. Marx

535

36.

Multiple E n d o c r i n e Neoplasia Type 2 Robert E Gagel

585

37.

Familial Forms of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . Lawrence Mallette and Robert Marcus

38.

Familial Benign Hypocalciuric Hypercalcemia and Neonatal Severe Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghada E1-Hajj Fuleihan and Hunter Heath III

35.

...................

451

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601

607

Section IV: Secondary Hyperparathyroidism 39.

The Parathyroids in Renal Disease: Pathophysiology . . . . . . . . . . . . . . . . . . . Kevin J. Martin, Esther A. Gonzdlez, and Eduardo Slatopolsky

625

40.

Renal Bone Diseases: Clinical Features, Diagnosis, and M a n a g e m e n t . . . . . . Jack W. Coburn and Isidro B. Salusky

635

Section V: Special Considerations 41.

Evaluation of the Hypercalcemic Patient: Differential Diagnosis . . . . . . . . . David Heath

663

42.

Hypercalcemia Due to P T H r P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard Kremer and David Goltzman

671

43.

O t h e r Local and Ectopic H o r m o n e Syndromes Associated with Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory R. Mundy and Babatunde Oyajobi

691

Genetic Disorders Caused by Mutations in the P T H / P T H r P Receptor: Jansen's Metaphyseal Chondrodysplasia and Blomstrand Lethal Chondrodysplasia . . . . Caroline Silve and HaraldJi2ppner

707

44.

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vii

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45. 46.

Acute M a n a g e m e n t of Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

729

Jean E. Mulder and John P. Bilezikian Primary H y p e r p a r a t h y r o i d i s m a n d O t h e r Causes of Hypercalcemia in Children a n d Adolescents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

743

Emily L. Germain-Lee and Michael A. Levine

Section VI: The Hypoparathyroid States 47.

H y p o p a r a t h y r o i d i s m in the Differential Diagnosis of Hypocalcemia . . . . . . .

48.

Magnesium Deficiency in Parathyroid Function . . . . . . . . . . . . . . . . . . . . . . .

755

Robert W. Downs 763

Robert K. Rude 49.

T h e Molecular Genetics of H y p o p a r a t h y r o i d i s m . . . . . . . . . . . . . . . . . . . . . .

779

R. V. Thakker

50.

A u t o i m m u n e Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51.

Pseudohypoparathyroidism: Clinical, Biochemical, and Molecular Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

791

Michael P. Whyte 807

Suzanne M. Jan de Beur and Michael A. Levine

52.

T r e a t m e n t of Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

827

Marc K. Drezner

Section VII: The Parathyroids in Osteoporosis 53.

Parathyroid Function in the N o r m a l Aging Process . . . . . . . . . . . . . . . . . . . .

54.

Parathyroid Function and Responsiveness in Osteoporosis . . . . . . . . . . . . . .

55.

Parathyroid H o r m o n e and Growth H o r m o n e in the T r e a t m e n t of Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

835

Sundeep Khosla, L.J. Melton III, and B. L. Riggs 843

ShonniJ. Silverberg and John P. Bilezikian 853

Robert Marcus Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

865

Contributors Harvard Medical School Boston, Massachusetts 02114

Joseph M. Alexander (53)* Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

Arthur E. Broadus (261) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510

Andrew Arnold (331) Center for Molecular Medicine and Division of Endocrinology and Metabolism University of Connecticut School of Medicine Farmington, Connecticut 06030

Edward M. Brown (127, 167) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

Jane E. Aubin (199) Department of Anatomy and Cell Biology and Department of Medical Biophysics Faculty of Medicine University of Toronto Toronto, Ontario, Canada M5S 1A8

Ernesto Canalis (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030; and Departments of Research and Medicine Saint Francis Hospital and Medical Center Hartford, Connecticut 06105

Cristina Bandeira (375) Endocrine Unit Hospital dos Servidores do Estado, and Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Gustav Celdas (375) University of Brazil 0020-020 Recife-PE, Brazil

Francisco Bandeira (375) Endocrine Unit Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Naibedya Chattopadhyay (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

John P. Bilezikian (349, 375, 387, 423, 729, 843) Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York 10032

Michael Chorev (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

E Richard Bringhurst (17, 227) Endocrine Unit Massachusetts General Hospital, and Department of Medicine *Numbers in parentheses indicate the pages on which authors'contributions begin. ix

x

/ Contributors

Thomas L. Clemens (261) Division of Endocrinology and Metabolism University of Cincinnati College of Medicine Cincinnati, Ohio 45267 Jack W. Coburn (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Arthur Conigrave (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115 L.J. Deftos (143) Department of Medicine University of California, San Diego, and San Diego VA Medical Center LaJoUa, California 92161 David W. Dempster (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032; and Regional Bone Center Helen Hayes Hospital West Haverstraw New York, New York 10993 Gerard M. Doherty (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 John L. Doppman (475)* Diagnostic Radiology Department National Institutes of Health Bethesda, Maryland 20892

Murray J. Favus (437) Department of Medicine University of Chicago Pritzker School of Medicine Chicago, Illinois 60637 Lorraine A. Fitzpatrick (527) Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Ghada EI-Hajj Fuleihan (607) Calcium Metabolism and OsteoporosisProgram American University of Beirut Medical Center Beirut 113-6044, Lebanon Robert F. Gagel (585)' Division of Internal Medicine University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 Paul G. Gauger (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Emily L. Germain-Lee (743) Division of Pediatric Endocrinology Department of Pediatrics The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 David Goltzman (671) Departments of Medicine and Physiology McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Esther A. GonzAlez (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110

Robert W. Downs (755) Division of Endocrinology and Metabolism Department of Internal Medicine Virginia Commonwealth University School of Medicine Richmond, Virginia 23298

Andrew Grey (411) Department of Medicine University of Auckland 92019 Auckland, New Zealand

Marc K. Drezner (827) University of Wisconsin-Madison Madison, Wisconsin 53792

Luiz Griz (375) University of Brazil 0020-020 Recife-PE, Brazil

*Deceased

Contributors David Heath (663) Department of Medicine SeUy Oak Hospital Birmingham B29 6JD United Kingdom Hunter Heath III (607) United States Medical Division Eli Lilly and Company Indianapolis, Indiana 46285 Johan N. M. Heersche (199) Faculty of Dentistry University of Toronto Toronto, Ontario, Canada M5G 1G6 Janet M. Hock (183) Department of Periodontics Indiana University School of Dentistry Indianapolis, Indiana 46202

Karl Insogna (411) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 Suzanne M. Jan de Beur (807) Division of Endocrinology Department of Medicine and Metabolism The Johns Hopkins University School of Medicine Baltimore, Maryland 2128 7 Harald Jiippner (707) Endocrine Unit Department of Medicine and Children's Service Massachusetts General Hospital, and Harvard Medical School Boston, Massachusetts 02114 Andrew C. Karaplis (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montreal, Quebec Canada H3T 1E2 Sundeep Khosla (835) Mayo Clinic and Foundation Rochest~ Minnesota 55905 Michael Kleerekoper (451) Department of Medicine Wayne State University Detroit, Michigan 48201

Vanessa A. Klugman (437) West Suburban Hospital Oak Park, Illinois 60302 Richard Kremer (671) Department of Medicine McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Henry M. Kronenberg (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Kaechoong Lee (245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Michael A. Levine (117, 451,743, 807) Departments of Pediatrics, Medicine, and Pathology The Johns Hopkins University School of Medicine Baltimore, Maryland 21287

Virginia A. LiVolsi (1) Department of Pathology and Laboratory Medicine University of Pennsylvania Medical Center Philadelphia, Pennsylvania 19104 Sverker Ljunghall (361) Global Clinical Sciences AstraZeneca Research and Development S-431 83 M61ndal, Sweden Ewa Lundgren (361) Department of Surgery Endocrine Unit University Hospital S-751 85 Uppsala, Sweden Geisa Macedo (375) University of Brazil 0020-020 Recife-PE, Brazil

/

xi

xii

/ Contributors

Lawrence Mallette (601) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 Robert Marcus (459, 601,853) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 John T. Martin St. Vincent's Institute of Medical Research Fitzroy, VIC3065 Australia Kevin J. Martin (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Stephen J. Marx (535) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 Urszula Masiuldewicz (411 ) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 L.J. Melton III (835) Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Xunwu Meng (375) Peking University Medical College Hospital 100730 Beijing, China Saroj K. Mishra (375) Department of Surgery Sanjay Gandhi Post Graduate Institute of Medical Sciences 226 O14 Lucknow, India Ambrish Mithal (375) Indraprastha Apollo Hospital 110044 New Delhi, India Maryann Mitnick (411) Mineral Metabolism Laboratory

Yale University School of Medicine New Haven, Connecticut 06520 Jean E. Mulder (729) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Gregory R. Mundy (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Inaam A. Nakchbandi (411) Mannheim Faculty of Medicine University of Heidelberg 68135 Mannheim, Germany M. T. Audrey Nguyen (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montrgal, Quebec Canada H 3 T 1E2 Robert A. Nissenson (93) Endocrine Unit San Francisco VA Medical Centeg, and Departments of Medicine and Physiology University of California, San Francisco San Francisco, California 94121 Babatunde Oyajobi (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Charles Y. C. Pak (437) Department of Internal Medicine University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75390 A. Michael Parfitt (293) Division of Endocrinology and Centerfor Osteoporosis and Metabolic Bone Disease University of Arkansas for Medical Sciences Little Rock, Arkansas 72205 May Parisien (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032

Contributors William M. Philbrick (31) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06520 John T. Potts, Jr. (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Lawrence G. Raisz (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030 D. Sudhaker Rao (375) Bone and Mineral Metabolism Department of Medicine Henry Ford Health System Detroit, Michigan 48202 Jonas Rastad (361) Department of Surgery Endocrine Unit • University Hospital S-751 85 Uppsala, Sweden B. L. Riggs (835) Mayo Clinic and Foundation Rochester, Minnesota 55905 Michael Rosenblatt (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02115 Robert K. Rude (763) University of Southern California School of Medicine Los Angeles, California 90089 Isidro B. Salusky (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Markus J. Seibel (399) Division of Endocrinology and Metabolism Department of Internal Medicine I

University of Heidelberg 69115 Heidelberg, Germany

Gino V. Segre (17, 245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Elizabeth Shane (423, 515) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Yifan Shi (375) Peking University Medical College Hospital 100730 Beijing, China Caroline Silve (707) INSERM U. 42 6 Facult~ de M~decine Xavier Bichat 75018 Paris, France Shormi J. Silverberg (349, 375, 387, 843) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Eduardo Slatopolsky (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Andrew E Stewart University of Pittsburgh Medical Center Pittsburgh, Pennsylvania 15213 John L. Stock (459) Department of Medicine University of Massachusetts Medical School Worcester, Massachusetts O1605; and Eli Lilly and Company Indianapolis, Indiana 46285 Gordon J. Strewler (213) VA Boston Healthcare System West Roxbury, Massachusetts 02132; and Department of Medicine Harvard Medical School Boston, Massachusetts 02114

/

xiii

xiv

/ Contributors

R. V. Thakker (779) Nuffield Department of Clinical Medicine University of Oxford Headington, Oxford OX3 9DU United Kingdom Norman W. Thompson (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Robert Udelsman (451) ~ Department of Surgery The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 Lee S. Weinstein (117) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases

National Institutes of Health Bethesda, Maryland 20892 Samuel A. Wells, Jr. (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 Michael P. Whyte (791) Centerfor Metabolic Bone Disease and Molecular Research Shriners Hospitals for Children St. Louis, Missouri 63131; and Division of Bone and Mineral Diseases Washington University School of Medicine at Barnes-Jewish Hospital St. Louis, Missouri 63110 John J. Wysolmerski Yale University School of Medicine New Haven, Connecticut 06520

Current affiliation: Department of Surgery, Yale University, New Haven, Connecticut 06520.

Preface to the S e c o n d Edition The first edition of The Parathyroids was published in 1994. It marked a milestone in the field, carrying on the tradition of Albright and Reifenstein whose 1948 classic The Parathyroid Glands and Metabolic Disease established a key role for the parathyroids in calcium homeostasis and metabolic bone disease. In The Parathyroids, we assembled a body of knowledge that had been accumulating over a 30-year period. The spectacular pace of discovery placed the tiny parathyroid glands at an epicenter of an enormous research effort in metabolic bone disease. The first edition was used widely and filled an essential gap in reference literature. Over the past seven years, as this field has continued to grow, with newer and greater appreciation of the role of the parathyroids in the overall governance of calcium homeostasis, a second edition appears to be particularly apt. The second edition of The Parathyroids contains chapters that have been extensively revised and expanded and many new chapters as well. The chapters d o c u m e n t our new knowledge about virtually every facet of this field and reexamine classic precepts that have stood the test of time. We understand better than ever before the structure and function of the parathyroid h o r m o n e gene and protein as well as the regulatory control of parathyroid h o r m o n e synthesis and secretion, the physiological and pathophysiological aspects of parathyroid hormone-related protein (PTHrP), the mechanisms of parathyroid h o r m o n e and PTHrP action, and the cell biology of PTH and PTHrE With regard to primary hyperparathyroidism, we now appreciate a spectrum of clinical presentations according to where in the world it is detected. Information about the course of primary hyperparathyroidism with and without parathyroid surgery is also new, as are the molecular genetics, biochemical, and histomorphometric dynamics of primary hyperparathyroidism. Advances in preoperative localization of parathyroid tissue and newer operative approaches to parathyroid gland surgery are noteworthy. The hypoparathyroid disorders are understood better with regard to their molecular genetics, pathophysiology, and mechanism. Finally, newer information is available about how parathyroid h o r m o n e can be both a catabolic and anabolic h o r m o n e for bone. This newer knowledge has fueled provocative ideas about the pathophysiology of osteoporosis and is heralding a new era in the therapeutics of osteoporosis. The second edition, thus, is still a comprehensive examination of basic and clinical concepts of the parathyroids. It is intended for students, teachers, practitioners, and investigators. In light of these newer developments in the field, the second edition has been reorganized to provide the reader with information that follows best the changing scientific logic. Fifty-five chapters are divided into seven sections. In Section I, nine chapters are devoted to basic concepts of parathyroid h o r m o n e and PTHrP, covering embryology, anatomy, and pathology of parathyroid tissue; gene structure, biosynthesis, and metabolism of PTH and PTHrP; receptors, nuclear targeting, and signal transduction for PTH, PTHrP, and calcium ion; and a comprehensive review of the immunoassays for PTH and PTHrE In Section II, eight chapters are devoted to the physiological aspects of calcium metabolism and the anabolic and catabolic effects of PTH at the level of bone and bone cells. Five chapters cover in detail all aspects of PTH and PTHrP with regard to traditional and nontraditional target organs. In Section III, 21 chapters are devoted to clinical aspects of primary hyperparathyroidism. Chapters on the growth of normal and abnormal parathyroid cells and the molecular genetics of primary hyperparathyroidism are followed by three chapters that describe different clinical presentations of primary hyperparathyroidism t h r o u g h o u t the world. Detailed coverage of bone dynamics and stone disease is followed by information relevant to the medical and surgical m a n a g e m e n t of primary hyperparathyroidism. Also covered are other presentations of primary

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/ Preface to the Second Edition hyperparathyroidism: as a malignancy, as an acutely hypercalcemic disorder, and in association with the multiple endocrine syndromes I and II. A chapter on familial hypocalciuric hypercalcemia completes this section. Two chapters in Section IV cover the parathyroids in renal disease. These are followed by six chapters in Section V that focus on special considerations. The first three chapters review the differential diagnosis of hypercalcemia, including syndromes caused by the local and systemic production of hypercalcemic factors such as PTHrR Jansen's disease, the acute m a n a g e m e n t of hypercalcemia, and hypercalcemia in children are considered in separate chapters. In Section VI, the hypoparathyroid states are reviewed in six chapters, which cover molecular, ionic, and immunological defects in the hypoparathyroid states and the role of hypoparathyroidism in the differential diagnosis of hypocalcemia. In Section VII, the role of parathyroid function in osteoporosis is covered in three chapters describing changes in parathyroid function with aging, parathyroid function and responsiveness in osteoporosis, and the potential of parathyroid h o r m o n e as a therapy for osteoporosis. As was true for the first edition, we recognize that this book is not likely to be read from cover to cover. Thus, each chapter has been written to provide a body of knowledge that can stand alone. The chapters, however, are also liberally cross-referenced to help the reader continue reading more directly related material if desired. The first edition of this book was dedicated to the m e m o r y of Gerald D. Aurbach, whose untimely and tragic death was its catalyst and inspiration. Virtually all the principal authors of the first edition had known and worked with Jerry. We r e m e m b e r e d him then for his "wisdom, scientific acumen, investigative skills, and daring insights." We r e m e m b e r him now in m u c h the same way. We were and still are mindful of the role Jerry had not only for us but also for the entire field, which he helped to create. We were his scientific progeny. It is 10 years since Jerry's death, virtually a generation in the world of science. As a result, some of the leading figures in this field have e m e r g e d without having had the special privilege of working with or knowing Jerry. The authorship of the second edition has been broadened, therefore, to include the very best in our field, recognizing that although Jerry's legacy is still alive, it now extends to an even broader cross section of the field. We wish to thank Jasna Markovac of Academic Press, who was instrumental in both the first and current editions of The Parathyroids. Mica Haley of Academic Press was also most helpful in attending to the many details required to ensure a rapid t u r n a r o u n d time to final publication. Enjoy the book.

John P. Bilezikian Robert Marcus Michael A. Levine

Preface to the First E d i t i o n One of us (JPB), d r e a m e d of this book about five years ago. It seemed then that advances in our knowledge of the parathyroids represented nothing less than a 30-year revolution of spectacular progress. We gained knowledge over this period at an explosive pace with a concomitant new appreciation of the basic and clinical ramifications of these four tiny endocrine glands. The major secretory product, parathyroid h o r m o n e (PTH), was isolated, sequenced, assayed, and cloned. PTH became one of the first h o r m o n e s to be shown to utilize cAMP as a second messenger. Regulation of PTH synthesis and secretion by calcium and 1,25-dihydroxyvitamin D was appreciated, as well as the cellular effects of PTH on its two major target organs, bone and kidney. The discovery of parathyroid hormone-related protein (PTHrP) as a cause of hypercalcemia of malignancy and a more general appreciation of PTHrP and PTH as polypurpose factors with many diverse biological effects represent exciting new advances in our field. The recent cloning of a bona fide receptor for both PTH and PTHrP is a tremendous achievement, as is the thinking that both PTH and PTHrP may utilize more than one second messenger pathway, and perhaps interact with more than one receptor. At the clinical level, we have seen a remarkable evolution in the presentation of primary hyperparathyroidism and are beginning to understand molecular features of this disease. Pseudohypoparathyroidism is now appreciated, in its classical form, to be a G protein deficiency disease. A u t o i m m u n e and molecular features of hypoparathyroidism have been identified and studied. New knowledge of the pathophysiology of secondary hyperparathyroidism associated with renal failure has had direct impact on m a n a g e m e n t and clinical outcome. PTH is now appreciated to have important anabolic properties in bone that may have implications for its use as a therapeutic agent in osteoporosis. This incomplete summary argues persuasively for how fast and how far this field has advanced. This is not to say that we were in the dark ages before Aurbach isolated parathyroid h o r m o n e . Certainly, it was Fuller Albright who in 1948 correctly pointed out that "back in the dark ages of endocrinology, in the early 1920s, hyperparathyroidism was an u n k n o w n fact." It was also Albright who r e m i n d e d us of the work of Sandstrom, who in 1880, 40 years before the first known cases of hyperparathyroidism wrote, "The existence of a hitherto u n k n o w n gland in animals that have so often been a subject of anatomical examination called for a t h o r o u g h approach to the region a r o u n d the thyroid gland even in man. Although the probability of finding something hitherto unrecognized seemed so small that it was exclusively with the purpose of completing the investigations rather than with the hope of finding something new that I began a careful examination of this region, so m u c h the greater was my astonishment therefore when in the first individual I examined, I found on both sides at the inferior b o r d e r of the thyroid gland an organ of the size of a small pea, which j u d g i n g from its exterior, did not appear to be a lymph gland, or an accessory thyroid gland, and u p o n histological examination showed a rather peculiar structure." The first chapters on the parathyroids were indeed written by Albright and a band of spectacular clinical investigators of the 1920s, 1930s, and 1940s. These chapters are recorded in the Albright and Reifenstein classic The Parathyroid Glands and Metabolic Disease. We r e c o m m e n d this insightful 45-year-old book as important and provocative reading. The Parathyroids is designed to follow the Albright and Reifenstein text. Certainly all endocrinology reference texts routinely include a section on the subject matter of this book. O t h e r texts that are more focused on calcium metabolism provide more information than the standard endocrinology texts on the parathyroids. However, there is no book that is exclusively devoted to a comprehensive examination of basic and clinical concepts

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/ Preface to the First Edition of the parathyroids. As indicated by the size and scope of The Parathyroids, it is clear that a book devoted to this subject is worthy and long overdue. It is time for such a book to stand on the endocrine shelf near its anatomical partner, the thyroid gland, which in Werner and Ingbar's The Thyroid has had its own literary repository since 1955. This book is intended for students, teachers, practitioners, and investigators of this field. It covers in a current and concise yet complete m a n n e r virtually all that we know about the parathyroids. Thus, it is both a basic and a clinical text. The 51 chapters are divided into a presentation of basic knowledge of the parathyroids and the clinical disorders associated with dysfunction of these glands. Section I, Basic Concepts of the Parathyroids, consists of 22 chapters. Chapters 1-7 cover the embryology, anatomy, and pathology of the parathyroid glands; calcium homeostasis; regulation of parathyroid hormone by dietary calcium and vitamin D; anabolic and catabolic effects of parathyroid hormone; cellular actions of parathyroid hormone on osteoblast and osteoclast function; autocrine and paracrine functions of parathyroid tissue; and the chemistry and biology of parathyroid h o r m o n e secretory protein. In Chapters 8-16, parathyroid hormone is considered with respect to the discovery by Aurbach of one of its second messengers, cAMP; regulation of it biosynthesis and metabolism; the parathyroid hormone gene; structure-function analysis of parathyroid hormone and parathyroid hormone-related protein; measurement of parathyroid hormone in the circulation; parathyroid hormone and parathyroid hormone-related protein as polyhormones; receptors for parathyroid hormone and parathyroid hormone-related protein; G proteins as transducers of parathyroid hormone action; biochemical mechanisms of parathyroid hormone action. The book proceeds in Chapters 17-20 to a consideration of PTHrP: its structure, physiological processing, and actions; its causative role in hypercalcemia of malignancy; it skeletal and renal actions; and its measu r e m e n t in the circulation. Other causes of hypercalcemia, besides PTHrP, and the management of PTH and PTHrP-dependent hypercalcemia complete this section (Chapters 21-22). Section II, Clinical Concepts of the Parathyroids, begins with an 18-chapter section on primary hyperparathyroidism (Chapters 23-40). This segment is a full exploration of the hyperparathyroid state from theoretical aspects of parathyroid cell growth to the molecular basis of primary hyperparathyroidism. A discussion of the spectrum of parathyroid tumors leads to a consideration of its modern clinical presentations and the course of primary hyperparathyroidism. The change in clinical presentation of primary hyperparthyroidism from a disease of bones and stones and groans to a relatively asymptomatic disorder does not lose sight of a major clinical complication, nephrolithiasis, which is still seen in patients on a regular basis. A chapter devoted to newer markers of bone turnover in primary hyperparathyroidism is followed by a discussion of the histomorphometric features of the disease. Medical and surgical management of primary hyperparathyroidism and the role of preoperative localization techniques are covered completely. Unusual manisfestations of primary hyperparathyroidism include separate discussions of parathyroid carcinoma and acute primary hyperparathyroidism. The MEN syndromes I and II focus on the parathyroids, as does the chapter on familial hypocalciuric hypercalcemia. In Chapters 41 and 42, the parathyroids in renal disease are reviewed with respect to pathophysiology, clinical profile, and management. Chapters 43-47 cover the hypoparathyroid states with respect to differential diagnosis, autoimmune etiologies, molecular genetics, and a special consideration of the clinical, biochemical, and molecular features of pseudohypoparathyroidism. A separate chapter is devoted to the therapy of hypoparathyroidism. The last four chapters of the book, Chapters 48-51, cover unusual aspects of the parathyroids: parathyroid function in the pathophysiology of osteoporosis and parathyroid hormone as a potential therapy of osteoporosis. Parathyroid functions in Paget's disease of bone and in magnesium deficiency complete the treatise. We recognize that few readers will read this book from cover to cover, although many of the chapters are closely interrelated. In order to permit virtually all chapters to "stand alone" but also to be connected to the rest of the book, we have liberally included cross-references to other chapters where appropriate. The reader can thus easily refer to other chapters for more information on a given subject. This design also necessarily calls for some interdigitation between chapters so that the reader in not always required to refer to another chapter but, rather, can get a brief summary in the chapter being read of an area that is covered more completely elsewhere.

Preface to the First Edition If it was true that we n e e d e d a b o o k on this subject five years ago w h e n the idea was first germinating, why did it take so long to get it d o n e a n d what was the impetus for finally accomplishing the task? T h e first of these two questions has a simple answer. Ideas for books are r a t h e r easy to develop but it is quite a n o t h e r m a t t e r to mobilize an army of over 90 experts to bring that idea to reality. As is true for so m a n y things, this idea was p u t on the shelf to be a d m i r e d for its own sake a n d to be c o m p l e t e d later. T h e mobilizing impetus a n d the inspiration for this effort eventually did come. Regrettably, it came in the f o r m of a tragic event in o u r lives, the death of Gerald D. Aurbach. T h e death of Jerry on a street in Charlottesville, Virginia, on N o v e m b e r 4, 1991, was r a n d o m , senseless, a n d violent. At 64 years of age, J e r r y was still alive with love for his work, his family, a n d his friends. In a m o m e n t , we suddenly lost a m a n who g u i d e d the very definition of o u r field for over 30 years. We lost a m a n who was o u r t e a c h e r a n d o u r friend. We lost a brilliant scientist who was involved in most of the major advances in this field over the past three decades. We lost a m a n who trained an e x t r a o r d i n a r y n u m b e r of us for successful careers in basic a n d clinical investigation of the parathyroids. We lost a gentle m a n who consistently b r o u g h t out the best of us. A s u m m a r y of the m a n y a c c o m p l i s h m e n t s that came f r o m Jerry's laboratory a n d the trainees, collaborators, a n d associates who worked with h i m is depicted in the time-line on pages xxvi-xxvii of this book. It is an e x t r a o r d i n a r y legacy. T h e two IN MEMORIA, by Bilezikian (Journal of Bone and Mineral Research 7:ix-x, 1992) a n d by Potts a n d Spiegel (Journal of Clinical Endocrinology and Metabolism 75:1386-1388, 1992), speak volumes to his career, to his accomplishments, a n d to his persona. In a flash, the d r e a m shelved in the recesses of consciousness a n d relegated to "when I get to it" b e c a m e an u r g e n t need. The Parathyroids h a d to be written in the m e m o r y a n d h o n o r of Gerald D. Aurbach, a n d it s e e m e d altogether fitting that it be written by those who were close to Jerry. We who knew h i m so well a n d respected h i m so m u c h would write a volume for the field. Virtually all of the principal authors of this text fit into that category. Maurice Attie, who also belongs in this book, was tragically killed in a bicycle accident in Philadelphia only a few m o n t h s after Jerry's death. We r e m e m b e r Maurice a n d wish that he too were still with us. It is e x t r a o r d i n a r y that a b o o k designed to be as c o m p r e h e n s i v e as this could be assembled by a collective a u t h o r s h i p whose scientific roots were established by Jerry. His contributions to this field are r e p r e s e n t e d not only by his science b u t also by his scientific p r o g e n y who are the n e x t g e n e r a t i o n of investigators to study a n d write a b o u t it. We took up this task with time in mind. The Parathyroids h a d to be published with a short lag time because the b o o k is a timely dedication to Jerry's memory. It h a d to be published soon because this field is in "fast forward" a n d if one used the n o r m a l publication time for a b o o k of this m a g n i t u d e , it would r u n the risk of rapidly b e c o m i n g outdated. To the credit a n d thanks to all the authors, virtually all 51 chapters were submitted within a six-month p e r i o d of time. T h e dedication of the authors to this task is gratefully acknowledged by us. We also are grateful to J a s n a Markovac of Raven Press, who h e l p e d to ensure that the process ran as efficiently as possible a n d whose efforts also were i n s t r u m e n t a l in ensuring a rapid t u r n a r o u n d time to final publication.

John P. Bilezikian Robert Marcus Michael A. Levine

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Th e Parathyroids Basic and Clinical Concepts

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Para

thyr o l"ds

Morphology and Pathology

VIRGINIA A. LIVOLSI Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Centeg, Philadelphia, Pennsylvania 19104

INTRODUCTION

The morphologic abnormalities seen in the parathyroid glands are predominantly those related to hyperfunction, i.e., primary hyperparathyroidism. Thus the focus here is on this aspect of parathyroid pathology, because almost all surgical specimens of parathyroid lesions are derived from patients with hyperparathyroidism. Because morphologic abnormalities are an important factor in the surgical treatment of this disease, a review of parathyroid embryologic development, anatomy, and normal histology is included. A brief discussion of parathyroid pathology in h y p o p a r a t h y roidism is also included, as is discussion of the pathology of the glands in humoral hypercalcemia of malignancy.

D E V E L O P M E N T O F P A R A T H Y R O I D GLANDS In the 8- to 10-mm embryo, the parathyroids begin to develop from the third and fourth branchial pouches. The third branchial pouch gives rise to the thymus and the parathyroid complex. The parathyroids migrate to and remain at the lower poles of the thyroid. Thus, in the usual case, the inferior parathyroids migrating with the thymus come to rest below the parathyroid derived from branchial pouch four (1). Embryologic studies in animals have demonstrated that ablation of the ventral half of the third branchial arch leads to nonformation of the upper parathyroid gland (2). Hoxa3 mutant homozygotes show defects in development and migraThe Parathyroids, Second Edition

tion pathways of thymus, thyroid, and parathyroid glands; the molecular events underlying the actions of the Hoxa3 genes remain to be determined (3). The fourth branchial pouch, or the fourth-fifth pharyngeal complex, gives rise to the superior parathyroid glands and via the ultimobranchial body to the parafollicular or C cells in the lateral thyroid. The superior parathyroids lie adjacent to the upper poles of the thyroid.

A N A T O M Y O F P A R A T H Y R O I D GLANDS Both the number and the location of the parathyroid glands vary in normal individuals. Variation in location of the glands can lead to problems during surgical exploration of the neck. For example, there may be difficulty in locating the diseased, abnormal parathyroid tissue in patients with hypercalcemia; conversely, surgery on the neck for other reasons, such as thyroid or laryngeal disease, may inadvertently cause trauma or removal of parathyroid glands because of the normal variability in their anatomic position (1,4-7). A report by Lee et al. indicates that almost 12% of patients undergoing thyroid resection have one parathyroid gland removed inadvertently (8). Although from one to twelve parathyroid glands can be found, (1), 84% of normal adults have four parathyroids (4). From 1 to 7% of adults have three glands and 3 to 13% have five glands (1,4-7). The variability of the location of the parathyroid glands is usually greater in the lower parathyroids. The superior parathyroids may be found close to the thyroid capsule or actually within Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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the thyroid capsule, but they may also be located behind the pharynx or the esophagus, lateral to the larynx, or behind any part of the thyroid. The lower glands, which usually lie near the lower pole of the thyroid, may be found behind the thyroid, in the paratracheal area, or close to or within the thymus in the superior mediastinum. The glands tend to be bilaterally symmetrical in location, with approximately 75% of cases showing such symmetry (4,5). The parathyroid glands measure between 2 and 7 m m in length, 2 and 4 m m in width, and 0.5 and 2 m m in thickness. They are reniform, soft, and brown to rust in color. However, color varies with fat content, the degree of vascular congestion, and the n u m b e r of oxyphil cells present (5,9,10). Parathyroid tissue weight varies with sex, race, and overall nutritional status of the individual (11). The combined weight of all parathyroid tissues in a normal adult male is a r o u n d 120 mg; in females combined tissue weight is around 145 mg. Weights of individual glands range from 3 to 75 mg, with averages of a r o u n d 35 to 55 mg (5,9-11).

HISTOLOGY OF PARATHYROID GLANDS Microscopic examination shows that each parathyroid gland is invested by a thin connective tissue capsule that extends into the parenchyma as fibrous septae, dividing the gland into lobules. A rich capillary vascular network is s u r r o u n d e d by nests and cords of

parenchymal cells. Small clusters of cells are interspersed with foci of adipose tissue (Fig. 1). However, there is variability in the location and interrelationships between the fat and the parenchymal cells in the parathyroid gland, so that biopsies from specific areas of the parathyroid may be predominantly fat, predominantly parenchyma, or a mixture of these two. In the adult, the parathyroid is composed of chief and oxyphil cells, fibrous stroma that is usually thin and delicate, and variable amounts of fat. Historically, the ratio of 50:50 cells:fat has been accepted as normal for adults. However, numerous studies have indicated that individuals dying without h o r m o n a l dysfunction of any type have parathyroids in which the stromal fat content is significantly less than 50% in most cases. It may be as little as 10%. In fact, n u m e r o u s studies (11-14) have shown that an approximately 17% fat content is normal in an adult parathyroid gland. Indeed, cell:fat ratios in terms of stromal fat serve little purpose in microscopic interpretation of functional status. Densitometry measurements concur, indicating that parenchymal cell mass accounts for 74% of parathyroid weight (4,14). The cells that make up the parathyroid glands include chief cells, oxyphils, and clear cells (Fig. 2). These variable cell groups probably represent different morphologic expressions of the same parenchymal cell. The chief cell is polyhedral in shape, poorly outlined, and measures 6 to 8 nm in diameter (15). It has an amphophilic to slightly eosinophilic cytoplasm, a

FIG. 1 Normal parathyroid gland adjacent to thyroid (lower left). Note the cellularity of the gland and the relative paucity of fat (f clear spaces) in this section. Hematoxylin and eosin, x50.

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FIG. 2 Parathyroid chief cells (dark cells), oxyphils (larger cells), and fat in normal adult gland. Hematoxylin and eosin, × 150.

sharp nuclear membrane, and well-defined, abundant nuclear chromatin. Clear cells represent chief cells in which there is an excessive amount of glycogen in the cytoplasm. Oxyphils, which tend to be found initially around the time of puberty and rarely in childhood, apparently increase in n u m b e r with age and may form small micro-

scopic nodules. The oxyphil cell in the parathyroid, as in other organs, is large, measuring approximately 10 nm in diameter, has a well-demarcated cell membrane, and has eosinophilic granular cytoplasm (Fig. 3). This reflects a marked mitochondrial content (9,10,15). In contrast to stromal fat content, intracellular fat content may be helpful in defining functional status. Thus, in

FI6.3 Cluster of oxyphils in normal parathyroid gland. Hematoxylin and eosin, x250.

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chief cells, which are the predominant cells in the parathyroid, intracellular fat, i.e., intracytoplasmic fat, is found in the overwhelming majority of cells in the euparathyroid state (approximately 80% of cells) (11-14). Ultrastructurally, the chief cells undergo a cyclic process during synthesis and secretion of parathyroid hormones, with the hormone being synthesized on Golgi apparatus-associated membrane-bound secretory granules. These cells eventually secrete these particles of h o r m o n e into the surrounding milieu. Little lipid is present in the active parathyroid cell, which in the euparathyroid state is approximately 20% of the parenchymal cell population (15).

DISEASES OF THE PARATHYROIDmPATHOPHYSIOLOGY Surgical pathologists dealing with the parathyroids almost always evaluate parathyroid tissue in patients who have hypercalcemia. The predominant effect of parathyroid hormone, as noted, is to increase serum calcium. The usual clinical problem is not to distinguish normal from hypercalcemic patients, but rather to distinguish those who have hypercalcemia caused by hyperparathyroidism from those who have hypercalcemia arising from other causes. Primary hyperparathyroidism is defined as the disease in which, in the absence of a known stimulus, one or more parathyroid glands secrete excess parathyroid hormone, producing hypercalcemia. Serum calcium ranges from 11 to 18 mg/dl, with most asymptomatic patients found in the lower end of the spectrum (16). The prevalence of primary hyperparathyroidism in the United States is estimated to be 1-5 cases per 1000 adults (16). The etiology of the disease is unknown. In a certain number of individuals a history of irradiation to the head and neck may be found, although the magnitude and significance of this association are not clear (17,18). Prinz et al. (18) found that 67% of patients in their series with combined thyroid and parathyroid tumors gave a history of irradiation. In some patients, genetics plays a role [multiple endocrine neoplasia (MEN) syndromes; see also Chapter 19] (19-25). Mutations of the M E N - 1 gene (menin) have been identified in some irradiated patients with hyperparathyroidism (22-24).

Pathology of the Parathyroid Glands in Primary Hyperparathyroidism Three subgroups of pathologic lesions are found in patients with primary hyperparathyroidism: adenoma, multigland hyperplasia, and, rarely, carcinoma.

Parathyroid Adenoma The parathyroid adenoma is responsible for hyperparathyroidism in 30-90% of cases. The wide range of variation indicates both pathologic interpretation and surgical interpretation of the disease (9,10,26-29). Most researchers believe that 75-80% of primary hyperparathyroidism is caused by a solitary adenoma (9,10,26-29). Evidence supports a clonal origin for parathyroid adenomas. Although older studies using protein polymorphisms indicated that parathyroid adenomas were polyclonal (30,31), many studies (25,32-36) using the techniques of molecular biology show that sporadic parathyroid lesions are monoclonal neoplasms. Grossly, parathyroid adenomas tend to be located more commonly in the lower glands than in the upper glands. Typically, the adenoma is an oval red-brown nodule that is smooth, circumscribed, or encapsulated. The lesion, which often replaces one parathyroid gland, may show areas of hemorrhage and, if large, cystic degeneration. Occasionally in small adenomas, a grossly visible rim of normal yellow-brown parathyroid tissue may be seen. Weights of adenomas vary from 300 mg to several grams. The size ranges from 1 to over 3 cm (9,10,27,29). Microscopically, adenomas are usually encapsulated lesions composed of parathyroid chief cells arranged with a delicate capillary network, recapitulating endocrine tumors in general (Figs. 4 and 5). Rarely, lobules are seen, and sometimes nodules may be formed. Stromal fat is usually absent. Unless they are very large, about 50% of adenomas will appear to have a normal rim or even atrophic parathyroid tissue outside the adenoma capsule. The cells in the rim tend to be smaller and more uniform, with stromal and cytoplasmic fat abundant in the rim but absent in the adenoma (9,10,27,29,37). However, the absence of a rim does not preclude the diagnosis of adenoma, because large tumors may have overgrown the preexisting normal gland or the rim may have been lost during sectioning. In large tumors, zones of fibrosis may be found in addition to hemorrhage, cholesterol clefts, and hemosiderin, as well as occasional areas of calcification. Rarely, lymphocytes will be noted within an adenoma (38). Thymic tissue may be found in association with an adenoma or an adenoma may be found within the thymus. There may be atypical cells in an adenoma. Most cells comprising the lesion have relatively small, uniform, dark nuclei. Usually focally, bizarre multinucleated cells with dark, crinkled nuclei can be seen. These nuclei probably represent degenerative changes rather than malignant or premalignant potential. It has been stated that mitotic activity is never found in a parathyroid adenoma and that such activity should suggest the

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FIG. 4 Parathyroid adenoma. Almost all chief cells; no fat present. Hematoxylin and eosin, x200.

possibility of a malignant neoplasm. This particular diagnostic area, however, is fraught with difficulty and is u n d e r debate at the present time. The n o n a d e n o m a tous glands in a patient with a parathyroid a d e n o m a may show normal to increased cytoplasmic fat content and normal weight (9,10,13,14). In about 10% of cases microscopic examination of biopsies from "normal" glands will show areas of hypercellularity, so-called microscopic hyperplasia. Although

this may represent a true parenchymal cell increase, the difficulty in defining "normal," or more likely sampling errors, probably accounts for this (39-41). Oxyphilic or oncocytic adenomas do occur and can function. These tumors tend to be larger than chief cell adenomas and the serum calcium levels tend to be minimally elevated (42-47). Because of the embryologic migration patterns, parathyroid adenomas can occur in ectopic locations.

FIG. 5 Parathyroid adenoma with follicle formation; rarely, this is mistaken for thyroid tissue, especially on frozen section. Hematoxylin and eosin, x300.

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Thus, when hyperparathyroidism occurs in such an individual and no a d e n o m a or abnormal glands are identified in the neck, ectopic locations that should be considered include the mediastinum, with or without associated thymic tissue, b e h i n d the esophagus, or even intrathyroidal (48-54). Double adenomas, if they occur, are very rare (55-57). Most patients who have socalled double adenomas will, over a period of time, have recurrent hyperparathyroidism and in fact have four-gland hyperplasia. The diagnosis of double aden o m a can be made only if two glands are enlarged and histologically abnormal; the remaining glands are normal, there is no family history of parathyroid disease, and p e r m a n e n t cure of hypercalcemia follows excision of only two enlarged glands (55-65). Indeed, heterogeneous size of four glands in primary hyperplasia may account for some cases interpreted as "double adenomas" (65).

Primary Parathyroid Hyperplasia Primary parathyroid hyperplasia is divided into two main groups: chief cell hyperplasia, which is common, and water clear cell hyperplasia, which occurs less commonly (9,10,29). Chief cell hyperplasia accounts for 15% of hyperparathyroidism in most series, although some reports indicate that about half of primary hyperparathyroidism is produced by hyperplasia. The reasons for this probably lie in discrepancies in pathologic interpretation. About 30% of patients with chief cell hyperplasia have familial hyperparathyroidism or one of the syndromes of multiple endocrine neoplasia (9,10,29,66-73). Grossly, all four glands are enlarged equally or nonequally. If unequal in size, the lower glands are usually larger. Occasionally one gland will be much larger than the others and will convey the surgical impression of an adenoma. The weight of all four glands ranges from 150 mg to over 20 g, but usually is in the range of I to 3 g (9,10). Microscopically, diffuse chief cell hyperplasia may be characterized by solid masses of cells with minimal to no fat. Usually almost all cells are chief cells, with rare oxyphils. Nodular or pseudoadenomatous hyperplasia consists of circumscribed nodules of chief, transitional, or oxyphil cells, each nodule devoid of fat, and with there being little fat in the intervening stroma. Usually in hyperplasia there is no rim of normal tissue. Bizarre nuclei are rarely found in primary hyperplasia. Mitoses may occasionally, however, be identified (9). Therapy in this disease is directed to the removal of all parathyroid tissue, with or without autotransplantation. Clear cell (water clear cell) hyperplasia is very rare and is the only condition of the parathyroid in which the superior glands are larger than the lower. Total weights of such parathyroids always exceed 1 g and usu-

ally range from 5 to 10 g. The glands are irregular and show pseudopods and cysts; a distinct mahogany color is seen grossly. Histologically, the glands are composed of diffuse sheets of clear cells without any mixture of other cell types. No rim is present (9,10,74-76). An interesting association of clear cell hyperplasia with the blood group O allele has been reported (77).

Parathyroid Carcinoma Parathyroid carcinoma accounts for approximately 1% of primary hyperparathyroidism (78-94). There is clinically an unusual scenario with an almost equal sex ratio, which is u n c o m m o n in parathyroid adenomas and usual hyperplasias, in which women predominate. The incidence of benign hyperparathyroidism appears to increase with age; however, patients with parathyroid carcinoma tend to be somewhat younger and are almost always symptomatic with very high levels of serum calcium. Very rarely, parathyroid carcinoma can occur in the setting of familial endocrine disease (95-99) or as a complication of secondary parathyroid hyperplasia (100-104). Most of the latter cases occur in patients with renal failure (12 cases were d o c u m e n t e d in 1999) (104). Clinically, patients with parathyroid carcinoma show high calcium levels (up to 15 m g / d l ) . Many have polyuria, polydypsia, nausea, vomiting, weight loss, and constipation. They may also have bone pain, renal stones, and other symptoms related to hypercalcemia. An important clinical clue is the presence of a palpable mass in the neck on physical examination. The mass may be clinically thought to be an a d e n o m a of the thyroid (9,10,78-94). Parathyroid carcinomas tend to be large tumors (average weight 12 g) and characteristically show a histology with trabecular a r r a n g e m e n t of tumor cells divided by thick fibrous bands, with capsular and blood vessel invasion in the presence of mitotic figures (Fig. 6) (9,10,105). The cytology may be clear or rarely oxyphilic; nuclear atypia may be seen or may be absent (9,10,102,106). Because mitotic figures are almost never found in a benign parathyroid adenoma, their presence in tumor cells should raise the suspicion of malignancy. However, this has been called into question and parathyroid tumors with mitotic activity may in fact be benign. As a note of caution, long-term follow-up in the reported series is quite limited, and there is a long natural history to parathyroid carcinoma, so the answers are not all in yet (107,108). Mitotic activity in secondary hyperparathyroidism is not to be equated with malignancy, and mitotic activity may occasionally be found in primary hyperparathyroidism as well (9). The presence of capsular invasion is not equated with malignancy because large parathyroid adenomas

PARATHYROIDS: MORX'HOLOGYAND PATHOLOGY /

7

FI6. 6 Parathyroid carcinoma; note mitosis (+). This tumor recurred three times locally and eventually metastasized to the lungs. Hematoxylin and eosin, x300.

may have u n d e r g o n e prior hemorrhage, with consequent fibrosis and trapping of tumor cells within the capsule. Vascular invasion is difficult to define except if seen outside the vicinity of the neoplasm. An important clue to the diagnosis of parathyroid carcinoma is the surgical finding of adherence a n d / o r invasion into local structures, which should raise the suspicion of a carcinoma (9,10,78-94,102). Metastases at the time of presentation are unusual, but may be found in the regional lymph nodes. There may also be local invasion into nerves, soft tissue, and the esophagus. Rarely, nonfunctioning parathyroid carcinomas have been described. These lesions tend to be large and composed of clear or oxyphil cells (83,109,110). The prognosis of parathyroid carcinoma is usually one of an indolent malignancy. Metastases may occur in up to one-third of cases and are found in regional lymph nodes, bone, lung, and liver. Many patients survive long periods of time, however. Multiple recurrences are known to occur over a 15- to 20-year period (9,10,78-94,102). The severity of the symptoms due to metastatic disease is directly related to tumor burden, because this is related to parathyroid h o r m o n e p r o d u c e d (111). Some solitary parathyroid tumors show features that suggest carcinoma, such as large size, fibrous bands, etc., but not all of the characteristics of malignancy are present. We use the term "atypical adenoma" for these lesions; short-term follow-up suggests they are benign, but long-term studies are needed in this subset of lesions.

Multiple Endocrine Neoplasia Syndromes The syndromes of MEN-1 (Wermer's syndrome) and MEN-2 (Sipple syndrome) are associated with pathologic changes in the parathyroids. In MEN-l, pathologic changes similar to adenomatous or p s e u d o a d e n o m a t o u s chief cell hyperplasia as described above are found (9,10). I n MEN-2, the parathyroids tend to show a diffuse hyperplasia, but occasionally one gland is involved, suggesting an "adenoma." In this syndrome the hyperparathyroidism is considered to represent a genetically determined event and not a response to hypercalcitoninemia (9,10). Parathyroid abnormalities are m u c h less c o m m o n in other variants of MEN-2 syndromes. Familial hyperparathyroidism shows the pathologic alterations of chief cell hyperplasia similar to Wermer's syndrome; in familial hypercalciuric hypercalcemia, mild parathyroid hyperplasia has been described (9,10).

Unusual Lesions of the Parathyroid

Parathyroid Cysts Cysts of the parathyroid glands are unusual and may present and be misinterpreted clinically as thyroid nodules (112-124). They occur more frequently in women than in men, usually are large, ranging from 1 to 6 cm, and may be located in any parathyroid gland, although most are found in the lower glands. Occasionally they may be found in the mediastinum, mimicking super i o r / a n t e r i o r mediastinal masses (114,121,123).

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1

may be functional or nonfunctional and usually is circumscribed but rarely encapsulated. In unusual examples a rim of normal parathyroid tissue is present at the periphery. In some instances at least one other histologically normal parathyroid has been recognized. Also, in some, there is an unusual myxomatous stroma, and other mesenchymal elements including metaplastic bone may be found. Wolff and Goodman (125) suggest the term "parathyroid adenomas with stromal component." More than three-quarters of the reported cases functioned, although with relatively low levels of hypercalcemia. We have studied a woman presenting with an orbital brown tumor due to a parathyroid lipoadenoma that weighed over 10 g (128).

Grossly, these cysts are almost always unilocular and smooth walled and contain water fluid with a high parathyroid hormone content. Histologically, they are lined by one layer of clear epithelium containing glycogen. The cyst wall is fibrous, with fragments of smooth muscle and nests of normal parathyroid tissue. It is unclear how these cysts arise. Microcysts are found in about half of normal parathyroids and might possibly enlarge by accumulation of secretions, or may fuse and produce grossly visible cysts. The cysts may arise from embryologic remnants of pharyngeal pouches in the neck undergoing cystic degeneration and entrapping portions of parathyroid tissue. Many investigators believe, however, that parathyroid cysts represent degenerated parathyroid adenomas, and in some cases, in fact, that the cysts are associated with hyperparathyroidism (Fig. 7) (117-119). However, this is u n c o m m o n and only a few functional cysts have been reported. It may be that different parathyroid cysts have different origins, although pathologically they resemble one another. Cytologists may encounter parathyroid cysts during attempts to aspirate thyroid nodules (112,124). The cyst fluid can be assayed biochemically for parathyroid hormone to confirm the diagnosis.

Parathyromatosis In rare instances of hyperparathyroidism due to primary hyperplasia, nests of hyperplastic parathyroid cells are found in the neck, outside of hyperplastic glands (129-131). In the individuals for which this has been reported, these nests were discovered at the first neck exploration, so that spillage during prior surgery could be excluded. In each of these patients there was no evidence of malignancy. It has been postulated that during embryologic development nests of pharyngeal tissue containing parathyroid cells might be scattered throughout the adipose tissue of the neck and mediastinum. Normally these nests are inconspicuous. However, in the process of diffuse hyperplasia of the parathyroids, all functioning tissue may become hyperplastic and appear as separate fragments on histologic evaluation.

Lipoadenoma-Hamartoma of the Parathyroid These tumors present as masses that histologically are composed of parathyroid cells arranged in nests, similar to normal parathyroid but intimately associated with large areas of adipose tissue (125-128). The lesion

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FIG. 7 Parathyroid cyst. This tumor presented with hypercalcemia and extended from the lower neck to the upper mediastinum. It was 6 cm in size, but most of the lesion was cyst. However, about 10-15% was solid parathyroid tissue, making this a cystic adenoma. Hematoxylin and eosin, x 150.

PARATHYROIDS: MORPHOLOGYAND PATHOLOGY /

Infarction of Parathyroid Adenomas Twelve d o c u m e n t e d cases (132) of adenomas that spontaneously infarcted have been reported. This phen o m e n o n is associated with remission of hypercalcemia. The etiology of the infarction is unclear in most cases, although some have been associated with the intake of certain drugs that may predispose to vascular damage, thrombosis, or hemorrhage. Therapeutic infarction can also result in cure of the metabolic abnormalities (85).

INTRAOPERATIVE ASSESSMENT OF PARATHYROIDS--THE BANE OF THE SURGICAL PATHOLOGIST In normal parathyroid glands, 80% of the cells are in the nonsecretory phase and contain intracytoplasmic fat (12,13,15). Therefore, is the fat stain useful in distinguishing hyperplasia from adenoma, because all hyperfunctioning glands should be fat depleted? The advocacy of fat stains (Sudan IV or Oil Red O) on parathyroid tissue removed at surgery has come into vogue. The scenario is as follows. A sample of an enlarged parathyroid gland is sent for frozen section and by hematoxylin and eosin stain it is hypercellular with little or no stromal fat. Thus it either represents an a d e n o m a or a hyperplastic gland and is not normal. A biopsy of a second parathyroid is frozen and is normocellular or minimally hypercellular. Fat stain shows a b u n d a n t cytoplasmic fat in the latter biopsy; hence this is a normal gland. The enlarged gland, which shows minimal to no fat, represents an adenoma. Many authors have cautioned, however, that the fat stain cannot be the sole procedure on which to base a diagnosis, because although the fat stain is helpful, it is helpful in only about 80% of cases and must be considered as an adjunctive technique in light of gross findings, gland weight, and size, and cannot be relied on by itself (133-141). We have found it useful to perform a r a p i d (30-second) toluidine blue stain on frozen sections of parathyroid tissue. The intracellular fat is well defined by this stain and it is faster to perform and interpret compared to Oil Red O (Lyle S, et al., unpublished observations, 2000). Another rapid technique that may prove useful for intraoperative assessment is density gradient measurements (142). There is an almost linear relationship between density and parenchymal content of parathyroid tissue and thus such a technique can assess parenchymal cell mass. The technique is to take a sample of the gland and weigh it, and take a small piece from the center and a piece from the rim, determining their densities in a 25% mannitol solution. Abnormal parathyroid tissue sinks because of decreased fat and

9

high parenchymal mass. Wang and Ryder (142) have found that this is a simple test to be used by the surgeon in the operating room for distinguishing normal from abnormal glands. In the intraoperative assessment of parathyroid pathology it cannot be stated strongly enough that there must be close communication between the surgeon and pathologist during the operation. The pathologist needs to be apprised of the gross findings and cannot work in a vacuum. What is r e c o m m e n d e d is as follows: the largest parathyroid gland found is resected in toto, then the pathologist weighs it, measures it, and examines it histologically. If the gland shows diffuse growth of chief cells and perhaps a normal-appearing rim, a lack of fat, and bizarre nuclei, a diagnosis of presumed a d e n o m a can be rendered. If the histology is that of hypercellularity but criteria for a d e n o m a are not seen, biopsy of at least one more gland is needed, and, in fact, in many centers pathologists prefer to have the largest abnormal gland and at least a biopsy of one more gland. Weight ratio of parenchymal cells to fat, and normal or a b u n d a n t intracytoplasmic fat content in the second gland, strongly support that the first gland is an a d e n o m a (133-141). The success rate of identifying parathyroid tissue by frozen section is over 99% (143); distinguishing one-gland from multigland disease is much more problematic.

OTHER TYPES OF HYPERPARATHYROIDISM

Secondary Hyperparathyroidism Secondary hyperparathyroidism is usually due to renal disease and is relatively c o m m o n in the age of hemodialysis and renal transplantation. The role of the surgical pathologist in the evaluation of secondary hyperparathyroidism is basically to identify parathyroid tissue at the time of frozen section to allow for the surgeon to remove portions of this tissue for autotransplantation. Secondary hyperparathyroidism is really no different histopathologically from primary hyperparathyroidism (144-146). Mitotic activity may occasionally be found in such glands. Usually all four glands are enlarged, although one or two glands may be of very great size. Transplantation of parathyroid tissue is successful in the majority of cases and occasionally part of this tissue may be removed if hyperfunction again becomes a problem (147,148). Such lesions will have small nests and islands of vascularized parathyroid tissue growing in muscle or fat, usually having been implanted in the arm (149,150).

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Tertiary Hyperparathyroidism Although the existence of tertiary hyperparathyroidism has been questioned, most authors believe it represents the autonomous function of one parathyroid gland that develops in the face of long-standing secondary hyperparathyroidism (151). The pathology resembles that of secondary hyperparathyroidism, although one of the four glands is usually disproportionately enlarged.

Familial Hyperparathyroidism In addition to the multiple endocrine neoplasia syndromes, in which hyperparathyroidism is often a prominent clinical problem, familial parathyroid hyperplasia without other endocrine lesions has been reported. The lesions in all of these patients resemble those of primary chief cell hyperplasia (9,10,152-154), although a ribbon pattern of cell growth may be prominent in MEN-1.

Familial Hypocalciuric Hypercalcemia Familial hypocalciuric hypercalcemia, inherited as an autosomal dominant gene, is manifested clinically by familial occurrence, moderate to minimally elevated serum calcium, and reduced urinary calcium excretion (see Chapter 41). The parathyroid glands appear normal to mildly hypercellular, and subtotal parathyroidectomy fails to reverse the hypercalcemia. The defect appears not to be in the parathyroid glands (155,156).

SPECIAL STUDIES AND THE PARATHYROID Cytology Because most parathyroid lesions are not palpable, direct biopsy of a parathyroid tumor by fine-needle aspiration (FNA) is unusual. However, on occasion, parathyroid lesions present clinically as thyroid nodules or are large enough to be clinically evident. The FNA features of parathyroid adenoma include cellular fragments of epithelial cells arranged around vascular cores, an organoid or trabecular architecture, and microacini. Parathyroid chief cells contain uniform round nuclei; groups of oxyphilic cells are helpful in defining the tissue as parathyroid. If available, immunostains for parathyroid h o r m o n e may help (157).

Proliferative Markers Attempts at using immunocytochemical markers (158-164) of proliferation index (MIB1 for cell cycle-

associated Ki-67 antigen) for distinguishing between parathyroid adenomas and hyperplasia have met with varied success (159). Whereas statistically significant differences are found between normal (suppressed "rim") parathyroid tissue and hyperfunctioning glands, similar proliferative indices are noted between adenomas and hyperplasias (159,160). Loda et al. (160) identified higher numbers of labeled nuclei in adenomas than in hyperplasias by proliferating cell nuclear antigen (PCNA) immunostaining. The labeling index of individual cases of parathyroid tumors shows so much overlap that it cannot be used to distinguish benign from malignant lesions (161-164).

Flow Cytometry and the Parathyroid Several studies of DNA content have shown that aneuploidy may be found in parathyroid adenomas, and even in hyperplasia, as well as in carcinomas. Approximately 70% of parathyroid carcinomas, 30% of adenomas, and 30-50% of chief cell hyperplasia glands have aneuploid DNA populations (165-171). As in proliferations of other endocrine organs, the finding of aneuploid cell populations does not ensure a diagnosis of malignancy (169-173).

Clonality Modern molecular biology techniques, primarily using restriction fragment-length polymorphisms, have shown that most (if not all) parathyroid adenomas are monoclonal proliferations (25). In addition, about 40% of primary hyperplasias and 60% of secondary hyperplasia (secondary to chronic renal disease) are clonal. Different laboratories utilizing different probes as markers confirm these findings (25,174-176). The biologic meaning of these results is unclear.

Genetics The P R A D 1 oncogene has been implicated in parathyroid tumorigenesis. PRAD1 (for parathyroid adenoma), which encodes cyclin D1, results from a chromosome inversion that occurs as a dominant clonal event in some parathyroid adenomas. The inversion is created by a break in the vicinity of the parathyroid gene on the short arm of chromosome 11 (band 1 lp15), another break in the long arm (band 1 lq13), rotation of the center piece around the axis of the centromere, and rejoining (177). Cyclin D overexpression can be detected immunohistochemically in 18-38% of parathyroid adenomas, and in 91% of carcinomas (178,179). The retinoblastoma (Rb) gene is a tumor suppression gene that has growth inhibitory effects in the cell cycle. Inactivation of the Rb gene has been associated

PARATHYROIDS: MORPHOLOGY AND PATHOLOGY

with loss of an Rb allele by molecular analysis, and immunostaining for Rb protein may assist in the distinction between parathyroid adenomas and carcinomas (179-183). However, caution must be used in interpretation of the results, because some parathyroid carcinomas do not show loss of Rb protein and a few adenomas do (181,182). Studies of parathyroid neoplasms (benign and malignant) have not shown p53 mutations in such lesions (184). In another study of parathyroid tissues, there were significant differences between p27 protein expression in parathyroid hyperplasia, adenomas, and carcinomas, suggesting that this cell cycle protein may be useful in distinguishing between these two conditions (185,186).

HUMORAL HYPERCALCEMIA O F MALIGNANCY, O R ECTOPIC PARATHYROIDISM Hypercalcemia without bone metastasis in nonparathyroid malignancies may be found in association with a malignant tumor. Hypercalcemia is relieved by excision of the tumor and returns with its recurrence. This paraneoplastic endocrine syndrome is due in many cases to a peptide that resembles parathyroid hormone but is distinctly different. The factor responsible for the syndrome of humoral hypercalcemia of malignancy, which is due to parathyroid hormone-related protein (PTHrP), is discussed in other chapters in this volume. PTHrP binds to parathyroid h o r m o n e receptors on bone and kidney and mimics the actions of parathyroid hormone. The tumors most commonly associated with this syndrome include squamous carcinomas arising in a number of primary sites, including lung, vulva, esophagus, and head and neck, and clear cell cancers, especially of renal and ovarian origin (187-191). The parathyroid glands appear normal or atrophic histologically.

HYPOPARATHYROIDISM The most common parathyroid pathology found in patients with hypoparathyroidism is four normal glands. Unfortunately, they often have been surgically removed from the patient! Accidental excision of normal parathyroid glands during the course of neck surgery, especially thyroid surgery, is an u n c o m m o n but unfortunately not a rare event (8). In addition to actual excision of the glands, injury to their vascular supply may cause their infarction, or they may be so damaged that they become functionally absent.

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Infiltration Impaired parathyroid function caused by infiltration of parathyroid glands has been described in hemochromatosis, amyloidosis, and metastatic carcinomas. These are all rare causes of hypoparathyroidism (192).

Radiation Rarely, patients are reported who have developed hypoparathyroidism after radioactive iodine treatment for hyperthyroidism. The presumed mechanism is radiation damage to and fibrosis of the parathyroids (193).

Autoimmune Parathyroid Destruction Lymphocytic infiltration of parathyroid tissue, with subsequent autoimmune destruction of the glands, is probably the most common cause of hypoparathyroidism (noniatrogenic cause). It may occur as an isolated event or in association with autoimmune diseases of other endocrine organs, i.e., thyroid, adrenal, or ovary (194-197).

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41. Harrison TS, Duarte B, Reitz RE, et al. Primary hyperparathyroidism: Four to eight year postoperative follow-up demonstrating persistent functional insignificance of microscopic parathyroid hyperplasia and decreased autonomy of parathyroid hormone release. Ann Surg 1981;194:429-437. 42. McGregor DH, Lotuaio LG, Chu LH. Functioning oxyphil adenoma of parathyroid gland. An ultrastructural and biochemical study. A m J Patho11978;92:691-703. 43. Ordonez NG, Ibanez ML, MacKay B, et al. Functional oxyphil cell adenomas of parathyroid gland: Evidence of hormonal activity in oxyphil cells. A m J Clin Patho11982;78:681-689. 44. Rodriquez FH, Sarma DE Lunseth JH, Guileyardo JM. Primary hyperparathyroidism due to an oxyphil adenoma. Am J Clin Pathol 1983;80:878-880. 45. Bedetti CD, Dekker A, Watson CG. Functioning oxyphil cell adenoma of the parathyroid gland: A clinicopathologic study of ten patients with hyperparathyroidism. Hum Pathol 1984;15:1121-1126. 46. Jones SH, Dietler E Oxyphil cell adenoma as a cause of hyperparathyroidism. Am J Surg 1981 ;141:744-745. 47. Baloch ZW, LiVolsi VA. Oncocytic lesions of the neuroendocrine system. Semin Diagn Patho11999;16:190-199. 48. Nathaniels EK, Nathaniels AM, Wang CA. Mediastinal parathyroid tumors: A clinical and pathological study of 84 cases. Ann Surg 1970;171:165-170. 49. Russell CE Edis AJ, Scholz DA, et al. Mediastinal parathyroid tumors: Experience with 38 tumors requiring mediastinotomy for removal. Ann Surg 1981;193:805-809. 50. Russell CF, Grant CS, vanHeerden JA. Hyperfunctioning supernumerary parathyroid glands: An occasional cause of hyperparathyroidism. Mayo Clin Proc 1982;57:121-124. 51. Edis AJ, Purnell DC, vanHeerden JA. The undescended "parathymus": An occasional cause of failed neck exploration for hyperparathyroidism. Ann Surg 1979;190:64-68. 52. Sloane JA, Moody HC. Parathyroid adenoma in submucosa of esophagus. Arch Pathol Lab Med 1978;102:242-243. 53. Spiegel AM, Marx SJ, Doppmann JL, et al. Intrathyroidal parathyroid adenoma or hyperplasia. JAMA 1975;234:1029-1033. 54. Kobayashi T, Man IM, Shin E, et al. Hyperfunctioning intrathyroidal parathyroid adenoma: Report of two cases. Surgery Today 1999;29:766-768. 55. Schwindt WD. Multiple parathyroid adenomas. JAMA 1967;199:945-946. 56. Verdon CA, Edis AJ. Parathyroid "double adenomas." Fact or fiction? Surgery 1981;90:523-526. 57. Harness JK, Ramsbury SR, Nishiyama RH, Thompson NW. Multiple adenomas of the parathyroids; do they exist? Arch Surg 1979;114:468-474. 58. Seyfar AE, Sigdestad JB, Hirata RM. Surgical considerations in hyperparathyroidism: Reappraisal of the need for multigland biopsy. A m J Surg 1976;132:338-340.

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109. Merlano M, Conte P, Scarsi P, et al. Nonfunctioning parathyroid carcinoma. A case report. Tumori 1985;71:193-196. 110. Yamashita H, Noguchi S, Nakayama I, et al. Light and electron microscopic study of nonfunctioning parathyroid carcinoma. Acta PatholJpn 1984;34:123-132. 111. Zisman E, Buckle RM, Deftos LJ, et al. Production of parathyroid hormone by metastatic parathyroid carcinoma. Am J Med 1971 ;45:1068:619-623. 112. Wang CA, Vickery AL, Maloof E Large parathyroid cysts mimicking thyroid nodules. Ann Surg 1972;175:448-453. 113. Ginsberg J, Young JEM, Walfish PG. Parathyroid cysts. JAMA 1978;240:1506-1507. 114. Thacker WC, Wells VH, Hall ER. Parathyroid cysts of the mediastinum. Ann Surg 1971;174:969-975. 115. Hoehn JG, Beahrs OH, Woolner LB. Unusual surgical lesions of the parathyroid gland. A m J Surg 1969;118:770-778. 116. Troster M, Chiu HE McLarty TD. Parathyroid cysts: Report of a case with ultrastructural; observations. Surgery 1978;83:238-242. 117. Earll JM, Cohen A, Lundberg GD. Functional cystic parathyroid adenoma. Am J Surg 1969; 118:100-103. 118. Albertson DA, Marshall RB, Jarman WT. Hypercalcemic crisis secondary to a functioning parathyroid cyst. Am J Surg 1981;141:175-177. 119. Clark OH. Hyperparathyroidism due to primary cystic parathyroid hyperplasia. Arch Surg 1978;113:748-750. 120. SilvermanJE Khazanie PG, Norris T, Fore WW. Parathyroid hormone (PTH) assay of parathyroid cysts examined by fine needle aspiration biopsy. A m J Clin Patho11986;86:708-776. 121. Marco V, Carrasco MA, Marco C, Bauza A. Cytomorphology of a mediastinal parathyroid cyst. Acta Cyto11983;27:688-692. 122. Gough IR. Parathyroid cysts. Aust N Z J Surg 1999;69:404-406. 123. Shields TW, Immerman SC. Mediastinal parathyroid cysts revisited. Ann Thorac Surg 1999;67:581-590. 124. Shi B, Guo H, Tang N. Treatment of parathyroid cysts with fine needle aspiration. Lancet 1999;2:797-798. 125. Wolff M, Goodman EN. Functioning lipoadenoma of supernumerary parathyroid gland in the mediastinum. Head Neck Surg 1980;2:302-307. 126. Grimelius L, Johansson H, Lindquist B. A case of unusual stromal development in a parathyroid adenoma. Acta Chir Scand 1972; 138:628-629. 127. Ober WB, Kaiser GA. Hamartoma of the parathyroid. Cancer 1958; 11:601-606. 128. Perosio P, Brooks JJ, LiVolsi VA. Orbital brown tumor as initial manifestation of parathyroid lipoadenoma. Surg Pathol 1988;1:77-82. 129. Reddick RL, Costa JC, Marx sJ. Parathyroid hyperplasia and parathyromatosis. Lancet 1977; 1:549. 130. Fitko R, Roth SI, Hines JR, et al. Parathyromatosis in hyperparathyroidism. Hum Patho11990;21:234-237. 131. Kollmorgen CF, Aust MR, FerreiroJA, et al. Parathyromatosis: A rare yet important cause of persistent or recurrent hyperparathyroidism. Surgery 1994;116:111-115. 132. Kovacs KA, Gay JDL. Remission of primary hyperparathyroidism due to spontaneous infarction of a parathyroid adenoma: Case report and review of the literature. Medicine 1998;77:398-402. 133. Roth SI, Wang CA, Potts JT. The team approach to primary hyperparathyroidism. Hum Pathol 1975;6:645-658. 134. LiVolsi VA, Hamilton R. Introperative assessment of parathyroid gland pathology. A common view from the surgeons and the pathologist. A m J Clin Pathol 1994;102:365-373. 135. Dufour DR, Durkowski C. Sudan IV staining: Its limitations in evaluating parathyroid functional status. Arch Pathol Lab Med 1987;106:224-227.

136. King DT, Hirose FM. Chief cell intracytoplasmic fat used to evaluate parathyroid disease by frozen section. Arch Pathol Lab Med 1979;103:609-612. 137. Kasden EJ, Cohen RB, Rosen S, Silen W. Surgical pathology of hyperparathyroidism: Usefulness of fat stains and problems in interpretation. Am J Surg Pathol 1981 ;5:381-384. 138. Ljungberg O, Tibblin S. Perioperative fat staining of frozen sections in primary hyperparathyroidism. AmJPatho11979;95:633-642. 139. Dekker A, Watson CG, Barnes EL. The pathologic assessment of primary hyperparathyroidism and its impact on therapy: A prospective evaluation of 50 cases with oil-red-O stain. Ann Surg 1979;190:671-675. 140. Monchik JM, Farrugia R, Teplitz C, Brown S. Parathyroid surgery: The role of chief cell intracellular fat staining with osmium carmine in the intraoperative management of patients with hyperparathyroidism. Surgery 1983;94:877-886. 141. Bondeson AG, Bondeson L, Ljundberg O, Tibblin S. Fat staining in parathyroid disease mdiagnostic value and impact on surgical strategy. Hum Pathol 1985;16:1255-1263. 142. Wang CA, Ryder SV. A density test for the intraoperative differentiation of parathyroid hyperplasia from neoplasia. Ann Surg 1978;187:63-67. 143. Westra WH, Pritchett DD, Udelsman R. Intraoperative confirmation of parathyroid tissue during parathyroid exploration. Am J Surg Pathol 1998;22:538-544. 144. Roth SI, Marshall RB. Pathology and ultrastructure of human parathyroid glands in chronic renal failure. Arch Intern Med 1969;124:397-407. 145. Malmaeus J, Grimelius L, Johansson H, et al. Parathyroid pathology in hyperparathyroidism secondary to chronic renal failure. Scan J Urol Nephrol 1984;18:75-84. 146. Akerstrom G, Malmaeus J, et al. Histological changes in parathyroid glands in subclinical and clinical renal disease. ScandJ Urol Nephro11984; 18: 75-84. 147. Rattner DW, Marrone GC, Kasdon E, Silen W. Recurrent hyperparathyroidism due to implantation of parathyroid tissue. Am J Surg 1985;149:745-748. 148. Akerstrom G, Rudberg C, Grimelius L, Rastad J. Recurrent hyperparathyroidism due to preoperative seeding of neoplastic or hyperplastic parathyroid tissue. Acta Chir Scand 1988;154-219. 149. Jansson S, Tisell LE. Autotransplantation of diseased parathyroid glands into subcutaneous abdominal adipose tissue. Surgery 1987;101:549-556. 150. Max MH, Flint LM, Richardson JD, et al. Total parathyroidectomy and parathyroid autotransplantation in patients with chronic renal failure. Surg Obstet Gyneco11981;153:177-180. 151. Krause MW, Hedinger CE. Pathologic study of parathyroid glands in tertiary hyperparathyroidism. Hum Pathol 1985;16:772-784. 152. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990;108:1006-1013. 153. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987;107:54-60. 154. Harach HR, Jasane B. Parathyroid hyperplasia in multiple endocrine neoplasia type 1. Histopathology 1992;20:305-313. 155. Law WM, Carney JA, Heath H. Parathyroid glands in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). A m J M e d 1989;76:1021-1026. 156. Thorgeirsson U, Costa J, Marx SJ. The parathyroid glands in familial hypocalciuric hypercalcemia. Hum Pathol 1981; 12:229-237. 157. Abati A, Skarulis MC, Shawker T, Solomion D. Ultrasoundguided fine needle aspiration of parathyroid lesions. A mor-

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CHAPTER2 Parathyroid H o r m o n e Biosynthesis and Metabolism

HENRY M. KRONENBERG, E RICHARD BRINGHURST, GINO V. SEGRE, AND JOHN T. POTTS, JR. Endocrine Unit,

Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

BI O SYNTHESIS OF PARATHYROID H O R M O N E

INTRODUCTION The parathyroid h o r m o n e gene has many jobs. It must encode a peptide that can bind to and activate receptors on target tissues. Equally importantly, the a m o u n t of p a r a t h y r o i d h o r m o n e (PTH) p r o d u c e d must be carefully controlled to maintain the blood level of calcium within a narrow range. Nature's solution to these problems has involved the specific synthesis of PTH primarily in the parathyroid chief cell, a cell designed to sense the blood level of calcium. In the chief cell, synthesis and secretion of the h o r m o n e can be carefully regulated. Furthermore, the structure of the h o r m o n e is designed for rapid metabolic degradation, even in the absence of receptor binding. In this way, the rapid turnover of the h o r m o n e can assure that blood levels of h o r m o n e change quickly in response to changes in h o r m o n e secretory rate. This rapid metabolism of h o r m o n e is required of a system designed to respond quickly to sudden changes in the amounts of calcium entering and leaving the bloodstream. Studies over the past two decades have shown that the sequences of PTH and its precursors are designed to steer the h o r m o n e through the chief cell's secretory pathway, to direct the h o r m o n e ' s binding to receptors, and to assure rapid metabolism of the hormone. More recent studies have begun to unravel the mechanisms whereby synthesis of PTH is regulated in the chief cell. Descriptions of the structure of the PTH gene and a summary of the current understanding of how this structure allows the gene to accomplish its multiple functions are presented in this chapter. The Parathyroids, Second Edition

PTH is synthesized as part of the larger precursor molecule, preproparathyroid h o r m o n e (preproPTH). Only trace amounts of this full-length precursor are found in parathyroid chief cells, because the "pre," or signal, sequence is cleaved from the amino terminus while the protein is being synthesized (see Fig. 1). As the signal sequence emerges from the ribosome, it binds to a signal recognition particle, an RNA-protein complex that recognizes signal sequences on most secreted proteins. The signal recognition particle then binds to a receptor on the rough endoplasmic reticulum (docking protein) and directs the nascent p r e p r o P T H molecule to a protein-lined channel, through which the p r e p r o P T H molecule is transported. A signal peptidase located on the inner surface of the m e m b r a n e of the endoplasmic reticulum then cleaves off the signal sequence, leaving the intermediate precursor, proparathyroid h o r m o n e (proPTH) in the cisternae of the endoplasmic reticulum. P r o P T H then travels via a series of vesicles to and through the Golgi apparatus (see Fig. 2). In the Golgi, the short, aminoterminal "pro" sequence is removed, leaving the mature PTH molecule. PTH is then concentrated in dense core secretory vesicles; these vesicles fuse with the plasma m e m b r a n e and release PTH in response to a decrease in extracellular calcium. The h o r m o n e secreted is predominantly the intact 84-residue PTH molecule, though a variable fraction made up of carboxy-terminal PTH fragments is secreted, as well.

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CHAPTER2

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®

O P

1. SRP

BINDING

O

2. DOCKING PROTEIN BINDING

4. MEMBRANE TRANSPORT, PEPTIDE CLEAVAGE

3. MEMBRANE INSERTION

5. TRANSPORT COMPLETED

,

I

FIG. 1 The signal, or pre, sequence directs the nascent polypeptide to the apparatus for transport across the membrane of the endoplasmic reticulum. SRP, Signal recognition particle.

Function of the Pre (Signal) Sequence The specific sequences of each of the three portions of the preproPTH molecule are responsible for directing the h o r m o n e through the complicated pathway of transport and cleavage. The known preproPTH sequences from h u m a n (1), bovine (2), rat (3), pig (4), chicken (5,6), and dog (7) tissues share a 25-residue pre sequence and a 6-residue pro sequence (see Fig. 3). Each pre sequence contains a hydrophobic stretch of

amino acids preceded by a positively charged residue. The signal sequence ends with a small amino acid at the last and third-to-last positions. These characteristics are typical of most signal sequences. The preproPTH signal sequence was first discovered (8) when parathyroid gland mRNA was translated in a cell-free extract devoid of endoplasmic reticulum. Directed mutations have demonstrated the importance of each of the regions of

AA, AAA

PTH "-------

FIG. 2 Multiple cleavages occur during the intracellular transport of PTH.

PTH BIOSYNTHESISAND METABOLISM /

human bovine porcine rat canine chicken

PRE $ PRO $ -31 -6 MIPAKDMAKVMIVNLAICFLTKSDG KSVKKR MMSAKDMVKVMIVNLAICFLARSDG KSVKKR MMSAKDTVKVMVVNLAICFLARSDG KPIKKR MMSASTMAKVMILMLAVCLLTQADG KPVKKR MMSAKDMVKVMIVMFAICFLAKSDG KPVKKR MTSTKNLAKAIVILYAICFFTNSDG RPMNKR

PTH +i +i0 SVSEIQLMHN AVSEIQFMHN SVSEIQLMHN AVSEIQLPIHN SVSEIQFMHN SVSEMQLMHN

human bovine porcine rat canine chicken

+20 +30 +40 +50 LGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG SQNPRK L G K H L S S M N R V E W L R K K L Q D V H N F V A L G A S IA Y R D G S S Q N P R K LGKHL S SLNNVEWLRKKLQDVHNFVALGAS IVHRDGG SQRPRK LGKHLASVERMQWLRKKLQDVH FVSLGVQMAAREGSYQNPTK L G K H L S S M N N V E W L R K K L Q D V H N F V A L G A P IA H R D G S S Q N P L K L G E H R H T V E N Q D W L Q M K L Q D V H . . S A L E ...... D A R T Q R P R N

human bovine porcine rat canine chicken

+60 +70 +80 DKADVNVLTKAKSQ KEDNVLVE...SHEKSLGEA K E D N V L V E . . . S H Q K S L G E A .......... D K A D V D V L I K A K P Q K E D N V L V E . . . S H Q K S L G E A .......... D K A A V D V L I K A K P Q K E E N V L V D . . . G N S K S L G E G .......... D K A D V D V L V K A K S Q K E D N V L V E . . . S Y Q K S L G E A .......... D K A D V D V L T K A K S Q KEDIVLGEIRNRRLLPEHLRAAVQKKSIDLDKAYMNVLFKTKP. .

.

.

.

.

.

.

.

.

.

FIG. 3 Amino acid sequences of preproPTH from mammalian and avian species. Residues -31 to - 7 constitute the pre sequences; residues - 6 to -1 constitute the pro sequences. Dots represent residues found in chicken PTH without corresponding residues in the mammalian sequences. Amino acids are indicated by the single-letter code: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; Q, Gin; E, Glu; G, Gly; H, His; I, lie, L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; V, Val.

the preproPTH signal sequence for normal signal function (9-11). Further, when a synthetic prepro peptide was added to a cell-free extract, it blocked the transport and cleavage of preproPTH by microsomal membranes (12). Most strikingly, a point mutation was found in the signal sequence of a preproPTH gene in a family with inherited hypoparathyroidism (13). A point mutation at residue 18 changed the cysteine to arginine and thereby inserted a charged residue into the hydrophobic core of the signal sequence. When this mutant preproPTH was expressed in cell-free extracts or in cultured cells, the precursor was inefficiently transported and cleaved (14).

Function of the Pro Sequence The signal sequence of preproPTH, thus, resembles the signal sequences of other secreted proteins and performs the important role of directing the protein across the membrane of the endoplasmic reticulum and into the secretory pathway. The function of the pro sequence is less well established. In all known preproPTH sequences, the pro sequence is six residues long. The first is always positively charged, the third is hydrophobic, and the last two residues are Lys-Arg. This pattern closely resembles that found in rat proalbumin

19

(Arg-Gly-Val-Phe-Arg-Arg) and that predicted to be present in the pro sequence of preproparathyroid hormone-related peptide (Arg-Arg-Leu-Lys-Arg). ProPTH was first discovered as a large PTH-related molecule that was the predominant form of the h o r m o n e found in parathyroid cells after pulse labeling with radioactive amino acids (15,16). Subsequent chase incubations demonstrated that the proPTH was converted to PTH in about 15 minutes; this correlated in time with transport to the Golgi (17). After this time, no trace of the pro peptide or possible fragments could be found in the cell or medium (18). These data strongly suggest that the pro sequence serves an exclusively intracellular function, probably involved in movement through the secretory pathway. Wiren et al. (19) tested this hypothesis by deleting the DNA sequences encoding the pro hexapeptide from cloned cDNA encoding h u m a n preproPTH and by subsequently expressing the cDNA in cell-free protein-synthesizing extracts and in intact rat pituitary GH4 cells. The mutant precursor functioned abnormally in both expression systems. The precursor crossed the membrane of the endoplasmic reticulum inefficiently, and, consequently, the subsequent cleavage of the signal sequence was inefficient. Cells secreted PTH but also secreted a molecule slightly bigger than PTH. Sequence analysis showed that the abnormal protein included the last two residues of the signal sequence. Thus, the removal of the pro sequence resulted in imprecise and inefficient function of the signal sequence. The pro sequence of preproPTH should be considered part of the functional unit responsible for transport and cleavage of the precursor on its entry into the secretory pathway. This result is not surprising. In other precursor proteins, the sequences immediately distal to the signal sequence can affect signal sequence function. One can speculate that the constraints on this region conflict with the constraints on the amino terminus of the mature PTH molecule. The PTH receptor, for example, requires very specific residues at the amino terminus of PTH for subsequent activation of adenylyl cyclase. The experiments of Wiren et al. show that these residues cannot be placed immediately distal to the signal sequence. The pro sequence can be considered a linker region that allows efficient signal sequence function and physically separates the signal sequence from the mature h o r m o n e sequence, which has its own and separate evolutionary constraints. The possibility that the pro sequence has additional functions, such as the promotion of proper folding of the PTH molecule in the endoplasmic reticulum, has not been rigorously examined. The enzyme responsible for cleavage of the pro sequence of proPTH has not yet been characterized, but a n u m b e r of arguments suggest that the protease, furin (or a close relative), is the cleavage enzyme (20).

20

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CI4AeTV.R2

Furin is a subtilisin-like enzyme that is located in the Golgi cisternae of probably all mammalian cells. The enzyme cleaves sequences like the pro sequence of rat proalbumin, which ends in dibasic residues and is preceded by other basic residues. Unlike the related PC2 and PC1 proteases, which are found in cells with secretory granules, furin cleaves precursors in cells like hepatocytes, which have no secretory granules. Cleavage by furin probably explains why proPTH, in contrast to proinsulin, for example, is cleaved normally when the hormone is synthesized in all sorts of cells, from parathyroid chief cells to fibroblasts and kidney cells (21,22). One can only speculate as to why proPTH, which is normally synthesized virtually exclusively in specialized parathyroid chief cells, uses an enzyme designed for cleavage of proteins secreted from nonendocrine cells. One plausible explanation is an evolutionary argument. The parathyroid hormone gene may well be derived from the gene encoding parathyroid hormone-related peptide (PTHrP). The PTHrP gene is widely expressed, both in cells with secretory granules, such as parathyroid chief cells and neurons, and in cells without secretory granules, such as smooth muscle cells. Therefore, it would be expected that the pro sequence of proPTHrP would be designed for cleavage by an enzyme expressed in most cells. The pro sequence of proPTH may well share this property because of its evolutionary heritage, even though proPTH is normally expressed only in cells with a secretory granule apparatus.

Intracellular Roles of the Mature PTH Sequence Like the prepro sequence, portions of the mature PTH molecule serve to facilitate intracellular handling of PTH (23). Shortened versions of preproPTH are not stable in transfected cells. When the h u m a n preproPTH cDNA was modified to encode preproPTH(1-40) (in which the numbers refer to the mature PTH sequence), the signal sequence functioned, and proPTH(1-40) was produced in transfected cells. The proPTH (1-40) was not further cleaved to PTH(1-40), however. Instead, it was degraded intracellularly; no PTH peptides were secreted from the cells. A similar, though less dramatic, defect in secretion was exhibited by preproPTH(1-52). These short precursors were long enough for the signal sequence to direct them into the secretory pathway, but they were unstable and were not transported through the entire pathway. These results may partly explain the role of the carboxy-terminal portion of the PTH molecule. One function of the full 84-residue protein may be to allow stable and efficient transport through the secretory apparatus. Because all secreted peptides are syn-

thesized as rather large precursors, this need for a minimal length of translation product may be a general one for secreted proteins. Of course, this "length" requirement for PTH does not preclude other functions for the carboxy-terminal portion of PTH, such as binding to a distinct PTH receptor (24). Even the 84-residue PTH molecule is not completely stable in the parathyroid chief cell. PTH(1-84) is concentrated in secretory vesicles and granules that contain the proteases, i.e., cathepsins B and H (25,26). This colocalization of proteases and PTH may explain the observation that the hormone secreted by calves in vivo under conditions of hypercalcemia consists largely of carboxy-terminal fragments of PTH (27). Secretion of fragments of PTH was studied in detail by Habener et al. (28) and Chu et al. (29). These workers noted that the degradation of newly synthesized PTH is influenced by the level of extracellular calcium. Few fragments were secreted when the gland was stimulated in vitro by medium containing low levels of calcium. In contrast, most of the hormone secreted under conditions of hypercalcemic suppression consisted of fragments. Thus, calcium regulated the amount of available intact PTH by causing the intracellular degradation of hormone. This effect could have been caused by the activation of a PTH-degrading pathway. Alternatively, the intracellular degradation rate might have been constant; the decrease in total degradation of PTH associated with low calcium levels might simply have resulted from rapid secretion of hormone and the concomitant shorter time of exposure to the intracellular degradation mechanism. Phorbol ester treatment of parathyroid cells in vitro has also been shown to result in the secretion of an increased fraction of PTH fragments, both in high and low calcium concentration conditions (30). Phorbols are either activating a proteolytic mechanism or may be selectively stimulating secretion from secretory granules containing a high proportion of PTH fragments. The physiologic correlate in vivo of this action of phorbol esters has not yet been established. In any case, the parathyroid gland has the capability of varying the fraction of PTH secreted as the biologically active, intact molecule. This seemingly wasteful capability makes it possible for the gland to vary quickly and dramatically the amount of biologically active hormone secreted. This regulatory capability provides a rationale for the intracellular instability of the hormone. To sum up, it can be seen that all portions of the preproPTH molecule have intracellular functions. The prepro region is required for efficient introduction of the hormone to the secretory pathway. The carboxyterminal region of the mature hormone is required for efficient and stable transport of PTH through the secre-

PTH BIOSYNTHESISAND METABOLISM / tory pathway. I n h e r e n t instability of even the full-length h o r m o n e provides a regulatory mechanism that allows extracellular calcium to alter rapidly the a m o u n t of active h o r m o n e available for secretion.

THE PARATHYROID HORMONE GENE The genomic DNAs encoding h u m a n (31), bovine (32), rat (33), and chicken (34) p r e p r o P T H have been cloned; the complete sequences of the h u m a n (35) and bovine (32) genes have been determined. Each gene contains three exons separated by two introns (see Fig. 4). The introns vary in size from species to species, though the first intron is invariably large, and the second intron in the human, bovine, and rat genes is about 100 base pairs in length. This length is close to the m i n i m u m length that can be recognized by the splicing machinery. The introns interrupt the sequences encoding mRNA at precisely the same locations in each species. The first exon contains most of the 5' n o n c o d i n g sequence. The second exon encodes most of the prepro sequence; the second intron comes in the middle of the triplet encoding the lysine residue that precedes the dibasic cleavage sequence Lys-Arg found at the end of the known pro sequences. The third exon encodes the Lys-Arg sequence, the mature PTH sequence, and the 3' noncoding region of the gene. The h u m a n and bovine genes are preceded by two functional TATA boxes that determine the two closely spaced start sites of the h u m a n and bovine transcripts. The rat and chicken genes are preceded only by one TATA box, found in a position equivalent to the second TATA box in the h u m a n and bovine genes. T h o u g h both start sites of transcription are used in the h u m a n and bovine genes, no conditions have been found that favor the use of one start site over the other. No data suggest that the two transcripts have importantly different stabilities or translatability, but such questions have not been exhaustively studied. The 5' n o n c o d i n g regions of each gene extend approximately 120 base pairs. The 3' n o n c o d i n g

iii !il!i iiiii!!i!iiiiii!iii! !iiiii!iiii!iiii!i~i!i~ii ii i liiii i!i iililiii ~~ii~!iiii

iiii!i i~i!iilililiiii i iiiiiii!illi!i!!ii!iiiiiii ii!i!iiiiiiiil iiliiiii ii!iil iiili!iiiiil!iiiiiiiiiii!i~iiiii!iiiiiil

ii

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!!!!!!!!!!!!! !!

!!!ii i!ii!iiiiiiii!iii !iili!i!iiiiiiiii!i!i!i!iiiiiii!iiiiiiiiiiiiiiilii i!iiiii!i!iiiii!!i!i!!!!!!ii i i i!i i~i~~ii~iii! !ill!if!!i~!~ ili i! i!ii! i! !i!i!iiii!ii!!iiiii!ii! i iiiiiii ii!iil FIG. 4 The parathyroid hormone gene. NC, Noncoding.

21

regions of each gene vary substantially in length, from the bovine at 227 base pairs to the chicken at more than 1600 base pairs. The 3' n o n c o d i n g region binds proteins that may regulate the stability of the p r e p r o P T H mRNA (36,37). The human, rat, and bovine PTH genes are represented only once in the haploid genomes of each species. The h u m a n PTH gene is located on the short arm of chromosome 11 at band 1 lp15 (38-40). A series of restriction fragment length polymorphisms (41,42) have made it possible to show that the h u m a n PTH gene is linked to the genes encoding catalase, calcitonin, H-ras, insulin, and [3-globin (43). Two other polymorphisms have been identified through the use of denaturing gel electrophoresis (44). All of these polymorphisms have proved useful in defining the inheritance of specific alleles of the PTH gene in families with calcium disorders (45). Several features of the PTH gene suggest that the gene is related to that encoding PTHrP (46-48). Most importantly, the major coding exon of both genes starts precisely at the same nucleotide, one base before the codons encoding the Lys-Arg residues of the pro sequences of each hormone. After the Lys-Arg sequences, the PTH and PTHrP amino acid sequences are identical in 8 of the next 13 residues. Further, the PTHrP gene is located on c h r o m o s o m e 12, a chromosome known to encode many genes that resemble genes on c h r o m o s o m e 11; for this reason, the chromosomes are t h o u g h t to have arisen by an ancient duplication event (49). One can speculate that the PTH gene may represent a variation of the PTHrP gene; the PTH h o r m o n e takes advantage of the PTHrP receptor in order to regulate calcium metabolism. If this hypothesis is correct, then the gene had to change in order to assure expression primarily in the parathyroid chief cell and to assure appropriate regulation by modulators such as extracellular calcium and 1,25-dihydroxyvitamin D [ 1,25 (OH) zOo]. A hypothalamic peptide called TIP39 (50) has been found to activate the PTH2 receptor and to be distantly related in sequence to PTH and PTHrE The structure of the TIP39 gene has not yet been reported. This gene may represent a third member of the PTH gene family.

REGULATION OF PTH BIOSYNTHESIS The minute-to-minute stability of the level of blood calcium depends on the regulation of PTH secretion by calcium. Longer term homeostasis depends on several other levels of control (see Fig. 5). The n u m b e r of parathyroid chief cells is carefully regulated; when appropriately stimulated, the parathyroid glands can

22

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C~a'TER2

TRANSCRIPTION/mRNA STABILITY

Ca, 1,25 D, PO4 Ca

Ca, 1,25 D

P H SECRETION PROLIFERATION

FIG. 5

Levels of parathyroid cell regulation.

increase in size dramatically. The parathyroid chief cell is uniquely designed to express the PTH gene; the state of differentiation of the chief cell can, therefore, influence the rate of PTH biosynthesis. Specific blood-borne signals, most notably calcium and 1,25(OH)zD3, regulate the activity of the PTH gene, as well. These several levels of regulation of PTH biosynthesis are examined in the following discussions (see also Chapter 18).

Regulation of Parathyroid Cell Number Little is known about the regulation of parathyroid cell number. The relatively uniform morphology of chief cells suggests that all chief cells have the potential to divide, if appropriately stimulated, but the alternative hypothesis that a subset of chief cells has the unique, stem-cell-like capability to proliferate has not been evaluated. Further, there is the general impression that parathyroid cells are long-lived, because mitoses are seldom seen in normal glands of mature animals, because the observed rate of apoptosis is low, and because hyperplastic glands only slowly decrease in size after stimulation. Nevertheless, specific studies to define potential modulators of chief cell longevity have not been performed. Despite this paucity of information, the dramatic hyperplasia of parathyroid cells in patients and animals with renal failure demonstrates the likely roles of calcium, phosphate, and 1,25(OH)2D 3 in regulating parathyroid cell proliferation. Dietary manipulation alone can similarly lead to chief cell hyperplasia. Naveh-Many and Silver (51), for example, used flow cytometry to count parathyroid cells and showed that 3 weeks of a calcium- and vitamin D-deficient diet fed to weanling rats led to a 1.7-fold increase in parathyroid cell number. These investigators subsequently studied the mechanism of the increase in parathyroid cell number caused by hypocalcemia, hyperphosphatemia, vitamin D deficiency, and uremia in vivo (52). They found that hypocalcemia and

uremia led to increases in parathyroid cell proliferation, whereas hypophosphatemia led to decreases in parathyroid cell proliferation. Administration of 1,25(OH)2D 3 for 3 days had no effect on parathyroid cell proliferation. None of these conditions led to changes in the rate of parathyroid cell apoptosis. Further studies of the effects of calcium in the uremic model suggest that calcium works by acting on the same calcium-sensing rector that mediates the actions of calcium on PTH secretion. The calcimimetic compound NPS R-568, like calcium, suppressed parathyroid cell proliferation in uremic rats (53). The possibly i n d e p e n d e n t roles of calcium and 1,25(OH)2D 3 in the regulation of parathyroid cell proliferation have not been studied extensively. In vivo, these variables are difficult to manipulate independently in the intact animal. One particularly instructive in vivo model, the vitamin D receptor knockout mouse, has been studied, however (54,55). These mice develop hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism in the days and weeks after weaning. When the hypocalcemia and hypophosphatemia are prevented by a diet high in calcium, phosphate, and lactose, the hyperparathyroidism and parathyroid gland enlargement are prevented. Because these mice lack vitamin D receptors, they must be able to regulate parathyroid cell n u m b e r without using the genomic actions of 1,25 (OH) 2D~. Presumably, the direct effects of normal calcium and phosphate are sufficient to prevent parathyroid cell replication. In studies of cultured parathyroid chief cells, it has been possible to vary the levels of calcium and 1,25(OH)2D ~ separately. Several groups have shown that 1,25(OH)2D 3 can regulate parathyroid cell proliferation in vitro. Whether the cells were grown in the presence of serum (56,57) or serum-free growth factors (58), administration of 1,25(OH)2D 3 decreased their rate of proliferation. Studies of the effects of calcium on parathyroid cell proliferation in vitro have yielded differing results. Several studies (59-61) have shown that lowering of calcium leads to increased cellular proliferation. Other studies of dispersed, early-passage chief cells have demonstrated no effect of calcium on the rate of cell proliferation, however (57,58,62). Though extracellular levels of calcium and 1,25(OH)2D~ can be independently regulated in vitro, it is hard to be sure that parathyroid cells in culture respond to modulators of proliferation in this setting in the same way that they do in vivo. Thus, though the combined effects of low calcium and low levels of 1,25(OH)2D ~ to stimulate parathyroid cell proliferation are well established, the individual roles of calcium, phosphate, and 1,25 (OH)2D~ in vivo remain uncertain.

PTH BIOSYNTHESISAND METABOLISM /

Cell-Specific PTH Gene Expression Expression of the parathyroid h o r m o n e gene occurs almost exclusively in the parathyroid chief cell. [Expression has also been noted in the rat hypothalamus (63).] Thus, genes required for parathyroid chief cell differentiation are possible candidates for genes that might regulate the PTH gene as well. These genes, identified through the study of knockout mice, include hoxa3 (64,65), pax9 (66), and glial cells missing 2 (67). The hoxa3 and pax9 mutant mice lack a range of branchial arch derivatives, whereas the glial cell missing knockout mouse exhibits highly selective parathyroid cell deficit. When the chief cell is disrupted by neoplastic transformation, the regulation of PTH gene expression can be altered. For example, parathyroid cancers may stop synthesizing PTH completely (68). Presumably, specific DNA sequences associated with the PTH gene respond to the environment of the chief cell to activate gene expression. Because no well-differentiated cell line expressing the PTH gene has been established, it has been difficult to determine the sequences responsible for chief cell-specific PTH gene expression. Occasional "experiments of nature" have provided important clues, however. Very rarely, h u m a n nonparathyroid tumors have been found to produce PTH ectopically, for example. In one case that was studied carefully (69), the PTH regulatory region upstream from the gene was disrupted in tumor cells. Presumably, this gene r e a r r a n g e m e n t allowed the gene to be expressed in nonparathyroid cells by providing new regulatory signals or abolishing normal silencing mechanisms found upstream of the gene. Further, in a subset of parathyroid adenomas, the entire upstream portion of the PTH gene along with the first, noncoding exon are separated from the rest of the gene and rearranged adjacent to the PRAD1 gene (70). As a consequence of this rearrangement, the PRAD1 gene (encoding cyclin D1), a regulator of the cell cycle, is dramatically overexpressed. These observations suggest that the PTH gene upstream region contains sequences that stimulate gene transcription in parathyroid chief cells. Further analysis of the sequences that determine chief cell expression of the PTH gene m u s t await studies of transgenic animals or the establishment of welldifferentiated parathyroid chief cell lines.

Modulators of PTH Gene Expression The effects of calcium on PTH gene expression were first demonstrated in experiments using primary parathyroid cells in culture. Russell et al. (71) found that high levels of calcium resulted in a decrease in PTH mRNA levels over a several-day period. In those

23

studies, no difference was noted between the effects of low and normal levels of extracellular calcium. The decrease in PTH mRNA levels in response to high calcium levels could be reversed by lowering the calcium level; thus, the suppressive effect of calcium was not an irreversible, toxic effect. These in vitro observations have been confirmed by Brookman et al. (72), who noted a slight increase in PTH mRNA u n d e r low calcium level conditions at one time point. Subsequent studies by Russell et al. (73) showed that the rate of transcription of the PTH gene in nuclei of dispersed bovine parathyroid cells fell within 6 hours in response to high levels of extracellular calcium. The rate of transcription of the actin gene was unchanged; therefore, the effect of calcium was shown to be specific. The lack of parathyroid cell lines that produce PTH has h a m p e r e d the search for DNA sequences responsible for the transcriptional effects of calcium noted in cultured parathyroid cells. Okazaki et al. (74) have identified short sequences several thousand base pairs upstream from the start site of PTH gene transcription that may well be important for calcium regulation, however. These investigators identified the region by showing that several short sequences in the region could decrease gene transcription from many different promoters, including the PTH gene p r o m o t e r (75). Further, when the level of extracellular calcium was varied, after transfection of fusion genes containing a short oligonucleotide from this region, high calcium levels further suppressed transcription from genes containing the sequence but had no effect on control plasmids. Intriguingly, almost identical sequences were found in the gene encoding rat atrial natruiretic polypeptide, another gene negatively regulated by calcium. This DNA sequence could also confer calcium sensitivity to a fusion gene in fibroblast transfection experiments. T h o u g h these experiments are very suggestive, further studies will be required to show that the regulatory region can confer calcium sensitivity in its normal location far upstream from the PTH gene transcription start site. Ultimately, studies using welldifferentiated parathyroid cells will be required. Two groups have studied the acute effects of changes in blood calcium on PTH mRNA levels in the intact rat. Both showed that acute lowering of blood calcium (with phosphate, calcitonin, or EDTA) led to a p r o m p t increase in PTH mRNA levels (76,77). Elevations in blood calcium, in contrast, led to no change in PTH mRNA levels after 6 hours (76) and to a slight decrease in PTH mRNA levels after 48 hours (77). The parathyroid gland apparently, then, in the normal state, rests near the bottom of the calcium dose-response curve. The gland is well equipped to increase PTH production, but poorly prepared to decrease production in the

24

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CHAPTER2

face of hypercalcemia. Subsequent studies showed that changes in PTH mRNA caused by hypocalcemia in vivo are not caused by a transcriptional mechanism, but rather are caused by changes in mRNA stability (37). 1,25(OH)zD 3 has been shown to be an i m p o r t a n t regulator of PTH gene transcription in studies both in vitro and in vivo. Silver et al. (78) used primary parathyroid cells in culture to show that exposure to 1,25(OH)2D 3 led to a decrease in PTH mRNA levels. This work has been confirmed by studies of Karmali et al. (79) and Brown et al. (80). Russell et al. (81) then showed that 1,25(OH)zD 3 lowers the PTH gene transcription rate as early as 2 hours after exposure of cells to 1,25 (OH)zD 3. Similarly, in intact rats, intraperitoneal injections of 1,25(OH)zD 3 rapidly led to decreased transcription of the PTH gene and decreased PTH mRNA levels (82). The doses of 1,25(OH)zD 3 were so low that blood calcium did not change; the precise blood levels of 1,25(OH)zD 3 required to suppress PTH gene transcription acutely in vivo have not been established, however. The effects of low levels of 1,25(OH)zD 3 have not been studied extensively in intact animals. Such studies are difficult to interpret, because of confounding effects of vitamin D deficiency on blood calcium and parathyroid cell number. Weanling rats fed a vitamin Ddeficient diet for 3 weeks had a modest increase in their PTH mRNA levels (51). This increase occurred with no a p p a r e n t decrease in blood calcium levels. In the intact organism, calcium and 1,25(OH)zD 3 seld o m vary independently; consequently, the effects of changes in both parameters simultaneously have important physiologic relevance. When rats were made acutely hypocalcemic with phosphate and were at the same time given 1,25(OH)zD 3 intraperitoneally, the suppressive effect of 1,25(OH)2D 3 reversed the effect of hypocalcemia and led to a decrease in PTH mRNA (76). In contrast, when rats were fed a low-calcium diet for 3 weeks, blood calcium levels decreased and blood 1,25 (OH) 203 levels increased dramatically. In this setting, PTH mRNA levels rose severalfold; thus, the effects of low calcium levels were more influential than the effects of high 1,25(OH)zD 3 levels. The partial vitamin D resistance of the parathyroid gland in the setting of hypocalcemia makes sense physiologically: in that setting the action of vitamin D to increase intestinal calcium absorption is needed, but the action to inhibit PTH synthesis is not. Sela-Brown et al. (83) studied the mechanism of hypocalcemia-induced resistance to vitamin D action on the parathyroid gland. They showed that hypocalcemia in vivo induces nuclear accumulation of calreticulin, a calcium-binding protein, in parathyroid chief cells, and that calreticulin can interfere with the actions of the vitamin D receptor on a negative vitamin D response e l e m e n t in transfected cells in vitro.

In experimental uremia, the double stimulus of hypocalcemia and low levels of 1,25(OH)zD 3 has consistently led to increases in PTH mRNA (84,85). Administration of 1,25(OH)zD 3 could reverse this increase. This effect of 1,25(OH)zD 3 is likely to contribute importantly to the decrease in PTH blood levels seen in dogs with experimental uremia (86) and in dialysis patients (87). A series of transfection studies and DNA binding assays have been used to identify DNA sequences in the PTH gene responsible for modulating transcription of the PTH gene in response to 1,25(OH)2D 3. When a fusion gene containing 684 base pairs (bp) of DNA upstream of the h u m a n PTH gene was introduced stably into rat pituitary GH4 cells, expression of the gene was specifically suppressed by 1,25(OH)2D 3 (88). Three groups have identified DNA sequences upstream of the PTH gene that bind to 1,25(OH)zD 3 receptors in vitro. Filter binding assays showed that 1,25(OH)zD 3 receptors can bind to bovine PTH gene sequences between - 4 8 5 and - 1 0 0 bp upstream from the transcription start site (89). Subsequently, gel mobility-shift assays were used to identify a specific 26-bp sequence, located 125 bp upstream from the start site of transcription of the h u m a n PTH gene, that binds 1,25 (OH)203 receptors (90). When this short sequence was linked to a reporter gene and expressed in pituitary GH4 cells, 1,25(OH)zD 3 decreased expression of the reporter gene. This suppression of transcription was even greater when the n u m b e r of 1,25(OH)zD 3 receptors in the GH4 cells was increased by cotransfection of a 1,25(OH)zD 3 receptor expression vector. The h u m a n negative 1,25 (OH)203 (vitamin D) response e l e m e n t (VDRE) contains one copy of a motif found in two copies in the mouse osteopontin gene, a gene up-regulated by 1,25(OH)zD 3. Negative VDREs have also been identified in the chicken (91) and rat (92) PTH genes. These sequences closely resemble positive VDREs and have been shown to bind heterodimers of the vitamin D receptor and RXR, just as positive VDREs do. Subtle differences in binding interactions may explain why these particular VDREs in the PTH gene can act as negative VDREs with vitamin D receptor-RXR heterodimers (93). Until recently, the effects of phosphate on the parathyroid cell were t h o u g h t to be indirectly mediated by the hypocalcemia associated with increases in blood phosphate. The rapid actions of changes in phosphate levels in vivo on PTH secretion work through such a mechanism. However, studies using intact rat parathyroid glands in vitro demonstrate that changes in phosphate levels can, after several hours, lead to changes in PTH secretion (94,95). PTH mRNA did not change in these studies in vitro, but analogous studies p e r f o r m e d in intact rats d e m o n s t r a t e d that phosphate, in the setting of apparently constant levels of calcium and

PTH BIOSYNTHESISAND METABOLISM / 1,25(OH)2D ~, increases PTH mRNA by a posttranscriptional mechanism (96). Though calcium and 1,25(OH)zD 3 are certainly the most important physiologic regulators of PTH gene transcription, other circulating factors are likely to modulate PTH gene transcription as well. The PTH gene contains a consensus cyclic AMP response element that can function in the context of a fusion gene in transfection experiments (97). Thus, hormones that stimulate adenylyl cyclase may increase PTH gene transcription. Glucocorticoids have been shown to increase PTH mRNA in dispersed, hyperplastic h u m a n parathyroid cells (98) and to abolish the decrease in PTH mRNA in response to 1,25(OH)zD ~ in dispersed bovine parathyroid cells (79). These cell culture studies need to be confirmed by studies in vivo to determine their physiologic significance. In ovariectomized rats, estradiol administration led within 24 hours to a fourfold increase of PTH mRNA (99). Estrogen receptors were identified in rat parathyroids. These observations may have important implications for an understanding of postmenopausal osteoporosis and hyperparathyroidism. The possibility that the effect of estrogen on PTH mRNA levels is a direct effect on the parathyroid gland needs to be tested by studies using cultured parathyroid cells.

Peripheral Metabolism of PTH Intact PTH is rapidly cleared from the circulation with a disappearance half-time of approximately 2 minutes (100-103). Removal of PTH from the blood occurs mainly (60-70%) in the liver but also in the kidneys (20-30%) and, to a much lesser extent, in other organs (100,102,103). Clearance of PTH by the liver is mediated mainly by a high-capacity, nonsaturable uptake by Kupffer cells and is followed by rapid and extensive proteolysis (104). Renal clearance occurs almost entirely by glomerular filtration. The hormone is also reabsorbed by the renal tubules and then extensively degraded, so that little or no intact PTH appears in the final urine (102). A large membrane-bound protein, megalin, binds PTH (but not carboxy-terminal fragments of PTH) in the lumen of the proximal tubule to initiate this reabsorption (105). In both the liver and kidney, as in bone, some PTH is removed by high-affinity binding to cell surface receptors, but this constitutes only a small fraction (

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CHAPTER3

3' mRNA termini (and, therefore, of the three alternative PTHrP peptides as well) would be to employ transcription terminator sequences of graded efficiencies, so that, for example, 33% of transcripts terminate after exon 6, 50% of those remaining terminate following exon 7, and 100% of the rest terminate beyond exon 8. Splicing would then be perfunctory, and not regulatory, in nature, although what regulatory mechanisms for PTHrP gene expression might exist at the level of transcription termination is presently unknown. The complexity in structure and processing evident in the h u m a n PTHrP gene seems to represent a relatively recent p h e n o m e n o n in evolutionary terms, because the organization of the gene is much simpler in lower species. The PTHrP gene in the rodent (both rat and mouse) appears to employ only a single TATA p r o m o t e r and contains only four exons (23-25) (Fig. 3). Exon 1 encompasses most of the 5' untranslated region, exon 2 encodes the "prepro" region from amino acids - 3 6 to - 8 , exon 3 encodes the r e m a i n d e r of the pro sequence (amino acids - 7 to - 1 ) and the bulk of the mature peptide (137 residues in the mouse and 139 in the rat), and exon 4 encodes the final two amino acids, the stop codon, and the 3' untranslated region. As seen in Fig. 3, one possible implication of the a r r a n g e m e n t of exons and introns in the colinear PTHrP genes from rodents and humans is that h u m a n exon 8 (rodent exon 4) represents the ancestral splicing pathway and that the emergence of the splice site defining h u m a n exon 7 occurred at a point in evolution distal to the rodent branchpoint. Interestingly, though the h u m a n exon 7 equivalent is also absent in chickens, the PTHrP gene in this species does display alternative termination 3' to exon 3 (the h u m a n exon 6 equivalent) and consequently generates a 139-residue peptide isoform, a p h e n o m e n o n not seen in rodents (26). This suggests either that the development of this termination site predated mammalian evolution and has been subsequently lost in m o d e r n rodents but retained in the human, or that this mechanism evolved independently in chickens and humans.

PROTEIN STRUCTURE AND POSTTRANSLATIONAL PROCESSING Translation of the three human PTHrP mRNAs, each with alternative 3' termini, generates three protein products with distinct carboxy termini (19-22). Each of these three protein isoforms have 139 amino acids in common; the isoform that undergoes translation termination at a stop codon within exon 6 ends with residue 139, whereas the isoform terminating in exon 8 adds two additional residues for a total of 141 and the isoform terminating in exon 7 extends the 139-amino acid trans-

lated product a further 34 amino acids for a total of 173 (Figs. 2B and 4). Rat PTHrP derives its carboxy terminus from the equivalent of h u m a n exon 8 and is also 141 amino acids in length, whereas the mouse protein, although similarly derived, is only 139 amino acids long due to the deletion of codons 130 and 131 (23-25) (Fig. 4). PTHrP coding sequences are extremely well conserved across species, with the region encompassing residues 1-111 of the mature peptide exhibiting approximately 98% homology between chickens and humans (26). The primary translation products of the human PTHrP gene (and from other species as well) share a c o m m o n 36-amino acid prepro sequence (residues - 3 6 to - 1 , encoded in the h u m a n by exons 3 and 4) that is composed of an N-terminal segment with the typical structural features of a signal peptide (the pre sequence), followed by a short leader peptide (the pro sequence; Fig. 1), the removal of which is required to allow ligand function (i.e., binding to and activation of the P T H / P T H r P receptor) (7-9). By analogy with other secreted proteins, the signal sequence presumably mediates the attachment of the nascent peptide to the endoplasmic reticulum, where it is then cotranslationally removed by signal peptidase as the growing chain is extruded through the membrane. The precise cleavage site is unknown, but is predicted to be in the region of residues - 8 to - 7 . The remaining pro sequence is then presumably cleaved at a tetrabasic site (RRXKR), spanning residues - 5 through - 1 , by the action of furin or a furinlike p r o h o r m o n e convertase within the endoplasmic reticulum or Golgi apparatus (27), thus generating the mature, secreted form(s) of the protein, PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173) (7-9). The function of the pro sequence is unknown, but the sequence could be required for intracellular targeting or proper folding, or may simply serve as an inert spacer.

Amino-Terminal Peptides Early evidence for further posttranslational cleavage or fragmentation of native PTHrP emerged from the contradictory estimates of protein concentration obtained by standardized quantitative immunoassays that used antibodies generated against epitopes on specific regions of PTHrP (amino terminal, carboxy terminal, or midregion) versus two-site immunoassays designed to detect the complete PTHrP molecule. Inspection of the amino acid sequences of the PTHrP isoforms reveals a n u m b e r of potential sites for proteolytic cleavage, many of which appear to be functional in cultured cells or in vivo. There are frequent clusters of basic amino acids (arginine and lysine) across the length of the protein (Figs. 4 and Figs. 5); proteolytic processing at such sites is typical of neuroendocrine peptides

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FIG. 4 Amino acid sequence of the mature form(s) of parathyroid hormone-related protein. The top line is the sequence of the human 141-residue isoform, beginning with the first residue of the mature form and with the pre and pro segments removed. The alternative carboxy termini of the 139- and 173-isoforms are included for comparison. The sequences for mouse, rat, and chicken PTHrP are aligned with the human sequence. Amino acid substitutions are indicated, identity is designated by vertical lines, gaps or deletions are designated by dashes, and insertions are designated by an asterisk. From data in Mangin et aL (19,20,23), Suva et aL (21), Thiede et aL (22,26), Karaplis et aL (24), and Yasuda et aL (25).

such as insulin, proopiomelanocortin, gastrin, calcitonin, chromogranins, and many others (28,29). Cleavage at these sites is commonly carried out by prohormone convertases of the PC2, PC1/3 family located within the Golgi and in secretory granules. Another prominent cleavage site resides within the mature peptide adjacent to a monobasic arginine residue at position 37, thus generating a processed PTHrP peptide that consists of residues 1-36 (30). Although posttranslational processing of peptide hormones occurs more commonly at di- and multibasic sites, there are also numerous instances of cleavage at monobasic sites (31); among hormones processed in this manner are somatostatin, atrial natriuretic peptide, and glucagon. Mutagenesis of Arg-37 to Ala, Phe, or Lys effectively abolishes cleavage at this site (32). Using region-specific antibodies, aminoterminal species of PTHrP that do not contain carboxyterminal or midregion epitopes have been detected in media from cultured cell lines and in sera from hypercalcemic patients (8,9). More importantly, PTHrP(1-36) has been shown to be as potent as PTH (1-34) when analyzed in cell-based assays and in vivo (33). Keratinocytes appear to produce a longer, glycosylated, amino-terminal form of PTHrP (34); the core protein has an apparent molecular mass of 10 kDa; the fully glycosylated form has an apparent molecular mass of 18 kDa. This form has not been observed in other cell types thus far.

Midregion Peptides In addition to generating the 36-residue aminoterminal pepdde, cleavage of PTHrP at Arg-37 also gen-

erates a midregion peptide species with Ala-38 as its amino terminus (30) (Fig. 5). The amino acid sequence of PTHrP extending from residue 38 to 111 is remarkably well conserved among species, with only two substitutions in the h u m a n as compared to the rodent (23-25) (Fig. 4). This midregion peptide has been identified as an endogenous product in cultured cell lines from a n u m b e r of species and has been shown to be produced by rat insulinoma cell lines transfected with cDNAs encoding each of the three human PTHrP isoforms (35). Midregion PTHrP has been shown to be secreted in a regulated fashion by transfected neuroendocrine cells (36) and to be present in the circulation (37). The function of midregion PTHrP has not been well established, but studies in squamous epithelial cells provide evidence for a midregion-specific receptor that mediates the mobilization of cytosolic calcium and the formation of inositol phosphates through activation of the phospholipase C signaling pathway (38). Furthermore, studies in sheep have implicated a midregion peptide in establishing and maintaining the calcium gradient between the mother and fetus (39). This maternal-fetal calcium gradient is also abolished in PTHrP knockout mice, which have a hemochorial placenta as is found in humans. Placental calcium transport can be restored, however, by infusion of full-length or midregion fragments of PTHrP, but not by aminoterminal PTHrP or PTH (40). Finally, the natural secretory form(s) of midregion PTHrP have been only partially characterized; species with an amino terminus at residue 38 and carboxy termini at residues 94, 95, and 101 have been identified thus far (41), although

PTHrP:

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FIG. 5 Parathyroid hormone-related protein domains. (A) Potential multibasic cleavage sites in the three human PTHrP isoforms. The positions of arginine (R) and lysine (K) residues are marked. Numbering is relative to the first amino acid of the mature peptide. (B) Functional domains and sequence homologies in PTHrP. The signal peptide (SP) and propeptide (P) are removed during intracellular processing. The PTH-like nature of the first 36 amino acids of the mature peptide reflects both the correspondence in primary structure in residues 1 through 13 and the conformational similarity that mediates receptor activation. The region between residues 36 and 111 is highly conserved among all species examined thus far. The carboxy-terminal segment from residues 111 to 139 or 141 is the least conserved region of the peptide and the portion extending beyond residue 141 has thus far only been found in humans. (C) Processed forms of PTHrP. Secreted products include the active, amino-terminal fragment (1-36), a glycosylated amino-terminal fragment found in keratinocytes, and the various midregion fragments defined thus far. Potential carboxy-terminal species include the purported osteostatin peptide and remain largely undefined. Adapted from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

most experimental work testing for effects in cell culture and in vivo has been conducted with a peptide encompassing residues 67-86. Carboxy-Terminal Peptides A n u m b e r of multibasic proteolytic processing sites that may serve as substrates for furin/kexin can also be found in the carboxy terminus of PTHrP (at residues 88-91, 96-98, and 102-106) (42) and there is evidence that a carboxy-terminal species is generated as a native cellular product (Fig. 5). A carboxy-terminal

PTHrP fragment can be detected in the circulation of patients with renal failure (43) and is also found in the urine of normal h u m a n subjects (44). Similar fragments are secreted by h u m a n carcinoma cell lines and by cell lines transfected with PTHrP cDNAs (45). Based on the sequence conservation of PTHrP through amino acid 111 and t h e presence of the potential cleavage sites at residues 102-106, synthetic peptides containing amino acids 107-111 and 107-139 were tested for physiologic effects and were reported to inhibit osteoclastic bone resorption in vitro (46). To date, however, the effects of these

38

/

CHAPTER3

"osteostatin" peptides have not proved to be universally reproducible (47). Carboxy-terminal PTHrP has also been reported to inhibit the proliferation of cultured keratinocytes, whereas amino-terminal PTHrP had the opposite effect (48). Finally, immunoassays with antibodies directed against the unique 141-173 region of PTHrP that is derived from h u m a n exon 7 have suggested the presence of this fragment in fetal cord blood; h u m a n breast milk; cultured keratinocytes; renal, squamous and prostate carcinomas; and h u m a n amniotic m e m b r a n e s (49-51). However, as is the case with all carboxy-terminal species of PTHrP characterized thus far, the precise nature of the secretory form (s) has not been defined. Also contained within the multibasic proteolytic processing region that extends from residue 87 to residue 107 is a consensus bipartite nuclear processing signal, similar to that found in a n u m b e r of steroid h o r m o n e receptors and transcription factors (52-57). This targeting signal has been shown to be both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus and similarly to mediate localization of heterologous peptides. Though the PTHrP signal sequence normally directs the nascent peptide into the secretory pathway via the endoplasmic reticulum, there appear to be circumstances u n d e r which this signal can be overridden and the resultant cytosolic PTHrP can then be directed into the nucleus (52-57). One possible mechanism by which such an "intracrine" pathway might be exercised entails the use of alternative translational initiation sites that effectively prevent the generation of a functional signal sequence. Another potential route by which PTHrP may be directed to the nucleus involves the receptor-mediated internalization of secreted peptide, a pathway that also appears to be d e p e n d e n t on the presence of the nuclear targeting signal and may be mediated by a receptor distinct from the classic type 1 P T H / P T H r P receptor (55-57). Evidence suggests that the biologic effects of nuclear PTHrP can be quite dissimilar to those transduced by interaction with the type 1 receptor, even within the same cell type (53).

POSTTRANSLATIONAL AND SECRETION

MODIFICATION

Although PTHrP contains no consensus sites for N-glycosylation, numerous serine and threonine residues are present that may serve as sites for O-glycosylation. As noted above, a heavily glycosylated fragment of PTHrP has been shown to be secreted by keratinocytes, but has not yet been demonstrated in other cell types (41). PTHrP also contains consensus sites at residues 87-91 and 94-97 for carboxy-terminal amidation by the enzyme peptidyl e~-monooxygenase

(28,29,58). Because peptide amidation is relatively c o m m o n and there is considerable overlap between the tissues that express PTHrP and those that express the amidating enzyme, this particular posttranslational modification of PTHrP seems likely to occur, but has not been formally documented. PTHrP is expressed by a wide variety of cell types, representing both the regulated and constitutive pathways of protein secretion. In n e u r o e n d o c r i n e cell types such as those found in the pancreatic islet, adrenal medulla, pituitary, parathyroid, and central nervous system, PTHrP is packaged into secretory granules, and in the parathyroid gland, evidence suggests that PTHrP is cosecreted with PTH (59). Also, in transfected rat insulinoma cells, midregion and carboxy-terminal fragments, as well as amino-terminal PTHrP, have been shown to colocalize with insulin and their release can be induced by secretagogues such as potassium chloride and leucine (36). In cell types such as keratinocytes, osteoblasts, chondrocytes, renal tubular cells, and smooth muscle cells, however, PTHrP is secreted via the constitutive movement of monensinsensitive secretory vesicles, so that its rate of release is d e p e n d e n t on its rate of synthesis (60). Interestingly, a comparison of the products secreted by cell lines transfected with cDNAs encoding the three h u m a n PTHrP isoforms (1-139, 1-141, and 1-173) and representing either the regulated (rat insulinoma cells) or the constitutive (Chinese hamster ovary cells) secretion pathways suggests that processing of PTHrP peptides can be both cell and isoform specific: though all six cell lines generated an amino-terminal (1-36) product, the CHO cell lines also contained a distinct amino-terminal peptide that was absent in the RIN lines, and both CHO and RIN lines expressing the 1-139 isoform secreted a unique carboxy-terminal peptide that was not secreted from the lines expressing other isoforms (35).

METABOLISM AND DEGRADATION Much is known about the metabolism and degradation of PTH, but this is not the case with PTHrP, although it is likely that there is some overlap in the pathways involved. PTH is predominantly cleared from the circulation by glomerular filtration, uptake by multifunctional endocytic receptors in the renal proximal tubules, and subsequent lysosomal degradation. Given the low circulating levels of PTHrP, this would seem unlikely to be a quantitatively significant disposal route u n d e r normal circumstances, but could well prove critical in conditions of PTHrP overexpression, such as humoral hypercalcemia of malignancy. In n o n h u m a n mammals, exogenous PTHrP fragments encompassing residues 1-34 and 1-86 were rapidly removed from the

PTHrP: GENE STRUCTUREAND BIOSYNTHESIS / circulation, with metabolic clearance rates in the range of 1.25-7.5 m l / m i n per kg, or only slightly slower than that of intact PTH in man (61). There is evidence, however, suggesting that the kidney may play a role in the normal disposal of PTHrE In patients with chronic renal failure, elevated levels of a carboxy-terminal PTHrP fragment can be found in the circulation, presumably due to impaired clearance (43). Further, studies in vitro have implicated the metalloprotease, meprin, in the degradation of carboxy-terminal PTHrP by the kidney (62). Another potential clearance mechanism for PTH is specific binding by the P T H / P T H r P type 1 receptor followed by receptor-mediated endocytosis (55, 57). Because most of the actions carried out by PTHrP appear to be restricted to the microenvironment surrounding its site of secretion, such a mechanism could well be an important means of turning off signal transduction. Unexpectedly, evidence has suggested that although PTHrP is a secreted protein, the precursor propeptide in the endoplasmic reticulum can gain access to the cytoplasm and there serve as a target for ubiquitin conjugation and sub-sequent proteosome-mediated degradation (63,64). Neither the pro sequence nor a carboxy-terminal PEST degradation motif appeared to be required as cis-acting determinants, although carboxy-terminal sequences have been implicated in the intracellular degradation of the fulllength peptide (65). In cell culture studies, both native PTHrP secreted into the medium and added synthetic PTHrP peptides undergo rapid degradation even in the presence of multiple protease inhibitors, suggesting that many cell types secrete proteases capable of progressive cleavage and inactivation of this peptide (66). PTHrP secreted into the extracellular milieu in vivo may also be labile, which would be in keeping with the short half-life of its mRNA. Overexpression of PTHrP at 10 to 20 times the normal level in the epidermis of transgenic mice does not lead to hypercalcemia nor does it generate detectable levels of the peptide in the circulation (67). Proteases known to cleave aminoterminal PTHrP and thus abolish bioactivity include kexin and prostate-specific antigen (68,69).

CONTROL ELEMENTS AND REGULATION OF GENE TRANSCRIPTION

As might be expected, given the size and complexity of the human PTHrP gene, the 5' controlling region contains numerous consensus regulatory sequences in addition to the two TATA-containing promoters and the GC-rich initiator element (14-18) (Fig. 6). Included among these are the following sites: (1) two enhancer sequences (CAAT boxes) within the long intervening sequence between exons 2 and 3, (2) multiple regions (5' to exon 1, within intron 2, and in exons 3 and 4) that contain potential binding sites for the transcription factors, Spl, AP-1 and AP-2, (3) sequences resembling cyclic AMP response elements 5' to exon 1 and within exon 4, and (4) sequences equivalent to glucocorticoid responsive half-sites, located both 5' to and within exon 1, and within intron 1, intron 2, and exon 3. It should be emphasized, however, that many of these sites have been identified on the basis of sequence alone, and not by functional assays. Information about the mechanisms of PTHrP gene regulation has been obtained principally with two complementary approaches: (1) by analysis of biological systems that display aberrant or dysregulated expression, such as cancer cells, and (2) by testing the activity of reporter genes (such as chloramphenicol acetyltransferase or luciferase) under the control of progressive deletions of PTHrP gene promoter regions in transfected cell lines. In adult T cell leukemia/lymphoma (ATLL), infection of T lymphocytes with the HTLV-1 retrovirus results in humoral hypercalcemia of malignancy through the production of PTHrP by tumor cells (70-76). A viral protein required for replication, Tax1, has been shown to transactivate PTHrP gene expression from the P2 promoter by acting at through an upstream binding site for the transcription factor Ets-1 ( - 7 3 to - 6 5 relative to the P2 transcriptional initiation site). Effective up-regulation was dependent on the presence of both Ets-1 and Taxl (70). Subsequent work has also established that P2 basal activity requires binding of the transcription factor Spl at an adjacent site,

P1 " / / " AP1, AP2, SP1

//

CRE, GRE

I

I 500 bp

GRE

P2

GC

I

GRE

39

SPI, GRE

AP2, SP1, ETS, GRE

SP1

AP2, CRE

FIG. 6 Regulatory regions of the human PTHrP gene. Identified positive (+) and negative ( - ) regulatory regions are designated. Consensus binding sites for various transcription factors are indicated, some of which (CRE, GRE) represent imperfect matches to the consensus. Functionally defined sites are italicized. Exons are boxed and numbered, transcription initiation sites are marked by arrows, and promoter regions are designated by asterisks. From data in Mangin et aL (14,15), Suva et aL (16), Yasuda et aL (17), Campos et aL (18), and Vasavada et al. (86,87).

40

/

CHAPTER3

and that Ets-1 interacts directly with both Tax1 and Spl to form a cooperative ternary complex that interacts with the DNA binding site and then transcriptionally activates the promoter (71,72). Transactivation of the PTHrP gene was abolished by deletion of the carboxy terminus of the Tax protein and was also found to be d e p e n d e n t on an interaction between Ets-1 and a site within the amino terminus of Tax (72,73). Similarly, overlapping Ets-1 and Spl sites in the immediate upstream region of the mouse PTHrP gene ( - 8 8 to - 5 8 relative to the transcriptional start site) have been shown to be essential for retinoic acid-induced expression in embryonal carcinoma and embryonal stem cells (74). Additional studies with the h u m a n gene in this system have also shown up-regulation of expression by prostaglandin El, acting via a cAMP-dependent pathway, and by interleukin-2 (IL-2) and the transcription factor AP1/c-jun (73,75,76). Although PTHrP is a normal secretory product of a variety of squamous epithelia, including epidermal keratinocytes, only a subset of squamous carcinomas express the gene at levels sufficient to cause humoral hypercalcemia, raising the possibility that the nature of the transforming events could have an impact on PTHrP gene expression. Comparison of PTHrP expression levels with p53 functional status in a series of squamous carcinoma lines has revealed an association between the loss of p53 function and high levels of PTHrP mRNA (77). Evaluation of p53 isoforms with stabilizing mutations showed them to be capable of repressing PTHrP gene expression in a p53-negative squamous line and, correspondingly, inactivation of an endogenous, stabilized p53 gene product by the introduction of adenoviral E1B genes resulted in an increase in PTHrP expression. Conversely, mutant isoforms of p53 displaying a denatured, rather than stabilized, conformation were found to activate PTHrP gene expression (78). Both repression and activation of the human PTHrP gene by p53 appeared to occur primarily at the two TATA-based promoters. Finally, analysis of a spontaneously immortalized murine keratinocyte line trans-

q~

I

500 bp

formed with adenoviral 12S EIA has shown certain key domains within this E1A gene product to effect marked repression of PTHrP gene expression by acting directly on the mouse TATA promoter (79). Similar analysis with a 13S E1A product identified an additional domain that served as a potent activator of PTHrP gene expression by acting through an Ets-1 site residing approximately 70 bp upstream of the transcriptional start site. Other oncogenes have also been found to affect PTHrP gene expression directly. The direct introduction of activated H- or K-ras oncogenes into a variety of cell lines has been shown to result in overexpression of PTHrP and transfection of cells with a constitutively activated tyrosine kinase oncogene, Tpr-Met, has been shown to enhance PTHrP transcription through the ras signaling pathway (80,81). Furthermore, the induction mechanism appeared to be dependent on ras processing events such as isoprenylation and farnesylation (81,82). In another system, analysis of a panel of renal carcinoma cell lines revealed a series of four CpG dinucleotide sites within the 5' flanking region of the human PTHrP gene that were consistently unmethylated in all PTHrP-expressing lines and methylated in all PTHrP-nonexpressing lines examined (83) (Fig. 7). Together these sites appear to constitute a minimal, methylation-free zone of approximately 550 bp, which resides approximately 1 kb upstream of the GC-rich initiator element and serves as a critical control switch for transcription from all three promoters. Furthermore, treatment of PTHrP-nonexpressing lines with the nucleoside analog, 5-azacytidine, effectively demethylated the identified critical sites and concurrently activated PTHrP gene transcription, thus strongly reinforcing the concept of a regulatory relationship. Also in this system, a 900-bp CpG island was identified in the proximal promoter region overlapping the 5' end of exon 3, which remained methylation-free regardless of PTHrP expression status (83). Work in a squamous carcinoma line from human lung, however, showed persistence of PTHrP gene expression despite

q~

q~

7/

I

FIG. 7 Methylation pattern of the human PTHrP gene. Sites were identified in a panel of human renal carcinoma cells by using methylation-sensitive restriction enzymes. The half-filled circles correspond to CpG dinucleotides that were found to be unmethylated in cell lines expressing the PTHrP gene and methylated in cells not expressing the gene. Other CpG dinucleotides were found to be methylated (solid circle) or unmethylated (open circles), regardless of the status of PTHrP gene expression. The CpG island is indicated by a black bar. Modified from Holt et aL (83). Holt, EH., Vasavada, R., Broadus, AE., Philbrick, WM. Region-specific methylation of the PTH-related peptide gene determines its expression in human renal carcinoma lines. J Biol Chem 1993;268:20639-20645.

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

methylation of the distal two-thirds of the CpG island (84), leaving open the possibility that the methylation status of the 3' portion of the island may have regulatory implications. Control sequences that regulate the degree of basal expression have also been examined. Deletion experiments have identified a n u m b e r of negative regulatory elements operative in both normal and neoplastic cell lines in the regions from 3.8 to 2 kb and 1.1 to 0.35 kb upstream of exon 1 and 1.3 to 0.63 kb upstream of exon 3 (18). An additional regulatory element has been found within a segment from 0.63 to 0.34 kb upstream of exon 3 and appears to act as a repressor of the GC-rich initiator, because deletion of this region leads to increased levels of mRNA transcripts originating from this p r o m o t e r (85-87). Interestingly, this same region has been reported as a positive regulatory element when evaluated in constructs in which reporter gene expression is solely u n d e r the control of the downstream P2 p r o m o t e r (18), suggesting that this one region can differentially regulate two distinct promoters. Relative expression profiles of the three h u m a n PTHrP promoters both in context and in isolation suggest that the sequences within the GC-rich initiator element contribute to the activity of the two TATAcontaining promoters, as well as to the activity of the initiator element (86-88). Further dissection of the sequences within the GC-rich initiator element revealed a prototypical bipartite construction with an internal transcription initiation site s u r r o u n d e d by numerous Spl and AP-2 sites. The two halves of the core element appeared to be functionally equivalent, whereas a region extending from approximately 180 to 340 bp upstream of the GC-element transcriptional initiation site played a positive regulatory role (88). Despite the obvious potential for the generation of discrete, tissue-specific patterns of h u m a n PTHrP gene p r o m o t e r usage or alternative termination, this has not proved to be a c o m m o n finding in vivo. Examination of p r o m o t e r usage (P1 and P2) in a large n u m b e r of benign and malignant tissues (including esophagus, stomach, cecum, liver, pancreas, thyroid, parathyroid, adrenal, and kidney) found no consistent patterns (85,89). Relative differences in PTHrP p r o m o t e r usage do exist, however; in squamous carcinoma cells and keratinocytes, P1 was found to be more active than either P2 or the GC promoter; in renal carcinoma cells, the GC p r o m o t e r appears to be preferred; and in the uterus, P2 and the GC p r o m o t e r are used exclusively (87,88). Similarly, no consistent patterns in 3' exon usage have emerged, although exon 6 appears to be more prevalent in prostate cancer than in normal tissue (90), and has also been reported to be associated with breast cancer metastasis (91). Studies of PTHrP gene regulation have found two primary patterns of response, a rapid, transient

//

41

increase in PTHrP mRNA levels or a delayed, sustained up-regulation. Examples of the former include the response to serum and angiotensin II in smooth muscle cells (92,93), mechanical stimuli in aortic smooth muscle cells (94), serum factors in pancreatic islet cells (95) and keratinocytes (96), endotoxin in mouse spleen (97), and ischemia in rat kidney (98). In this response pattern, steady-state levels of PTHrP mRNA typically display a rapid increase within 1 or 2 hours after stimulation, reach a peak at 4 to 6 hours, and then quickly decline due to the short half-life of the mRNA. Examples of a delayed, sustained induction of PTHrP gene expression include the response to estrogen in rat myometrial cells (99); transforming growth factor [3 (TGF-[3) in h u m a n myometrial and endometrial cells in primary culture (100), mechanical stimuli in the bladder, rat uterus, and avian oviduct (101-104); and cAMP in embryonal cells and trophectoderm (105). The generation of this type of response would require a perpetuation of the transcriptional induction, an increase in the stability of the mRNA, or some combination of the two. A n u m b e r of hormones, cytokines, growth factors, and second messengers have been shown to induce PTHrP gene expression; more often then not, these effects tend to follow the rapid, transient response pattern (Table 1). Examples include estradiol in rat kidney, uterus, pituitary, and hypothalamus (106-108); estradiol and tamoxifen in the MCF-7 breast carcinoma cell line (109); calcitonin in lung carcinoma cell lines (110); TNFot and IL-113 in h u m a n umbilical vein endothelial cells (111); forskolin, cAMP, and IL-2 in HTLV-infected T cells (75,76); bradykinin, serotonin, endothelin, thrombin, and n o r e p i n e p h r i n e in smooth muscle cells (92,94); and prolactin in m a m m a r y tissue (112). As noted above, PTHrP gene expression has been shown to be induced by serum in a n u m b e r of cell types, including rat osteosarcoma (ROS) cells. Induction is rapid (peaking within 4 hours) and is mediated in part via a transcriptional mechanism (113), although a substantial effect on mRNA stability has also been observed in ROS cells (see Posttranscriptional Regulation, below). T h o u g h a prototypical serum response element of the c-fos/[3-actin type is not found in the rat PTHrP gene, the serumresponsive region has been localized to a segment extending from 0.3 to 1.05 kb upstream of the transcriptional start site (113). The effects of serum in this system may be mediated, at least in part, through insulin and epidermal growth factor, because both these factors have been shown to stimulate a rapid induction of PTHrP mRNA. Likewise, inhibition of the angiotensin II receptor greatly attenuated the seruminduced rise in PTHrP gene expression in vascular smooth muscle cells (94), suggesting a primary regulatory role for that factor. Phorbol esters, which activate

42

/

C~a'TF~R3

protein kinase C isozymes, have been reported to strongly up-regulate PTHrP mRNA in several cell types (114-116). Correspondingly, treatment of h u m a n myometrial smooth muscle cells with okadaic acid, an inhibitor of the serine/threonine protein phosphatases 1 and 2A, also effected a marked increase in PTHrP gene expression (116). In contrast to the more prevalent pattern of rapid induction, TGF-[3 has been found to provoke a slow, sustained rise in PTHrP mRNA levels that is maximal by 12 to 24 hours in several cell lines, including h u m a n keratinocytes, renal carcinoma cells, primary endometrial and myometrial cells, and mouse bone organ culture (100,117,118). Conversely, TGF-[3 has also been shown to decrease PTHrP gene expression in immortalized murine endochondral chondrocytes, a property shared by several bone morphogenetic proteins (BMP-2,-5 a n d - 7 ) and opposed by basic fibroblast growth factor (119). There are also examples of negative regulation of PTHrP gene expression (Table 1). The active vitamin D metabolite (1,25-dihydroxyvitamin D~) and two glucocorticoids (dexamethasone and triamcinolone) have been shown to decrease steady-state levels of PTHrP mRNA in a time- and dose-dependent m a n n e r in a human medullary thyroid carcinoma cell line and a lung carcinoid line (120,121). The glucocorticoid effect was completely blocked by the competitive antagonist, RU-486. Nuclear runoff and transcriptional inhibition experiments indicated that neither vitamin D nor glucocorticoids appeared to influence mRNA stability, but that these agents acted by repressing the rate of PTHrP gene transcription. Two noncalcemic analogs of vitamin D, EB1089 and 22-oxacalcitriol, were also found to suppress both basal and s e r u m - o r EGF-stimulated PTHrP gene expression in a lung squamous cancer cell line through a transcriptional mechanism, thus raising the possibility of therapeutic potential (122). In the rat PTHrP gene, two vitamin D-responsive elements (VDREs) have been localized approximately 1 kb upstream of the transcriptional start site; one element resembling a canonical positive regulatory VDRE was identified by both DNA-protein binding studies and functional assays, and a second element bearing similarity to the negative regulatory VDRE in the human PTH gene was identified by binding studies alone (123,124). Treatment with 9-cis-retinoic acid has also been shown to repress the transcriptional activity of the human PTHrP gene and mobility-shift experiments with the rat gene suggest that the effects of vitamin D may be mediated through the binding of a heterodimer composed of the vitamin D receptor and the retinoid X receptor (125). PTHrP has been localized to a n u m b e r of discrete cell populations in the central nervous system and evidence suggests that PTHrP gene expression in these neuronal cell types is up-regulated by excitation

(126,127). In primary cultures of cerebellar granule cells, for example, it has been shown that PTHrP gene expression can be induced by potassium ion-dependent membrane depolarization and the subsequent entry of extracellular C a 2+ into the cell through L-type voltagesensitive calcium channels; depolarization with sodium ionphores o r C a 2+ entry by other routes proved ineffective (128). The induction of PTHrP gene expression in this system appears to be mediated through the C a 2+ calmodulin kinase pathway in a m a n n e r similar to that for the c-fos gene. The L-type voltage-sensitive calcium channels (L-VSCCs) and the C a 2+ calmodulin kinase cascade also appear to be involved in excitation secretion coupling in neuroendocrine cells and in excitation-contraction coupling in skeletal, cardiac, and smooth muscle (129). Interestingly, the role played by PTHrP in the central nervous system appears to be neuroprotective in nature, based on experiments showing that the peptide serves as a highly effective inhibitor of L-VSCC-associated C a 2+ influx and consequent neuronal toxicity (see Chapter 16). PTHrP has also been shown to be expressed in smooth muscle types from a n u m b e r of organs, including the gastrointestinal tract, bladder, myometrium, vasculature, and chicken oviduct. In all of these sites, PTHrP gene expression appears to be induced by a variety of mechanical stimuli, such as balloon angioplasty in the aorta (94), atherosclerotic stenosis in the coronary arteries (130), stretch (131) or shear flow (94) in vascular smooth muscle cells in culture, distension of the uterus by fetal growth or balloon inflation (103), expansion of the chicken oviduct during egg transit (104), expansion of the bladder by urinary volume (101), and distension of the stomach by pyloric ligation and subsequent gastric filling (132) (Fig. 8). Accumulating evidence indicates that this mechanotransduction is mediated through the opening of stretch-activated cation channels, subsequent ion influx and membrane depolarization, and the resultant activation of L-type voltage sensitive calcium channels (133). PTHrP gene expression is thus induced and the secreted peptide then serves as a muscular relaxant and vasodilator, thereby constituting a feedback system to regulate muscular tone (see Chapter 16). The rapid induction-deinduction kinetics that typify PTHrP mRNA responses in many systems are reminiscent of the kinetics associated with so-called primary response or immediate early genes, which include many protooncogenes, cytokines, and growth factors (96,134). The transient nature of the induction serves to limit the translational yield of the resultant proteins and thus to restrict the biologic effects of these powerful regulatory molecules. The induction of PTHrP gene expression by 1713-estradiol in rat GH4C1 pituitary cells has been shown to display many of the features of the

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS //

TABLE 1 Regulation of PTHrP Gene Expression a Stimulus

Physiologic Suckling Uterine occupancy Stretch Stretch Stretch Egg-laying cycle Differentiation Differentiation Differentiation Differentiation Pharmocologic Glucocorticoids Glucocorticoids Glucocorticoids Glucocorticoids 1,25(OH)2D 1,25(OH)2D 1,25(OH)2D 22-oxa-1,25(OH)2D Estrogen Estrogen Estrogen Estrogen Estrogen Serum Serum Serum Serum Growth factors EGF EGF IGF-I TGF-13 TGF-13 TGF-13 TGF-13 Prolactin Cycloheximide Cycloheximide Cycloheximide Tax Forskolin Calcitonin Phorbol ester Phorbol ester Endothelin-I Thrombin Angiotensin II

Tissue/cell type

mRNA/protein

Lactating breast (rat) Myometrium (rat) Myometrium (rat) Urinary bladder (rat) Amnion (human) Oviduct (chicken) Insulinoma (rat) Embryonal carcinoma Keratinocyte (human) Trophoectoderm (mouse)

1" 1" 1" 1" T 1" T 1" T 1"

Carcinoid (human) Insulinoma (rat) Aortic smooth muscle (rat) Keratinocyte (rat) Medullary carcinoma (human) Keratinocyte (human) Keratinocyte (rat) T cell (human) Uterus (rat) Pituitary/hypothalamus (rat) Myometrial cell (rat) Pituitary GH4C1 (rat) Kidney (monkey) Insulinoma (rat) Keratinocyte (human) Keratinocyte (rat) Aortic smooth muscle Keratinocyte (human) Keratinocyte (rat) Mammary epithelial (human) Mammary epithelium (human) Renal carcinoma (human) Myometrial (human) Endometrial (human) Keratinocyte (rat) Mammary gland (rat) Multiple (rat and human) Osteosarcoma (human) Insulinoma (rat) T cells (human) T cell MT-2 (human) Squamous carcinoma (human) Osteosarcoma (human) T cells (human) Aortic smooth muscle (rat) Aortic smooth muscle (rat) Aortic smooth muscle (rat)

,I, $ $ $ J, $ ,1, $ T T 1" T T 1" 1" T 1" T 1" 1" 1" T T 1" T 1" T T 1" T 1" T T 1" 1" T T

aModified from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian The parathyroids. New York: Raven, 1994:259-294.

43

44

/

CHAPTER3

FIG. 8 Induction of PTHrP mRNA during gestation in rat uterus. Little PTHrP gene expression is detected in the nongravid uterus by Northern blot analysis. With progressive distention of the uterus during gestation, steady-state levels of PTHrP mRNA are markedly induced. After parturition on day 21, PTHrP gene expression declines precipitously and continues to fall throughout the postpartum period. When pregnancy is prolonged by the administration of progesterone, PTHrP mRNA levels continue to rise. Reproduced from Thiede et aL (103). Thiede MA, Daifotis AG, Weir EC, Brines ML, Burtis W J, Ikeda K, Dreyer BE, Garfield RE, Broadus AE. Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in pre-term rat myometrium. Proc Natl Acad Sci USA 87:6969-6973, 1990.

primary response pattern (134). Steady-state mRNA levels peaked at 1 to 2 hours due to a burst of transcription that was maximal at 20 to 40 minutes and declined thereafter. The 30-minute half-life of PTHrP mRNA in these cells, although unaffected by estradiol, was sufficiently short to mediate the rapid decline in steady-state mRNA levels. Inhibition of protein synthesis by treatm e n t with cycloheximide had no effect when used alone and failed to block the estradiol-mediated induction of PTHrP gene expression, but eliminated the transcriptional arrest, thus implicating the action of a labile transcriptional repressor protein that is estrogen inducible (134). As is the case for other primary response genes, the combination of a rapid post stimulation repression of gene transcription with a short mRNA half-life is essential to the generation of the transient response.

POSTTRANSCRIPTIONAL

REGULATION

A further measure of control over PTHrP gene expression appears to be exerted at the level of mRNA stability. The steady-state mRNA levels of all genes represent the p r o d u c t of both the rate of transcription and the rate of degradation. T h o u g h the transcription rate of the PTHrP gene is similar to that of the actin gene, steady-state levels of PTHrP mRNA are extremely low in most tissues (estimated to be from 0.001 to 0.01% of mRNA), as a result of rapid turnover (19,23,135-137). M e a s u r e m e n t of the half-life of PTHrP mRNA in a variety of tissues has ranged from 30 minutes to several

hours, kinetics that are similar to those for a n u m b e r of cytokines and protooncogenes. For such factors, a rapid rate of degradation serves to allow a rapid response to the transcriptional down-regulation of the gene, both in terms of mRNA levels and consequent biologic effects. The sequences responsible for mediating mRNA instability in many of these genes are AU-rich elements (AREs) typically found in the 3' untranslated regions of these genes (138). These motifs are also present in all PTHrP mRNAs characterized thus far; multiple iterations of the core element, AUUUA, can be found in all three alternative h u m a n PTHrP 3' transcriptional termini (exons 6, 7, and 8), as well as in the 3' untranslated regions (UTRs) of PTHrP mRNAs from the mouse, rat, and chicken. There is also some functional evidence to suggest that the relative stability of h u m a n PTHrP mRNAs is mediated through the 3' UTRs, with transcripts containing exon 8 typically displaying a more rapid degradation rate than those containing exons 6 or 7 (139,140). Transfection of fibroblasts or keratinocytes with each of the three alternative h u m a n PTHrP 3' UTRs fused to a luciferase reporter gene also showed transcripts containing exon 8 to be the most unstable, although this was d e p e n d e n t on the cell line used (141). A n u m b e r of studies looking at AU-rich elements in other genes, however, have indicated that the core AUUUA motif is insufficient by itself to mediate instability and that the m i n i m u m consensus element is an octamer or n o n a m e r (142,143), neither of which is found in PTHrP mRNAs. This implies that instability in this system may be mediated by non-AUUUA AREs or by elements other than AREs. Finally, there are data to suggest that the degradation rate of PTHrP mRNA is a regulated p h e n o m e n o n , because a n u m b e r of factors, including serum, TGF-[3 and epidermal growth factor (EGF), have been reported to affect stability (139,144-146). Preliminary evidence indicates that the TGF-[3-dependent stabilization of h u m a n PTHrP mRNAs may be mediated through cis-acting sequences that are not contained in the 3' UTR, but rather reside within the coding region (146). The inhibition of protein synthesis has been shown to result in the superinduction of PTHrP mRNA expression in a n u m b e r of h u m a n and rat cell lines (95,96,114,147). T h o u g h there is often a transcriptional c o m p o n e n t in this p h e n o m e n o n , the effect is mediated in large part at the posttranscriptional level and is presumed to reflect the reduced synthesis of critical components in the mRNA degradation/instability pathway.

DEVELOPMENTAL REGULATION The pattern of PTHrP gene expression in the adult is widespread and is even more so in the fetus (Table 2).

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS /

TABLE 2

45

PTHrP Gene Expression during Embryogenesisa

Chicken

Mouse and rat

Human

Days 3-10 Viscera Allantois Yolk sac Chorioallantoic membrane Day 15 Brain Heart Lung Liver Gizzard Intestine Skeletal muscle

Day 3mcompacted morula Day 7.5mtrophoblast Days 8-12mplacental decidua Days 13-14 Epidermis, skin appendages Skeletal, cardiac muscle Vascular smooth muscle Liver parenchyma Renal tubular epithelium Bronchiolar epithelium Gastrointestinal epithelium Choroid plexus, spinal cord, dorsal root ganglia, and eye Days 15-16 Lung epithelium Perichondrium Dental lamina, inner ear Day 18 Salivary ducts Pancreatic ducts, islets Day 20.5--keratinocytes

Weeks 7-8 Trophoblastic layers of chorionic villi Lung epithelium Liver parenchyma Pancreatic acini Stomach epithelium Hindgut epithelium Kidney Perichondrium Epidermis Otic placode Tooth bud Choroid plexus, spinal cord, dorsal ganglia Weeks 18-20 Cardiac, skeletal muscle Vascular smooth muscle Endochondral and intramembranous bone

aModified from Broadus AE, and Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

Cumulative data from a large n u m b e r of localization studies in fetal tissues allow the following generalizations: (1) PTHrP mRNA a n d / o r peptide can be found in almost every embryonic tissue examined, but are restricted to certain cell types within those tissues, (2) the types of tissues that express PTHrP encompass derivatives of all three germ layers, as well as extraembryonic sites, such as the amnion and trophoblast (148,149), (3) the levels a n d / o r locations of PTHrP gene expression are not static, but change as a function of developmental stage, and (4) at most sites of expression, both PTHrP mRNA and peptide are present in low abundance and require sensitive methods for detection. Indeed, it was precisely this pattern of near ubiquitous fetal expression, coupled with both spatial and temporal specificity, that first suggested that PTHrP was likely to be a factor involved in the regulation of growth and differentiation. The P T H / P T H r P type 1 receptor is also widely expressed during fetal life (150) and studies in the mouse suggest that PTHrP and the PTH1 receptor represent one of the earliest h o r m o n e receptor pairs operative in development (105). Mso, in most tissues, PTH and PTHrP are coordinately expressed in adjacent

cell layers (typically, PTHrP displays focal expression in the surface epithelium, but the receptor is expressed diffusely in the underlying mesenchyme), a pattern that is consistent with our emerging understanding of their paracrine interactions (150). Localization studies in preimplantation mouse embryos have shown that PTHrP can be detected as early as the compacted morula stage before the onset of e n d o d e r m a l differentiation (105) (Fig. 9) and that the peptide serves as an early marker for cells of the trop h e c t o d e r m lineage. PTHrP gene expression is also induced on differentiation of F9 embryonal carcinoma stem cells into parietal endoderm-like cells (137). Other studies with cultured cells, including keratinocytes and pancreatic islet tumor cells, have also shown up-regulation of PTHrP mRNA or protein when differentiation is stimulated (95,151). The temporal sequence of the tissue-specific acquisition of PTHrP gene expression during the fetal development of the rat has been carefully examined by in situ hybridization (150) (Fig. 10). By El5 to El6, PTHrP mRNA was found to be most highly expressed in the epidermis, hair follicles, and the enamel epithelium of the tooth

46

/

CHAPTER3

i~f ~ ~ ' "~....

~:~ ....

~:.:...~:~:~:,~...~a,,,

% ~ ~.......... ..

FIG. 9 Acquisition of PTHrP gene expression in preimplantation mouse embryos. Whole embryos were subjected to immunofluorescent staining with an anti-PTHrP antibody. (A) The 8-cell stage; (B) compacted morula; (C) blastocyst. Left panels: Nomarsky image. Right panels: fluorescent image. Preparations were viewed with confocal scanning laser microscopy. Reproduced from Van de Stolpe et aL (105), The Journal of Cell Biology, 1993, Vol. 120, pp. 235-243, by copyright permission of The Rockefeller University Press.

buds. Lower levels of expression were apparent in the epithelia of the inner ear and nasal cavity, the bronchial epithelium of the lung, the ependymal cells of the choroid plexus, and the endocardial cushion region of the heart. Diffuse mesenchymal expression could also be detected in some developing organ systems at this stage, but this expression diminished over time. Expression of PTHrP along the border of the intestinal epithelium could not be detected until E20. In the skeleton at E15-E16, hybridization was seen in the membranous bone forming within the mandible, in early chondrocytes of the cartilage primordia of endochondral bones such as the ribs and digits, and in the perichondrium of the sternum and the periosteum of the clavicle. By El8, expression could also be detected in the hypertrophic chondrocytes of the developing growth plates (150).

FIG. 10 Expression of PTHrP and the PTH/PTHrP receptor during development of the rat. Tissue sections were subjected to in situ hybridization and emulsion autoradiography. Darkfield views are shown. (Top) Whole rat fetus from day E15. (Bottom) Craniofacial portion of day E19 fetus. Labels: rP, PTHrP; R, PTH/PTHrP receptor; CP, choroid plexus; E, inner ear; TB, tooth bud; L, lung; H, heart; I, intestine; MC, Meckel's cartilage; Md, mandible; Mx, maxilla. Reproduced from Lee et aL (150). Lee K, Deeds JD, Segre GV. Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995; 136:453-463.

Longitudinal surveys have revealed a number of instances in which developmental progression is accompanied by changes in either the quantitative levels or the spatial patterns of PTHrP gene expression (150,152-158). In both the human and rat kidney, early expression of PTHrP is evident in the glomeruli and in the tubular epithelium of the mesonephros and metanephros. Soon after, however, glomerular expression begins to decline, and by midgestation, expression is limited to the proximal and distal tubules, the collecting duct, and the urothelium (152-155,158). As noted above, in the developing skeletal system of the rat, PTHrP is expressed in the early mesenchyme and cartilage primordia of the ribs, limbs, and vertebrae. As maturation proceeds, however, expression of the pep-

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

tide becomes increasingly restricted to hypertrophic chondrocytes, osteoblasts, and areas of the perichondrium and bone collar (150,152,154). Similar changes in PTHrP expression patterns have been observed to occur in the lung, liver, and dental lamina. The stage-specific acquisition of PTHrP gene expression in a number of organ systems, especially the developing epidermis, mammary gland, placenta, endochondral bone, tooth, and central nervous system, has been shown to correlate with distinct developmental functions, many of which are now becoming increasingly well understood. These varied roles of PTHrP in regulation of programmed differentiation and physiologic responses are detailed in succeeding chapters.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants DE12616, AR46032, and DK45735.

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gen and antiestrogens. Biochem Biophys Res Commun 1998;251:849-854. 110. Chilco PJ, Gerardi JM, Kaczmarczyk SJ, Chu S, Leopold V, Zajac JD. Calcitonin increases transcription of parathyroid hormone-related protein via cAME Mol Cell Endocrino11993;94:1-7. 111. Eto M, Akishita M, Ishikawa M, Kozaki K, Yoshizumi M, Hashimoto M, Ako J, Sugimoto N, Nagano K, Sudoh N, Toba K, Ouchi Y. Cytokine-induced expression of parathyroid hormonerelated peptide in cultured human vascular endothelial cells. Biochem Biophys Res Commun 1998;249:339-343. 112. Thiede MA. The mRNA encoding a parathyroid hormone-like peptide is produced in mammary tissue in response to elevations in serum prolactin. Mol Endocrino11989;3:1443-1447. 113. Falzon M. Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms. Endocrinology 1996; 137:3681-3688. 114. Rodan SB, Wesolowski G, Ianacone J, Thiede MA, Rodan GA. Production of parathyroid hormone-like peptide in a human osteosarcoma cell line: Stimulation by phorbol esters and epidermal growth factor. J Endocrinol 1989;122:219-227. 115. Brandt DN, Pandol SJ, Deftos LJ. Calcium-stimulated parathyroid hormone-like protein secretion: Potentiation through a protein kinase C pathway. Endocrinology 1991;128:2999-3004. 116. Morimoto T, Devora GA, Mibe M, Casey ML, MacDonald PC. Parathyroid hormone-related protein and human myometrial cells: Action and regulation. Mol Cell Endocrino11997;129:91-99. 117. Werkmeister JR, Blomme EA, Weckmann MT, Grone A, McCauley LK, Wade AB, O'Rourke J, Capen CC, Rosol TJ. Effect of transforming growth factor-betal on parathyroid hormonerelated protein secretion and mRNA expression by normal human keratinocytes in vitro. Endocrine 1998;8:291-299. 118. Serra R, Karaplis A, Sohn E Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation. J Cell Bio11999;145:783-794. 119. Terkeltaub RA, Johnson K, Rohnow D, Goomer R, Burton D, Deftos LJ. Bone morphogenetic proteins and bFGF exert opposing regulatory effects on PTHrP expression and inorganic pyrophosphate elaboration in immortalized murine endochondral hypertrophic chondrocytes (MCT cells). J Bone Miner Res 1998;13:931-941. 120. Ikeda K, Lu C, Weir EC, Mangin M, Broadus AE. Transcriptional regulation of the parathyroid hormone-related peptide gene by glucocorticoids and vitamin D in a human C-cell line. J Biol Chem 1989;264:15743-15746. 121. Lu C, Ikeda K, Deftos LJ, Gazdar AF, Mangin M, Broadus AE. Glucocorticoid regulation of parathyroid hormone-related peptide gene transcription in a human neuroendocrine cell line. Mol Endocrinol 1989;3:2034-2040. 122. Falzon M, ZongJ. The noncalcemic vitamin D analogs EB 1089 and 22-oxacalcitriol suppress serum-induced parathyroid hormone-related peptide gene expression in a lung cancer cell line. Endocrinology 1998; 139:1046-1053. 123. Kremer R, Sebag M, Champigny C, Meerovitch K, Hendy GN, White J, Goltzman D. Identification and characterization of 1,25-dihydroxyvitamin D3-responsive repressor sequences in the rat parathyroid hormone-related peptide gene. J Biol Chem 1996;271:16310-16316. 124. Falzon M. DNA sequences in the rat parathyroid hormonerelated peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression. Mol Endocrinol 1996;10:672-681. 125. Akeno N, Ohida S, Horiuchi N. Inhibitory effects of 1,25dihydroxyvitamin D 3 and 9-cis-retinoic acid on parathyroid

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

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of human parathyroid hormone-related peptide. Mol Endocrinol 1994;8:1656-1666. Knlys V, Huez G. Translational control of cytokine expression by 3' AU-rich sequences. Biochimie 1994;76:862-866. Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Bio11995;15:2219-2230. Zakalik D, Diep D, Hooks MA, Nissenson RA, Strewler GJ. Transforming growth factor beta increases stability of parathyroid hormone-related protein messenger RNA. J Bone Miner Res 1992;7:S118. Kirayama T, Gillespie MT, Glutz JA, Fukumoto S, Moseley JM, Martin TJ. Transforming growth factor beta stimulation of parathyroid hormone-related peptide: A paracrine regulator? Mol Cell Endocrinol 1993;92:55-62. Sellers RS, Tannehill-Gregg SH, Capen CC, Rosol TJ. Cis-acting elements in the 3' untranslated and coding regions of parathyroid hormone-related protein mRNA mediate transforming growth factor-J3 induced stability. J Bone Miner Res 1999; 14 (Suppl. 1) :$546. Ikeda K, Lu C, Weir EC, Mangin M, Broadus AE. Regulation of parathyroid hormone-related peptide gene expression by cycloheximide. J Biol Chem 1990;265:5398-5402. Beck E Tucci J, Senior PV. Expression of parathyroid hormonerelated protein mRNA by uterine tissues and extraembryonic membranes during gestation in rats. J Reprod Fertil 1993;99:343-352. Karperien M, Lanser P, DeLaat SW, Boonstra J, DeFize LHK. Parathyroid hormone-related peptide mRNA expression during murine postimplantation development: Evidence for involvement in multiple differentiation processes. Int J Dev Biol 1996;40:599-608. Lee K, Deeds JD, Segre GV. Expression of parathyroid hormonerelated peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453-463. Kremer R, Karaplis AC, Henderson J, Gulliver W, Banville D, Hendy GN, Goltzman D. Regulation of parathyroid hormonelike peptide in cultured normal human keratinocytes. J Clin Invest 1991;87:884-893. Burton PBJ, Moniz C, Quirke P, Malik A, Bui TD,J/ippner H, Segre GV, Knight DE. Parathyroid hormone-related peptide: Expression in fetal and neonatal development. JPath 1992;167:291-296. Campos RV, Asa SL, Drucker DJ. Immunocytochemical localization of parathyroid hormone-like peptide in the rat fetus. Cancer Res 1991;51:6351-6357. Moniz C, Burton PBJ, Malik AN, Dixit M, Banga JP, Nicolaides K, Quirke P, Knight PE, McGregor AM. Parathyroid hormonerelated peptide in normal human fetal development. J Mol Endocrinol 1990;5:259-266. MoseleyJM, HaymanJA, DanksJA, Alcorn D, Grill V, SouthbyJ, Horton MA. Immunochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 1991;73:478-484. Senior PV, Heath DA, Beck E Expression of parathyroid hormonerelated protein mRNA in the rat before birth: Demonstration by hybridization histochemistry. J Mol Endocrino11991 ;6:281-290. Schermer DT, Chan SDH, Bruce R, Nissenson RA, Wood WI, Strewler GS. Chicken parathyroid hormone-related protein and its expression during embryo loci development. JBone Miner Res 1991;6:149-155. Dunne FP, Ratcliffe WA, Mansour P, Heath DA. Parathyroid hormone-related protein (PTHrP) gene expression in fetal and extra-embryonic tissues of early pregnancy. Hum Reprod 1994;9:149-156.

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CHAPTER 4

Interactions of Parathyroid Hormone and Parathyroid Hormone-Related Protein with Their Receptors

MICHAEL CHOREV, JOSEPH M. ALEXANDER, AND MICHAEL ROSENBLATT

Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

INTRODUCTION

plex protein structure occupying three distinct phases: the intracellular loops and carboxyl terminus (located in the cytoplasm), the seven hydrophobic membranespanning helices, and the extracellularly oriented N terminus and loops. The receptor is posttranslationally modified by N-glycosylation and disulfide bond formation, and u n d e r certain circumstances it can also be phosphorylated. Heterotrimeric G proteins are composed of a unique ot subunit, which binds GDP or GTP with high affinity, and which is associated with a [3y complex. H o r m o n e binding to the GPCR increases its affinity for the GDP-bound heterotrimeric G protein complex, which in turn activates it and causes a "GTP for GDP" exchange. The GTP-bound G protein separates from the receptor, and the Got-GTP subunit dissociates from the [3y complex. Both the G0t-GTP and the [3y dimer are able to interact with effectors such as adenylyl cyclase and phospholipase C. Hydrolysis of GTP to GDP by the GTPase activity of the e¢ subunit results in the dissociation of the oL subunit from the effector molecule, allowing its reassociation with the [3y dimer. G protein signaling is thus governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. This system is highly dynamic. The hormone has more diversified conformations when in solution than when membrane bound, and its conformation changes on interaction with the receptor. On ligand binding, the receptor may change conformation to allow global movements of transmembrane domains that lead to changes in the conformation of the cytoplasmic portions of the receptor. This, in turn, increases affinity

Obtaining a detailed understanding of structurefunction relations of a h o r m o n e - r e c e p t o r complex at the most fundamental level currently requires an interdisciplinary approach; state-of-the-art techniques in peptide and protein biochemistry, as well as cellular and molecular biology, must be utilized. Traditionally, most or all of the insights regarding the h o r m o n e - r e c e p t o r complex have been obtained by correlating the effects of structural modifications on function of either the hormone or the receptor molecule alone. The detailed mechanisms that explain how structural changes in either hormone or receptor can alter the h o r m o n e - r e c e p t o r bimolecular interaction or later in signaling events are only beginning to be understood. The complexity of the parathyroid hormone ( P T H ) - r e c e p t o r system presents a significant challenge, but investigations of this system have yielded novel insights, some of which may be generalizable to many members of the superfamily of G protein-coupled receptors (GPCRs). The P T H - r e c e p t o r system is composed of at least three major constituents: the linear peptide hormone, heptahelical transmembrane receptors, and heterotrimeric guanine nucleotide binding proteins (G proteins). PTH, a fully active form of which is a linear peptide 34 amino acids long, is a highly flexible molecule. Therefore, an assortment of low-energy conformations exists in fast dynamic equilibrium. Only a subset of these conformations is thought to be acceptable to the receptor for binding. The receptor is acomThe Parathyroids, Second Edition

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toward the GDP-bound G protein. The formation of the hormone-receptor-G-protein ternary complex represents the initiation of the intracellular signaling cascade. In addition to h o r m o n e - r e c e p t o r - G protein interactions, there are important interactions of these components with other molecules, such as [3-arrestins, regulators of G-protein signaling (RGS) proteins that act as GTPase-activating proteins (GAPs), G proteincoupled receptor kinases (GRKs), and receptor activitymodifying proteins (RAMPs). Formation of GPCR homodimers and heterodimers may represent an additional mechanism for modulating receptor function. Several receptors, including the type 1 P T H / PTH-related protein (PTHrP) receptor (PTH1-Rc), can interact with more than one G protein (e.g., the PTH1Rc interacts with e i t h e r Gq o r G s proteins), but other GPCRs interact directly via their C-terminal domain with proteins containing primary decidual zones (PDZs) and Enable/vasodilator-stimulated phosphoprotein (VASP) homology-like domains. The complexity of the GPCR signaling system requires that studies of structure-function relations include direct analysis of multicomponent systems, such as the ligand-receptor complex, in order to achieve useful level of resolution. Analyzing the system at this level may provide insight into partially understood physiologic and pathophysiologic processes associated with the PTH/PTHrP-receptors system. These studies have the potential to identify new therapeutic targets and generate paradigms useful for the development of novel agents directed at ligand-receptor interactions. This chapter summarizes recent findings obtained from studies of either PTH ligands or receptors as single entities and the most recent data emerging from direct study of the complexes formed when PTH or PTHrP interact with the PTH1-Rc or PTH2-Rc. To provide a framework for understanding the most recent approach developed to study the PTH/PTHrP-receptors system, namely the "ligand-receptor-centered" approach, we review first the "ligand-centered" and "receptor-centered" approaches. Studying ligands and receptors separately also generates important insights. However, these concepts can only be validated when the hormone-receptor bimolecular interaction is examined directly.

LIGAND-CENTERED APPROACH Recent Advances in Structure-Activity Relations Extensive reviews have been published covering early work on structure-activity relations of PTH and PTHrP (1-3). It has been established for both these calciotrophic hormones that the N-terminal 1-34 amino

acid sequence of either is sufficient to induce the entire spectrum of in vitro and in vivo PTH1-Rc-mediated activities (4,5). PTH(1-34) and PTHrP(1-36) are equipotent for binding to the PTH1-Rc and for stimulating adenylyl cyclase and intracellular calcium transients in cells expressing the PTH1-Rc. Significant sequence homology is shared by residues 1-13 of both hormones (8 identical residues), though sequence homology is negligible for the 14-34 sequence. The assignment of "activation domain" to the homologous N-terminal sequences is based on demonstration that this region has a functional role in intracellular signaling, and that truncation of 2-6 residues from the N terminus converts an agonist peptide to an antagonist (6-8). The divergent mid- and C-terminal amino acid sequences contain the "binding domain" assigned to residues 14-34 (9-12). Based on these observations, it was hypothesized that N-terminal sequences comprising the activation and binding domains of both hormones share similar conformations despite their sequence differences (10,13). The early work on structure-activity relations of PTH and PTHrP has been reviewed previously (1,2); provided below is a summary of the more recent progress in this field.

Truncated Sequences Although amino-terminal fragments of PTH and PTHrP shorter than 1-27 were initially reported to be devoid of biological activity (14-17), recent efforts of Gardella and co-workers have focused on the activation domain represented by the amino terminus PTH(1-14) (18-20). In the search for small peptide and nonpeptide molecules with PTH activity as potential therapies for metabolic bone disorders, the marginally active PTH(1-14) was used as the starting point for structure-activity studies. The rationale was based on site-directed mutagenesis and chimera studies of PTH1-Rc (21-24), functional analysis of structural complementary between PTH1-Rc/calcitonin (CT) receptor chimera and P T H / C T hormone hybrids (25), and photoaffinity cross-linking studies between photoreactive PTH and PTHrP analogs and PTH1-Rc (26-30) or PTH2-Rc (31). These studies suggest that the activation domain of PTH interacts with the extracellular loops (ECLs) and the juxtamembrane portions of the transmembrane (TM) domains of receptor. These receptor sites are different than those involved in interacting with the binding domain of PTH, which is primarily within the receptor's N-terminal extracellular domain (N-ECD). Similar observations were reported for secretin (32,33), vasoactive intestinal peptide (VIP) (33,34), CT (35), and CT/glucagon chimera (36) receptors, all belonging to class II (or group B) of the GPCRs.

PTH/PTHrP/REcF~eTOR INTERACTIONS / PTH (1-14) stimulated cAMP levels with equipotency (EC50 ~ 100 IzM) via the intact rat (r) PTH1-Rc and the N-terminal truncated (missing residues 26-181 of the N-ECD) receptor (rANt), both transiently expressed in COS-7 cells. In contrast, PTH(1-34) was two orders of magnitude less potent in stimulating cAMP accumulation in the rANt than in the intact rPTH1-Rc (18). In addition, "Ala-scan" of PTH(1-14) revealed that the first nine N-terminal residues form the critical activation domain and are involved in ligand-receptor interaction rather than an intramolecular interaction with the C-terminal domain PTH(15-34), as was previously suggested (37-39). This study concludes that the N terminus of PTH interacts with binding determinants within the ECLs and the juxtamembrane portions of the TM domains of PTH1-Rc, Interestingly, some substitutions in the 10-14 sequence of the hormone were not only compatible with function, but also resulted in more potent peptides , such as [Ala3,10,12, Arg ~1] PTH (1-14) 1 and [Ala ~'1°, Arga1]PTH(1-11), which were 100- and 5-fold more active than PTH(1-14), respectively (19). In addition, increases in cAMP levels were observed following the insertion of His, a "Zn 2+ switch," into positions in the 10-13 sequence of PTH(1-14) (20). Taken together, Gardella and co-workers suggest that the C-terminal portion of PTH(1-14) contributes important interactions with the ECLs and TM domains, which are stabilized by complex formation with Zn 2+ salts. However, in the absence of demonstrable specific binding, the extremely small increases in cAMP production do not provide a high degree of confidence that the reported cAMP increases are mediated by a specific interaction between PTH(1-14) and PTH1-Rc. Substitutions within the Intact Sequence

Several independent studies comprehensively scanned either the entire or limited segment of PTH(1-34/36) by a multiplicity of substitutions. Extensive corroborating data were generated, as well as some new insights into the tolerance and significance of certain residues with regard to bioactivity (40-42). Both Gardella and Oldenburg and their co-workers used recombinant DNA methodologies to generate analogs either randomly mutated at codons 1-4 in hPTH (40) or with individual codon replacement with [ (A/G) (A/G)G] (coding for lysine, arginine, glummine, or glycine) (42). In addition, Gombert et al. used a parallel multisynthesis approach to generate D-Ala, L-Ala, and D-Xxx scans of hPTH (1-36) (41). 1To simplify the reference to amino acid residues in the ligand and the receptor, the amino acids of the ligand are denoted using the three-letter code, whereas the one-letter notation is used for the residues of the receptor.

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For the D-Ala scan, the largest decrease in binding affinity was observed for the segments 2-8 and 20-28, accompanied by the largest loss in efficacy for the latter segment, especially for Arg-20, Trp-23, Leu-24, and Leu-28 substitutions (41). A high correlation between binding and adenylyl cyclase activation was observed in the L-Ala scan, with the segment 2-8 suffering the largest loss in activity (41). Only substitutions of Lys-13, Asn-16, and Glu-19 yielded slightly more active analogs. The D-Xxx scan resulted in an overall loss of affinity and efficacy, with the greatest loss at the putative amphiphilic helical domain (residues 23-29), and a slightly better tolerance at the C-terminal segment (32-36) (41). Mutations of the evolutionarily conserved first four N-terminal residues in PTH were carried out by Gardella and co-workers (40). Residues Glu-4 and Val-2 were less tolerant of substitution, suggesting that they contain important determinants for receptor binding and activation (40). Conversely, Ser-1 and Ser-3 were more tolerant of substitution, suggesting that they play less critical roles in h o r m o n e activity. The most intriguing finding of this study was the divergent activity displayed by [Arg2, Tyr~4]PTH(1-34)NH2 in two different cell lines, both expressing the wild-type PTH1-Rc (40). This analog binds to ROS 17/2.8 cells, a rat osteosarcoma cell line, with twofold higher affinity than do OK cells, an opossum kidney cell line (40). Nevertheless, it is a weak partial agonist for stimulation of adenylyl cyclase in ROS 17/2.8 cells, whereas it is a full agonist for cAMP increases in the OK cell system (40). It remains to be determined whether the differences in activity were related to potential tissue-and speciesspecific effects across the two cell types. A latter study addressed some of these questions and will be discussed below (43). The highly conserved residues Ser-3 and Gln-6 in PTH and PTHrP contribute importantly to binding and activation (39). Substitution of Ser-3 by either Phe or Tyr and of Gln-6 by Phe and Ser generated partial agonists. Both [Phe3]hPTH(1-34) and [Phe6]hPTH(1-34) were found to inhibit competitively bPTH(1-34)- and PTHrP(1-34)-stimulated adenylyl cyclase activity. Taken together, the findings that substitutions within the "activation domain" may convert full agonists into partial antagonists provide new tools to design potent full-length antagonists (1-34). It also suggests that structural perturbation of the ligand-receptor bimolecular interactions at the N terminus of PTH(1-34) may interfere with the conformational changes required for coupling the ligand-occupied receptor to G proteins, thus inhibiting induction of intracellular signaling. Some provocative observations were reported by Oldenburg and co-workers following an extensive

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mutational study of h P T H ( 1 - 3 4 ) in UMR-106 rat osteosarcoma cells (42). Single nonconservative substitutions spanning the 11-30 sequence, Leu 11 Arg, Asn TM Lys, Glu 19 Arg, Glu 22 Arg, Asp 3° Arg, as well as a conserved one, Lys26 Arg, resulted in e n h a n c e m e n t of bioactivity. The C-terminal amino acid substitutions Asn 3~ Glu/Gly and Tyr 34 Lys/Glu also resulted in e n h a n c e d potency. One of the most interesting analogs is [Arg 19'22'3°, Lys 20, Hse34]hPTH(1-34), a peptide carrying a net charge of + 7 concentrated in the C-terminal amphiphilic helix. The analog is equipotent (EC50 --0.9 and Kd --- 1.5 nM) to bPTH. One possible explanation for the observation is that the amphiphilic nature of this helix is not critical per se, but rather the presence and disposition of the positive charges in this helix are critical for either intramolecular, ligand-receptor, or ligand-lipid interactions (42). Taken together, these results suggest that some favored paradigms, such as salt bridges stabilizing the bioactive conformation of PTH a n d / o r intramolecular stabilization between the amino- and carboxyl-terminal regions of PTH forming a U-shaped conformation, now need to be reexamined.

Receptor Subtype Specificity Switch The two homologous PTH receptors, PTH1-Rc and PTH2-Rc, display differing specifications for ligands: PTH1-Rc binds both PTH and PTHrP, whereas PTH2Rc binds only PTH. These two receptors provide a platform for probing the basis of molecular recognition of ligand and receptor (44,45). The finding that the N-truncated sequence, P T H r P ( 7 - 3 4 ) , can bind and weakly activate the PTH2-Rc suggests that the Nterminal sequence 1-6 of PTHrP must contain a structural element that disrupts P T H r P ( 1 - 3 4 ) - P T H 2 - R c interaction (45). Swapping the nonconserved residues in position 5 between PTH(1-34) and PTHrP(1-34) generates the single-point mutants [His 5, Nle s'ls, Tyr~4]bPTH(1-34)NH2 and [Ile5]PTHrP(1-34)NH2 (45). Indeed, in HEK293 cells stably transfected with

either hPTH1-Rc or hPTH2-Rc, the receptor specificity of these point hybrids is reversed when compared with their parent compounds. Therefore, His-5 is the specificity "switch" between these two highly homologous receptor subtypes (45). Gardella and co-workers conducted studies in COS-7 cells transiently expressing the PTH1-Rc and PTH2-Rc and reached a somewhat different conclusion (46). According to their study, two sites are responsible for the divergent specificity: position 5 determines signaling and position 23 determines receptor binding affinity. Swapping the residues in positions 5 and 23 between PTH and PTHrP results in [His 5, Phe 2~, Tyr34]PTH(1-34)NH2 (IC50 > 10,000 n M f o r both PTH1Rc and PTH2-Rc, and EC50 - 1.18 and > 1000 n M for PTH1-Rc and PTH2-Rc, respectively) and [Ile 5, Trp 2s, Tyr36]PTHrP(1-36)NH2 (IC50 = 16 and 10 nM, and EC50 = 0.21 and 0.5 n M for PTH1-Rc and PTH2-Rc, respectively). In that study [Trp 2~, Tyr36]PTHrP (1-36) NH 2 is an antagonist for the PTH2-Rc but a full agonist for the PTH1-Rc. The discrepancies between the two studies (45,46) may be related to differences in the experimental systems employed, such as stable versus transient transfections or homologous versus heterologous receptor-cell systems, and the use of different radioligands, e.g., rat- versus bovine-derived peptides. An endogenous ligand selective for PTH2-Rc, tuberoinfundibular peptide of 39 amino acids (TIP39), has been purified from bovine hypothalamic extracts (47). A homology search reveals that 9 out of the 39 residues of TIP39 are identical to b P T H (Fig. 1). Interestingly, TIP39 did not activate adenylyl cyclase in COS-7 cells transfected with either h u m a n or rat PTH1Rcs (47). The physiologic role of the TIP39-PTH2-Rc system remains to be established.

Search for the Putative Bioactive C o n f o r m a t i o n Identification of the peptide ligand conformation responsible for the recognition by, binding to, and activation of the GPCR is a major objective in structural

1

34

hPTH

SVSEIQLM

HNLGKHLNSM

~.RVEWLPKKL

QDVHNF ~~~

hPTHrP

AVSEHQLL

HDKGKSIQDL

RRRFFLHHLI

AEIHTA ~~~

SLALADDAAF

RERARLLAAL

~.RRHWLNSYM

HKLLVLDAP

TIP39

FI6. 1 Peptide Homology for human PTH, PTHrP, and TIP39. The functional N termini of hPTH and hPTHrP are shown [264-266], and are aligned with the complete human tuberoinfundibular peptide of 39 amino acids (TIP39) (47). Boldfaced amino acid residues shown in TIP39 have direct homology to the boldfaced ones shown in either PTH or PTHrP. The numbering at the top refers to PTH and PTHrP, and at the bottom, to TIP39.

PTH/PTHrP/REcEPTOR INTERACTIONS / biology. The ligand-receptor complex is the definitive system for study of the putative bioactive conformation. Unfortunately, for GPCRs this is currently an unattainable goal because no h o r m o n e - G proteincoupled receptor complex has been crystallized, probably because the receptor is embedded in the cell membrane. Based on GPCRs being embedded in the membrane, the hypothesis formulated by Schwyzer proposes that the initial conformation adapted by a ligand is induced by nonspecific interactions with the membrane (48-50). This membrane-induced conformation is the one recognized by the membrane-embedded GPCR. Therefore, study of conformations in the presence of membrane-mimetic milieu, like the micellar environment, is probably the best available approximation of the natural state. Secondary structure prediction methods (51-54) suggest that the N-terminal 1-34 sequences of both PTH and PTHrP assume helical structures at their N and C termini (39,55,56). These helical domains span residues 1-9 and 17-31 in PTH and 1-11 and 21-34 in PTHrP (39). Correlation between the receptor binding affinity and the extent of helicity was determined by circular dichroism (CD), a method that can assess the global conformational nature of a peptide (57). The same spectroscopic method estimated PTH(1-34) in water to have, on average, less than eight residues in the helical conformation. This n u m b e r was even smaller for PTHrP(1-34) (39,55,58-60). In the presence of 45% trifluoroethanol (TFE), a solvent that promotes secondary structure, the total helical content of bPTH(1-34) and hPTHrP(1-34) is 73% (39). Nevertheless, there is much controversy about the relevance of the conformation in TFE to the bioactive conformations. Early ~H nuclear magnetic resonance (NMR) studies in water demonstrate that PTH (1-34) is mostly random in structure, except for a short ordered region encompassing residues 20-24 (61-63). According to our recent findings, hPTH(1-34) in water is highly flexible, with some evidence of transient helical loops spanning the sequence 21-26 and 7-8 (64). Cohen and co-workers suggest that in TFE the amphiphilic helices located at the N and C termini of bPTH(1-34) and hPTHrP(1-34) interact to form a U-shaped tertiary structure with the hydrophobic residues facing inward to form a hydrophobic core. The hydrophilic residues orient outward and are exposed to the polar solvent (39). However, the lack of long-range interactions between the two helices in both hPTH(1-34) (65-67) do not support the notion of a U-shaped tertiary structure. Interestingly, the longrange p r o t o n - p r o t o n correlations between the two N-terminal helices (sequences 1-10 and 17-27) in full-

57

length recombinant hPTH(1-84) in aqueous TFE are d e p e n d e n t on interactions provided by the middle and C-terminal portion of the molecule (sequences 30-37 and 57-62, respectively) (68). In TFE, the low dielectric constant, which helps to stabilize helices, is also supposed to shield the side chains from hydrophobic interactions between the helices and, therefore, destabilizes alleged U-shaped tertiary structures. Marx and co-workers suggest that hPTH(1-37) in aqueous solution containing high salt concentration assumes a U-shaped structure (37). However, their reported long-range p r o t o n - p r o t o n correlations are limited to side chains of Leu-15 and Trp-23 located close to the bend forming the putative U-shaped structure, therefore leaving too much flexibility to define a stable U-shaped structure. The same researchers identified the loop region around Hisl4-Ser 17 and longrange p r o t o n - p r o t o n correlations between Leu 15 and Trp 23 found in hPTH (1-37) and in N-truncated analogs hPTH(2-37), hPTH(3-47), and hPTH(4-37) but did not interpret it to stabilize a U-shaped structure (69). Other studies of PTHrP analogs described interactions between the N- and C-terminal helical domains, in the presence of TFE, thus offering support for the U-shaped structure (70-74). However, current established understanding runs counter to the U-shaped structure as the predominant bioactive conformation of PTH(1-34) and PTHrP(1-34). In our studies (13,75) of PTHrP(1-34)-related analogs in aqueous solutions and in the presence of TFE, we could not confirm the presence of long-range helix-helix interactions (76). In addition, we studied a series of side chain to side chain-bridged monocyclic and bicyclic lactam-containing PTHrP analogs. These analogs are cyclized through side chain pairs AsplS-Lys17 and Lys26-Asps°, located at the putative N- and C-terminal helical domains, respectively (13,75,77-80). The i to i + 4 side chain to side chain cyclization is known to stabilize helical structures. Bioactivity in the agonist (1-34) and antagonist (7-34) series of lactamcontaining analogs was found to require well-defined N- and C-helical domains that are linked by two flexible hinges located around residues 12-13 and 19-20, the latter being associated with high bioactivity (Fig. 2) (13,75). Similar conclusions were reached by Gronwald and co-workers studying PTHrP(1-34) in water and in 50% TFE (81). In the presence of TFE, they observe two stable or-helical regions spanning residues 3 to 12 and 17 to 33, which are connected by a flexible linker. Their observations clearly exclude the possibility of any significant tertiary structure (81). Although Barden and Kemp mention the presence of a hinge at ArglO-Arg2° in [Ala°]PTHrP(1-34)NH2 and attribute to it a functional role in signal transduction, they also postulate

58 / CHAr'TR4 V, Antagonist C-Terminus

..... ~,~,~~'~ ................. i:~ .............!i! .................... ..........i'iiii~ .... '

........... .

i!ili ~'~ii"i~i~i iiiill................... Agonist

....-""

~

.2(

•

J..;::.

I ",

--!

}

..

{

"

/

,,, ....... b I~ ,~

Agomst

.....................

~'.';.........

M ..........~: ........~.......... . ~ , ".>,~ ...."~ ~[]q~

:~.>.~;.("',!"~ ...............:~,L-q "...... ~ '.~........!...... ......... :i-.~.

~;- ........-..":...

-..-.7~: .,,.... ....~,,. ....... ..............

.~

L;-lermlnus

..... ":~."" ....... i. 100 nM) to the uncoupled receptor (94). Therefore, the uncoupled PTH1-Rc in the cell-based binding assay binds RS-66271 with lower affinity compared to hPTH(1-34). The lower affinity to the uncoupled receptor may result from either less favorable interactions between the MAP sequence and the receptor or a compromised conformation of RS-66271. It is generally accepted that PTH(1-34) and PTHrP(1-34) contain two helical domains spanning sequences 13-18 and 20-34 (66,67,95,96). Introduction of side chain to side chain cyclizations between residues that are four amino acids apart and located across a single helical pitch (residue i to residue i + 4) has been demonstrated to be an effective way to stabilize a helical structure (97-101). Therefore, we undertook replacement of a potential ion pair, participating in or-helical stabilization, by a covalent lactam bridge, in an attempt to further stabilize the helices in these regions. The initial application of this approach generated c[Lysl~-Asp17]PTHrP(7-34)NH2, which was about 10-fold more potent than the linear parent antagonist (Kb = 18 and 170 nM, Ki = 17 and 80 nM, respectively, in SaOS-2/B10 cells) (77). Rigidification of the C-terminal helix in c[LysZ6-Asp3°]PTHrP(7-34)NH2 did not improve antagonist potency (79). However, combination of two 20membered lactam bridges, in both the N- and C-terminal helices, generated c[Lysl~-Asp17, Lys 26Asp~°]PTHrP(7-34)NH2, a potent (Kb = 95 n M and Ki = 130 nM, in SaOS-2/B10 cells) (79) highly conformationally constrained PTHrP-derived antagonist and a valuable tool for conformational studies (75). The same approach applied to the agonist PTHrP(1-34)NH 2 yielded the mono- and bicyclic analogs, 13 17 26 c[Lys13-Asp17]PTHrP(1-34)NH2 and c[Lys-Asp , L y s 3O Asp ]PTHrP(1-34)NH 2, which were equipotent to the linear parent compound (Kb = 3.2, 2.1 and 1 nM, Km = 0.17, 0.22, and 0.57 nM, respectively, in SaOS-2/B10 cells) (79). A similar approach was also applied to the signalingselective analogs, hPTH (1-31)NH 2 and the more potent [Leu 27]hPTH (1-31 ) NH 2. Both of these analogs stimulate

/

61

adenylyl cyclase but not the PLC/PKC signaling pathway (85). Though i to i + 4 lactam bridge formation between Glu-22 and Lys-26, as in c[Glu 22LysZ6,Leu27]hPTH (1-31) NH 2, results in about a fourfold increase in adenylyl cyclase activity, as compared to the linear parent peptide (EC50 = 3.3 and 11.5 nM, respectively, in ROS 17/2 cells), similar cyclization between Lys-26 and Asp-30 or i to i + 3 lactam bridge formation between Lys27 and Asp-30 results in cyclic analogs that are less potent than the corresponding linear parent peptides (85). Interestingly, the higher adenylyl cyclase activity in vitro observed for c[ Glu22-LysZ6,Leu 27] hPTH (1-31 ) ~qH2 compared to the linear peptide results in greater anabolic effect on trabecular bone growth in ovariectomized rats (102) and affords more effective protection than hPTH(1-34) against loss of femoral trabeculae in the same animal model (103). The retention of full ability to activate PKC (in ROS 17/2 cells) by the extensively N-terminally truncated linear fragment, [LysZ7]hPTH(20-34)NH2, and the structurally related lactam-bridged analog, c[Lys 26Asp~°]hPTH(20-34)NH2, was consistent with the stabilization of the amphiphilic helix at the C-terminus, implicating the helix as an important functional motif for binding to the PTH1-Rc (104). Taken together, the above studies provide important insights regarding the structural nature of the hormones PTH(1-34) and PTHrP(1-34) and help to better characterize conformational features important for PTH binding and bioactivity.

Signaling-Selective Ligands Activation of PTH1-Rc evokes dual signaling pathways, increasing both adenylyl cyclase/PKA via GsOLand PLC/IP~-DAG/cytosolic transients of [CaZ+]i/PKC via Gq (43,105-111). Dual signaling is observed in homologous and heterologous receptor/cell systems, which include rat, opossum, mouse, porcine, and h u m a n receptors and cells. In general, maximal signaling intensity through both pathways increases with receptor density. However, a larger n u m b e r of PTH1-Rcs per cell is needed to activate the PLC-associated pathway than is needed to stimulate adenylyl cyclase. PTH modulates downstream activities in osteoblasts, leading to regulation of cell growth, proliferation, and differentiation (112,113). PTH affects osteoclasts indirectly through its direct action on osteoblasts (114). Subcutaneous administration of PTH results in an immediate and transient expression of c-fos mRNA in PTH1-Rc-bearing cells (chondrocytes, osteoblasts, and spindle-shaped stromal cells), followed by a delayed expression in the majority of stromal cells and osteoclasts (115). This observation provides further support

62

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CHAPTER4

for the indirect action of PTH on osteoclasts, which may be mediated by osteoblasts, a n d / o r a subpopulation of stromal cells. In UMR cells, PTH rapidly and dose-dependently induces transcription of c-fos (116,117). Pearman and co-workers reported that the cAMP response element (CRE) in the c-fos p r o m o t e r is required for PTH-induction of c-fos in UMR cells and that the CRE binding protein (CREB) binds to this site, apparently as a homodimer, and is phosphorylated in a PTH-inducible fashion at Ser-133 (118). Therefore, c-fos appears to have pleiotropic and essential effects in bone, including mitogenesis a n d / o r differentiation in the skeletal system, as well as inhibition of osteocalcin expression by binding to the AP-1 site in the osteocalcin promoter, thereby suppressing the mature osteoblast phenotype (119). PTH-induced c-fos p r o m o t e r activity was completely inhibited in a concentration-dependent m a n n e r by transfection of a heat-stable inhibitor of PKA (120). This finding provides strong evidence that PKA is the enzyme responsible for phosphorylation of CREB at Ser-133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression. Nevertheless, the relationship between these signaling pathways and cellular and in vivo responses to PTH has not been completely elucidated. Understanding the role of cellular processes such as receptor inactivation, internalization, trafficking, and recycling in bone metabolism is only beginning to be elucidated. One of the major questions in the PTH field focuses on the mechanism responsible for catabolic versus anabolic actions of PTH induced by continuous versus intermittent administration of hormone, respectively. The linkage of one or both the signaling pathways to the anabolic activity of PTH remains to be established.

Search for the Ligand-Based Signaling Specificity Switch Much attention is directed toward identifying a signaling pathway that is specifically associated with the anabolic activity of PTH. One hypothesis is that this pathway or portions of it will be c o m m o n to all agents and treatments that have anabolic effects on bone. Studies carried out on bone cell and organ cultures suggest that residues 1-7 in PTH constitute the cAMP/PKA activation domain (121), whereas residues 28-34 in PTH comprise the PKC activation domain (122,123). The latter encompasses the region also associated with PTH mitogenic activity on cultured osteoblast-like cells (residues 30-34) (124,125). Cyclic AMP appears to be involved in the bone formation (126) and resorption activities (16) of PTH. PTH analogs that stimulate increases in cAMP levels have been shown either to inhibit (127-129) or stimulate (129-131) osteoblastic cell proliferation, d e p e n d i n g on species, the cell models used, and the experimental

conditions. However, N-terminally truncated fragments of PTH, which selectively activate PKC without affecting cAMP (121,132,133), are also mitogenic for osteoblastic cells (134). Because these truncated fragments do not stimulate bone resorption (16), they may be more effective "anabolic" analogs than are peptides with an intact N terminus. Truncation of two amino acids from the N terminus of PTH(1-34), e.g., PTH(3-34), reduces adenylyl cyclase activation without significantly affecting PKC activation or the mitogenic response in vitro (132). Similarly, the stimulation of TE-85 h u m a n osteosarcoma cell proliferation by PTH(1-34) is not associated with an increase in intracellular cAMP (135). Therefore, if stimulation of bone formation in vivo is related only to the mitogenic response in vitro, the bone formation response should be retained in the aminotruncated PTH fragments. Although PTH stimulation of bone resorption in vitro is mediated primarily through cAMP-dependent activation of PKA (136), it may not be the sole second messenger pathway involved in this activity (137,138). One of the current working hypotheses holds that dissociation between the two signaling pathways of PTH, adenylyl cyclase and PLC, will result in separation between the anabolic and catabolic activities of PTH in bone (14,126,132,139-143). If the stimulation of bone resorption in vivo is related to the bone resorption response in vitro, the in vivo response should be diminished in amino-terminal-truncated PTH fragments. However, neither PTH(3-34) nor PTH(3-38) (both PKC-selective, N-terminal-truncated analogs of PTH) are active in vivo as bone anabolic agents (4,122,126,143-146). Furthermore, desaminoPTH(1-34), which has drastically reduced ability to stimulate adenylyl cyclase but is equipotent to h P T H ( 1 - 3 4 ) in stimulating PKC, does not stimulate cortical or trabecular bone growth in ovariectomized rats (126). Surprisingly, hPTH (1-31 ) NH 2 (Ostabolin), an adenylyl cyclase-selective PTH agonist equipotent to PTH(1-34) in stimulating cAMP production in ROS 17/2 (57,123), strongly stimulates cortical and trabecular bone growth in ovariectomized rats (126,143,145-147). In this analog the putative PKC-signaling motif GlnZS-His32 is compromised by the elimination of His-32 (123). A second generation of adenylyl cyclase-selective analog, 22 26 27 c[Glu -Lys ,Leu ] hPTH (1-31) NH2, in which the helical nature of the C terminus was enhanced by the formation of a side-chain to side-chain lactam ring and the introduction of a hydrophobic residue at position 27, was 1.4to 2-fold stronger than the linear parent analog as a stimulator of femoral trabecular bone growth (102). Both 22 26 27 hPTH (1-31) NH 2 and c[Glu -Lys ,Leu ] hPTH (1-31)NH 2 were reported to prevent loss of vertebral trabecular bone in ovariectomized rats and to increase vertebral tra-

PTH/PTHrP/RECEPTOR INTERACTIONS / becular volume and thickness over those of control vehicle-injected sham-operated rats (147). The action of these analogs on vertebral bone was as effective as that of h P T H ( 1 - 3 4 ) N H 2. However, unlike h P T H ( 1 - 3 4 ) N H 2, their effects on pelvic BMD were equivocal. An alternative view has been offered regarding the structural determinants associated with signaling pathway activation. Replacement of Glu 19 --) Arg, a receptorbinding affinity-enhancing modification, generated [Arg19]PTH(1-28) as a potent and full stimulator of adenylyl cyclase and PLC. Interestingly, substituting 1 19 Gly-1 for Ala generated [Gly ,Arg ] h P T H ( 1 - 2 8 ) , which is an adenylyl cyclase-selective agonist (148). This study concluded that the extreme N terminus of h P T H constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially h P T H ( 2 8 - 3 1 ) , contributes to PLC activation through receptor binding, but the domain is not required for full PLC activation. The N-terminal determinants for adenylyl cyclase and PLC activation in h P T H ( 1 - 3 4 ) overlap but are not identical; subtle modifications in this region may dissociate activation of these two effectors. Another approach attempted to design target organspecific PTH analogs on the assumption that a boneselective analog would be a better bone anabolic agent. To this end, [HisS] - and [Leu~]hPTH(1-34) were generated and found to be partial agonists of adenylyl cyclase in a kidney cell line (50 and 20%, respectively), but full agonists in UMR-106 rat osteosarcoma cells (149). However, both analogs were less potent than native PTH(1-34) in vivo in the induction of bone formation. In the course of designing photoreactive PTHrP analogs for mapping the bimolecular ligand-receptor interface, we generated trB p a 1,Ile 5,Ar g 1113 ' ,T ry 361jPTHrP(1-36)MH 2 (29). This analog binds and stimulates adenylyl cyclase equipotently to the parent analog [Ile5,Arg11'a~,Try~6]PTHrP (1-36) NH 2 in HEK293/C-21 cells overexpressing the h u m a n PTH1-Rc (--400,000 receptors/cell), but does not elicit intracellular calcium transients. Moreover, it does not stimulate translocation of [3-arrestin2-green fluorescent protein (GFP) fusion protein, an effect that is PKC d e p e n d e n t (150). In summary, development of an effective and safe therapeutic modality that would stimulate the formation of new, mechanically competent bone and possibly reconstitute trabecular architecture in osteoporotic patients continues to be a worthy goal. This goal may be approached by analogs that interact with the PTH1-Rc in a signaling-selective manner.

Nuclear Localization o f PTHrP PTHrP has been shown to function in a second mode of action: as an intracrine factor with direct intracellular effects following translocation into the nucleus

63

a n d / o r nucleolus of the cell. Exogenous h u m a n PTHrP(1-108) is internalized specifically by UMR106.01 osteogenic sarcoma cells that express PTH1-Rc. The h o r m o n e accumulates in the nucleus and nucleolus (151). PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) homologous to SV40 T antigen. Deletion of the NLS, or mutation of the conserved GxKKxxK motif within the NLS, effectively prevents both cell surface binding and n u c l e a r / n u c l e o l a r accumulation of PTHrP(1-141) (152). In contrast to proteins containing conventional NLS motifs, which are actively transported by importinet[3 heterodimers (members of a family of structural molecules that mediate nuclear import of proteins containing NLS motifs), PTHrP is recognized exclusively by importin-[3 and the small GTPase, Ran, which together actively transport PTHrP to the nucleus i n d e p e n d e n t of importin-e~ (151). Thus, PTHrP appears to be actively transported to the nucleus via a novel mechanism that is i n d e p e n d e n t of importin-ot, although the biologic significance of this alternate nuclear targeting pathway is currently not understood. Synchronized cell culture studies have demonstrated that PTHrP localizes to the nucleus at the G 1phase of the cell cycle and is transported to the cytoplasm on initiation of mitosis (153). Scanning mutagenesis reveals that T-85 adjacent to the NLS of PTHrP was phosphorylated by CDC2-CDK2 in a cell cycle-dependent manner. Mutation of PTHrP, [As5]PTHrP, results in nuclear accumulation of PTHrE Mutation to [E85], which mimics a phosphorylated threonine residue, results in localization of PTHrP predominantly to the cytoplasm. This study concludes that phosphorylation of T-85 results in decreased nuclear accumulation of PTHrP, whereas the unphosphorylated state (e.g., [A-85] mutant) is preferentially nuclear localized. However, a potential role for PTHrP in regulating cellular phenotype in a cell cycled e p e n d e n t m a n n e r is currently not known. Although the precise role of PTHrP translocation to the nucleus is currently unknown, it may participate in the regulation of cell proliferation, differentiation, and apoptotic cell death during development. Future studies that characterize the nuclear actions of PTHrP would add significantly to our understanding of the role of PTHrP during embryonic skeletal development and as an oncoprotein whose expression in ~ many tumors may correlate with increased tumor aggressiveness and metastatic potential.

RECEPTOR-CENTERED A P P R O A C H The physiologic effects of PTH and PTHrP are largely mediated through the glycosylated PTH1-Rc receptor. PTH1-Rc is encoded by a single-copy gene that

64

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CHAPTER4

is expressed in PTH target tissues, including kidney, intestine, and bone, where it is essential for maintaining proper mineral ion homeostasis (154). In kidney, PTH1Rc mediates PTH-induced calcium resorption directly by the distal nephrons (155,156). It also inhibits phosphate resorption in the brush border membrane of proximal tubules by targeted lysosomal degradation of the sodium-dependent cotransporter Npt2 (157). In kidney and intestine, PTH1-Rc indirectly mediates vitamin D-dependent calcium absorption by regulating 10t-hydroxylase activity as well as vitamin D receptor biosynthesis (158,159). In bone, PTH1-Rc mediates acute release of calcium from mineralized matrix by activation of osteoclasts. It also plays a major role in modulating more long-standing calcium metabolism by osteoblasts and indirectly by osteoclasts. PTH1-Rc is expressed on osteoblasts as well as in many osteosarcoma cell lines, where it has been demonstrated to signal PTH-mediated changes in gene expression for a number of critical factors in bone homeostasis, including osteocalcin and osteoprotegerin (160). PTH1-Rc has been cloned from several diverse species, including human, rat, mouse, opossum, Xenopus, and zebrafish (27,110,154,161-163). It is a m e m b e r of the class II G protein-coupled receptors that include heptahelical transmembrane receptors for peptide hormones such as secretin, glucagon, and calcitonin (Fig. 5) (164). The 85-kDa PTH1-Rc contains many of the hallmark structural features of class II GPCRs, including a large extracellular domain amino terminus (N-ECD), eight conserved extracellular cysteine residues, and a large (150-190 amino acid residues) cytoplasmic C terminus (Fig. 6). Class II receptors are also identified by conserved cysteines in the first and second extracellular loops as well as by several homologous N-glycosylation sites within the N-ECD. However, though highly conserved, N-glycosylation of PTH1-Rc appears to have little or no influence on receptor expression, ligand binding, and intracellular signaling (165). Like several characterized class II GPCRs, PTH1-Rc is capable of activating multiple intracellular signaling cascades. On binding ligand, PTH1-Rc rapidly up-regulates activity of two distinct intracellular pathways, G, ot/adenylyl cyclase/protein kinase A (PKA) and Gq/phospholipase C/protein kinase C (PKC) (111,166-169). The activation of these signaling cascades gives rise to increased cytosolic cAMP or calcium, respectively. In addition to multiple PTH receptor genes, differential mRNA exon splicing is another cellular mechanism for generating receptors that have altered ligand specificity a n d / o r signaling capacity. For example, Northern blot analyses of human squamous cell lines and keratinocytes demonstrate expression of multiple PTH1-Rc mRNA transcripts that differ in size from the cloned human receptor mRNA (170). Further analysis using a

GIP-Rc Gluc-Rc GLP1-Rc PTHR1 PTHR2 PACAP-Rc VIP2-Rc Sec-Rc VIP1-Rc GHRH-Rc

CRH-Rc

t

msDH-Rc CTR-Rc

FIG. 5 Phylogenetic dendrogram of the human class II GPCR gene family. Thirteen related receptors are shown. GIP-Rc, Gastric inhibitory polypeptide receptor; Gluc-Rc, glucagon receptor; PACAP-Rc, pituitary adenylate cyclase activiating peptide receptor; VIP1- and VIP2-Rc, vasoactive intestinal peptide type 1 and type 2 receptor; Sec-Rc, secretin receptor; GHRH-Rc, growth hormone releasing hormone receptor; CRH-Rc, corticotropin releasing hormone receptor; msDH-Rc, Manduca sexta diuretic hormone receptor; CTR-Rc, calcitonin receptor.

polymerase chain reaction (PCR)-based strategy in human kidney as well as SaOS-2 osteoblast cell lines detected two variants of the PTH1-Rc mRNA that are created by alternative splicing of exons coding for the N-terminal receptor domain (171). One alternatively spliced receptor, designated the S-N3-E2 isoform, juxtaposes exon 1 encoding the signal peptide (S) to an inframe alternative 3 acceptor site within the N3 intron. This splicing event produces a novel receptor with an additional 12 amino acids in the N-terminal extracellular domain of the receptor. In a second characterized PTH1Rc isoform, S-E2, an entire exon is deleted, causing a shift in the reading frame and premature translational truncation of receptor protein. However, an N-terminal truncated receptor may be produced by reinitiation of translation at a downstream initiation codon. A recombinant cDNA encoding the S-N3-E2 alternatively spliced receptor isoform exhibited weak signaling, inducing a two- to threefold increase in cAMP content, but not intracellular calcium, after stimulation with human PTH(1-34). A recombinant cDNA encoding the truncated S-E2 isoform failed to activate either signaling

PTH/PTHrP/R~cF~pTOR INTERACTIONS /

1

S i gnu_!. Sequence ..........................

....

PGLALLLCCP PSLALLLCCP WGWLMLGSCL CGWLILRSCL

60

hPTHR1 rPTHRI hPTHR2 rPTHR2

~~~MGTARIA .....MGAARIA MAGLGASLHV MPWLEALPYI

VLSSAYALVD VLS SAYALVD L.. .ARAQLD L ....VGAQLD

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61 RPASIMESDK GWTSASTSGK P ~ K A S G K L YPESEEDKEA PTGSRYRGRP TAANIMESDK GWTPASTSGK PRKEKASGKF YPESKENKDV PTGSRRRGRP ITAQLQEGE ...................................... GN ...... ITAQFQEGE ...................................... GN .......

120 ~LP~HIL~ | t I l ~LP~NIV~ ~FPEWDGLI~ ~FPE~GLI~

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121 WPLGAPGEVV WPLGAPGEVV WPRGTVGKIS WPRGTAGKTS

180 SE~VKFLTNE S KFMTNE S LQPD SD~..FLQPD

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rPTHR1 S G ~ i ~ i ii~~ iiii~T~LRETNAGRCDTRQQYr~ii hPT~ AGDI~~ i~ iii~i ~I~=AV GHDTRKQ~i rP T H ~ A G D . ~ i i i i ~ ~ i ~ ~ I ' ~ T N A V GHDMRKQYN~

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FIG. 6 Alignment of amino acid sequences and assignment of TM domains (shaded segments) of human ' (h) and rat (r) PTH1-Rc and PTH2-Rc.

65

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CHAPTER4

pathway in response to ligand. However, studies utilizing either iodinated PTH or receptor-specific antibodies to evaluate the cell surface expression of receptor isoforms indicate that the low or absent responses to PTH stimulation for these alternatively spliced receptors were in part due to low surface expression of the S-N3-E2 and S-E2 isoforms. Therefore, these data suggest that exon E1 is critical for cell surface expression of PTH1-Rc, but the S-N3-E2 recombinant isoform lacking this exon is capable of PTH binding and ligand-induced intracellular signaling, albeit at low levels.

PTH1-Rc and Ligand Binding A great deal of information is available detailing the current model of PTH1-Rc-ligand interaction and the important regions of receptor that facilitate recognition and binding. Experimental approaches utilizing site-specific mutagenesis, receptor chimeras with other class II GPCRs, PTH and PTHrP ligand alterations as functional probes, and photoaffinity cross-linking of receptor-ligand interfaces all have contributed to the most current understanding of PTH1-Rc structure and function. Though each of these approaches have inherent limitations, the "receptor-centered" approach to PTH1-Rc structure/function continues to refine an overall working model of the receptor domains critical for both mediating ligand binding, pharmacology, and intracellular G protein activation. Moreover, structure-function analysis of PTH1-Rc using these techniques is especially important given that X-ray crystallography, which has been instrumental in dissecting enzyme-substrate interactions, is not available for GPCR transmembrane molecules because of the lack of suitable crystals for analysis.

Chimera Receptors and a General Model of Ligand-Receptor Interactions Studies examining the structure-function of recombinant chimeric receptors, cloned from two class II members, support a general model that relies on distinct extracellular interaction domains that act in concert to affect G protein binding and activation on the intracellular receptor surface. Cognate class II members are thought to be derived from a single ancestral precursor receptor, and therefore share a general mechanism for ligand binding and activation. In this model, the N terminus of the ligand binds to the extracellularjuxtamembrane regions of the transmembrane and extracellular loop regions of TM5, TM6, and TM7 as well as ECL3 and is responsible for G protein activation; the C terminus of the ligand is critical for specific binding to the receptor N terminus. Though class II GPGR receptor structure has diverged to allow for specificity of ligand binding and

receptor activation, chimeric receptor studies have revealed that the overall pattern of ligand-receptor interactions has remained similar for many members of this large receptor family of molecules. This "cognate receptor" model was directly tested using the porcine calcitonin receptor (CTR) and rat PTH1-Rc (25). Though CT and PTH share little homology, the N termini of both ligands have been shown to be critical for receptor activation, and the C termini for receptor-binding specificity. Though similar in structure, the CTR and PTH1-Rc class II receptor glycoproteins share only 42% homology and are selectively activated only by their respective ligands. Bergwitz et al. created reciprocal CT/PTH1-Rc chimeras in which the N-ECD was exchanged between the two receptors (25). Similarly, chimeric ligands were synthesized in which the ligand activation and binding domains of each ligand were exchanged to create C T / P T H hybrid peptides. Using a COS-7 mammalian expression system to assess ligand binding and cAMP accumulation, it was demonstrated that reciprocal hybrid ligands (CTl-11/PTH 15-34 and PTHl-13/CT12-32), which do not activate the normal CT or PTH1-Rc receptors, could activate P T H / C T and C T / P T H receptor chimeras, respectively. This interaction was dependent on the receptor N-ECD binding the appropriate ligand C terminus. Chimeric receptor was then activated by the common N terminus on each hybrid ligand. Similar studies using interspecies PTH1-Rc chimeras have defined receptor domains critical for ligand binding (21). The recombinant human PTH1-Rc binds several PTH ligands that lack the N-terminal activation domain, including bPTH(7-34), bPTH(15-34), and hPTH(10-34), with at least 50-fold higher affinity than does the rat PTH1-Rc homolog, whereas binding affinities for bPTH(1-34) are similar for both receptor homologs. Applying a similar approach to the CT/PTH1-Rc chimeric receptors, recombinant chimeric r a t / h u m a n PTH1-Rc receptors were cloned and expressed in COS-7 cells. All chimeras bound bPTH (1-34) with normal affinity. However, chimeras encoding the N-ECD of the hPTH1-Rc bound bPTH (7-34), bPTH ( 15-34), and hPTH (10-34) with high specificity, whereas chimeras expressing the rat N-ECD failed to bind those ligands. As in humans, the opossum PTH1-Rc homolog binds bPTH(7-34) with high specific affinity. Studies of rat/opossum PTH1-Rc chimeras confirm the importance of the N-ECD for bPTH (7-34) binding. Thus, studies utilizing chimeric receptors that are designed to exploit the differential binding of PTH analogs demonstrate consistently that a domain within the N-ECD region of the PTH1-Rc is a critical r e c e p t o r region in determining the binding affinity of amino-terminally truncated PTH analogs.

PTH/PTHrP/RECEPTOR I N T E R A C T I O N S Reciprocal receptor chimera studies have implicated the amino-terminal portion of each receptor in having a major role in observed differences in ligand binding affinity by specifically interacting with the C terminus of PTH(l-34). Another series of experiments investigating rat/opossum chimeras helped to elucidate the mechanism by which the N-terminally modified analog, [Arge]PTH(l-34), is an antagonist with rat PTH1-Rc but is an agonist when bound to opossum PTH1-Rc (24). Here, ligand activity was associated with extracellular juxtamembrane residues of TM5 and TM6 in the carboxyl terminus of PTH1-Rc. Site-specific mutagenesis further refined the residues critical for rat PTH1-Rc interactions with the Arg-2 sidechain a s S 370 and V~71 (TM5) and L 427 (TM6). Mutagenesis studies that replaced these residues in rat PTH1-Rc with corresponding residues in the opossum PTH1-Rc (S~7°A, V371I, and LazwT) resulted in an alteration in [ArgZ]PTH(1-34) binding toward that seen with wild-type opossum receptor, yet had no effect on the binding of PTH(1-34). One of these mutations in rat PTH1-Rc, $37°A, also conferred agonist activity to [ArgZ]PTH(1-34) in cAMP assays, whereas V371I 427 and L T failed to alter receptor activation by [ArgZ]PTH(1-34). Thus, these reciprocal mutations of specific residues confirmed results from chimeric receptors. In addition, specific mutagenesis pinpoints potential residues that are critical for local direct interactions with the amino terminus of the ligand as well as for the pharmacologic profile of [Arg 2]PTH(1-34). Thus, chimeric receptor studies have pointed to at least two distinct, independently functioning domains on the extracellular surface of the PTH1-Rc receptor: (1) the N-ECD, which largely determines binding specificity of ligand by interactions with the C terminus of PTH(1-34), and (2) the TM5/ECL3/TM6 region of the receptor, which interacts with the N-terminal activation domain in PTH. Taken together, receptor chimera-based studies indicate that these class II receptors share a similar overall structure with multiple functionally independent, ligand-specific domains. These domains are sufficiently different to permit synthetic hybrid ligands to bind and efficiently activate the complementary receptor chimeras.

Site-Specific Mutagenesis Identifies Residues Critical for Receptor-Ligand Interactions Chimera studies have provided a general model of PTH1-Rc-ligand interactions. Mutagenesis studies with intact receptors have provided more detailed insights into PTH1-Rc-ligand interactions (Fig. 7). Though receptor chimera studies have identified the juxtamembrane region composed of residues on TM5 and

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67

TM6 as an important interaction domain for the extreme amino terminus of PTH and PTHrP, scanning mutagenesis of the N-ECD has identified an important binding region in the rat PTH1-Rc (residues 182-190). Specifically, F184A, Rla6A, L187A, and I19°A located at the base of the N-ECD were demonstrated to be important determinants for maximum binding of 125I-labeled bovine PTH(l-34) and 125I-labeled bovine PTH(3-34) (172). Homologous substitutions further revealed that hydrophobicity at positions occupied by F TM and L 187 in the PTH1-Rc plays an important role in determining functional interaction with the 3-14 portion of PTH. Conversely, deletion or epitope tag substitutions of more distal N-ECD domains are welltolerated by PTH1-Rc in terms of expression efficiency, ligand-binding affinity, and specificity. Mutagenesis strategies have also identified polar residues within the hydrophobic transmembrane domains of PTH1-Rc as important determinants of

IT33A,Q37A [

Extracellular

R233H, I234N R227A,

Intracellular

12

T33A, Q37A P132L R186A H223R R227A, R230A, R233H I234N L289I, I363Y $370A, V371I T410P M425L L427T W437L Q440L Q451K I458R

PTH COOH-terminus binding Bloomstrand chondrodysplasia PTH Bpzl3 crosslinking site Jansen's metaphyseal chondrodysplasia Disruption of ligand binding, signaling Critical for ligand specificity PTH position 5 (I/H) selectivity [Arg2]PTH binding Jansen's metaphyseal chondrodysplasia PTH Bpal crosslinking site [Arg2]PTH binding PTH[1-34] binding PTH[1-34] binding Disruption of binding, signaling Jansen's metaphyseal

FIG. 7 Overview of mutational analysis of residues involved in ligand binding and receptor activation.

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CHAPTER4

receptor function (22,173). Random mutagenesis of two highly conserved polar sites resulting in conservative polar residue substitutions in the TM regions of the rat PTH1-Rc, R 233 in TM2 and Q451 in TM7, causes 17- to 200-fold reductions in the binding affinity of the agonist peptide PTH(1-34). Yet these residue changes failed to alter the binding affinity of the antagonist/partial agonist PTH (3-34). Furthermore, the double-mutant receptor (R23~/Q451) displays a binding affinity for PTH (1-34) nearly equal to that of the wild-type receptor. However, the double-mutant receptor fails to activate either GsoLor G e~ signaling. Mutation of three residues, S227, R 230, and S2%, predicted to be aligned on the same face of TM2, resulted in blunted PTH(1-34)-stimulated adenylyl cyclase response and lower binding affinity for the agonist despite efficient cell surface expression (22). The same mutation at the corresponding sites in the secretin receptor, another member of the class II GPCRs, resulted in a similar reduction in adenylyl cyclase activity. Taken together, Turner and co-workers suggest that this region in TM2 participates in the mechanism of signal transduction that is common to the class II subfamily (22). Another series of experiments confirms the important role of TM regions in ligand recognition as well as receptor structure. Mutation of a single amino acid (N~92I) in the second TM of the secretin receptor to the corresponding residue in the PTH receptor led to PTH binding and functional signaling by secretin receptor (174). The reciprocal mutation in the PTH1-Rc (I2~4N) led to a PTH1-Rc that was responsive to secretin. Neither mutation significantly altered the response of the receptors to their own ligands. The results suggest a model of specificity wherein TM residues near the extracellular surface of the receptor function as selectivity filters that block access of the wrong ligands to sites involved in receptor activation (174). Recombinant expression of portions of PTH1-Rc has also offered insights into the molecular mechanisms of ligand recognition and receptor activation. The studies described above offer a model of PTH(1-34) receptor binding whereby the extreme N terminus of the peptide interacts with binding determinants within the extracellular loops/juxtamembrane region of the receptor, but more C-terminal residues of the ligand interact with the amino-terminal extracellular domain of PTH1-Rc. Studies by Luck et al. of binding of PTH(1-14) also support this paradigm (18). Other receptor deletion mutant studies have eliminated the second extracellular loop without affecting receptor function and have shown that PTH1-Rc can accommodate a heterologous epitope tag replacement of a portion of that region and retain full binding and signaling capacity (175).

PTH2-Rc

A second class II GPCR, designated PTH2-Rc, selectively binds PTH, but not PTHrE It has been cloned from rat and h u m a n cDNA libraries (176). Immunocytochemical and in situ hybridization studies have identified a n u m b e r of endocrine cells expressing PTH2-Rc, incuding thyroid parafollicular cells, pancreatic islet D cells, and a subset of gastrointestinal peptide-synthesizing cells (177). However, little is known about the endocrine role of PTH2-Rc in these tissues. Though its tissue distribution, in particular its lack of expression in kidney and bone, suggests it has a limited physiologic role in mineral metabolism, its ligand specificity has provided insight into the current model of PTH ligand-receptor interactions. Several lines of evidence suggest that PTH is unlikely to be a physiologically important endogenous ligand for PTH2-Rc: (1) different ligand rank order of intrinsic activity of a series of PTH analogs in the human and rat PTH2-Rcs, (2) considerable lower intrinsic activities and relative potencies of PTH-like ligands at the rPTH2-Rc than at the hPTH2Rc, and (3) the partial agonist effect of PTH-based peptides when compared to bovine hypothalamic extracts (178). Receptor chimera studies, in which the extracellular domains of PTH2-Rc are selectively replaced with homologous portions of PTH1-Rc, have identified several binding domains that regulate selectivity between PTH and PTHrE For example, Turner et al. demonstrated that chimeras in which the N-ECD of PTH1-Rc was fused to the remaining PTH2-Rc at the TM1 extracellular surface permitted binding and cAMP accumulation by PTHrP (177). Similarly, PTH2-Rc N-ECD fused to PTH1-Rc altered ligand specificity and disrupted binding of PTHrE In addition, mutational analysis of PTH2-Rc residues within TM3 and TM7 demonstrated that residue changes of I244L in TM3 and both C3°7Yand F4°°L in TM7 altered specificity of PTH2-Rc and increased binding of PTHrE Based on these data, it was postulated that the extracellular juxtamembrane portion of the transmembrane domain bundle functions as a selectivity filter or barrier that prevents PTHrP from interacting with the PTH2-Rc. In addition, another study using PTH2-Rc demonstrated that the N-ECD and the ECL3, specifically residues R3O4Q and Q44°R of human PTH2-Rc and PTH1-Rc, respectively, interact similarly with PTH and that both domains contribute to differential interaction with PTHrP (179). Other chimeric studies have identified three single amino acids in PTH2-Rc, I TM in TM3, y318 in TM5, and C 307 in TM7, as being involved in the specificity switch for PTH and PTHrP (180).

PTH/PTHrP/RECEPTOR INTERACTIONS / A Third PTH Receptor Subtype Three PTH receptor genes, including a novel PTH3Rc, were cloned by genomic PCR from zebrafish (z) DNA. The zPTH1-Rc and zPTH3-Rc receptors exhibited 69% similarity (61% identity), but less homology with zPTH2-Rc. Zebrafish PTH1-Rc and zPTH3-Rc showed 76 and 67% amino acid sequence similarity with hPTH1-Rc, respectively; but similarity with hPTH2-Rc was only 59% for both teleost receptors. Recombinant zPTH1Rc bound a variety of PTH and PTHrP ligands with a high apparent affinity (IC50, 1.2-3.5 nM), including [Tyr34] hPTH- (1-34) NH2 (hPTH), [Tyr36]hPTHrP(1-36) NH 2 (hPTHrP), and [AlaZ9,Glu3°,Ala~4,Glu35,Tyr36] fugufish PTHrP(1-36)NH 2 (fuguPTHrP). In addition, zPTH1-Rc was efficiently activated by all three peptides (EC50, 1.1-1.7 nM). PTH3-Rc exhibited higher affinity for hPTHrP and fuguPTHrP (IC50, 2.1-11.1 nM) than for hPTH (IC50, 118.2-127.0 nM) and adenylyl cyclase was more efficiently stimulated by fugufish and human PTHrP (ECs0 = 0.47 _+/0.27 and 0.45 _+0.16 nM, respectively) than by hPTH (EC50 = 9.95 _+1.5 nM). Finally, total inositol phosphate accumulation by zPTH1-Rc was observed to increase after agonist administration; however, zPTH3-Rc failed to activate this signaling pathway. These studies suggest that PTH and PTH-like peptides may exert their effects via as yet uncharacterized receptors.

Receptor Mutations and Human Disease

Jansen'sMetaphysealChondrodysplasia Jansen's metaphyseal chondrodysplasia (JMC) (also see Chapter X) is a rare form of short-limb dwarfism associated with abnormalities in endochondral skeletal development, hypercalcemia, hypophosphatemia, and normal levels of PTH and PTHrP. Originally, two missense mutations in the PTH1-Rc coding region were discovered in patients with the disease (181,182). These mutations, H223R and T41°P, resulted in constitutive activation of the cAMP signaling pathway and are both located at the cytoplasmic base of TM2 and TM6, respectively. A third novel missense mutation was found (I458R) in anotherJMC patient, and is located at the cytoplasmic juxtamembrane region of TM7 (183). In COS-7 cells expressing the human I458RPTH1-Rc, basal cAMP accumulation was approximately eight times higher than in cells expressing the recombinant normal receptor. Furthermore, the I458Rmutant showed higher activation by PTH than by the normal receptor in assays measuring accumulation of downstream effectors, adenylyl cyclase and phospholipase C. Like the HZZ3R and the T41°p mutants, the I458Rmutant does not constitutively activate basal inositol phosphate accumulation. Interestingly,

69

these mutations all occur at TM regions near the intracellular loops of PTH1-Rc that are hypothesized to interact and activate intracellular G proteins and the subsequent signaling cascade. These mutations in PTH1Rc also have been utilized to screen for identification of PTH and PTHrP analogs with inverse agonist activity. Two peptides, [Leu 11, D-Trp12]hPTHrP(7-34)NH2 and [D-Trp12,Tyr34]bPTH (7-34)NH 2, exhibited inverse agonist activity in COS-7 cells expressing either mutant receptor (H22~Rand the T41°P), and reduced cAMP accumulation by 30-50% with an EC50 of approximately 50 nM (184). Such inverse agonist ligands someday may be useful tools for exploring the different conformational states of the receptor as well as leading to new approaches for treating human diseases with an underlying etiology of receptor-activating mutations.

Blomstrand Chondrodysplasia Blomstrand osteochondrodysplasia (BOCD) (also see Chapter 44) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. The phenotype of BOCD is strikingly similar to PTH1-Rc knockout mice in which PTH1-Rc-ablated mice display prominent pathology in the growth plate (185). In both human disease and the PTH1-Rc-ablated mouse model, the growth plate is reduced in size due to a lack of columnar architecture of proliferating chondrocytes, as well as a greatly reduced zone of resting cartilage. This overall similarity of phenotype suggests an inactivating mutation of PTH1-Rc as a possible underlying genetic defect causing BOCD. To date, two types of inactivating mutations have been documented in BOCD (186,187). The first is a single homozygous nucleotide exchange in exon E3 of the PTH1-Rc gene. This alteration changes a proline residue to leucine at position 132 in the receptor's amino-terminal extracellular domain. Proline 132 is conserved in all mammalian class II G proteincoupled receptors. COS-7 cells expressing a green fluorescent protein-tagged mutant receptor do not accumulate cAMP in response to PTH or PTHrP and do not bind radiolabeled ligand, despite being expressed at levels comparable to GFP-tagged wild-type PTH1-Rc. Thus, while full-length PTH1-Rc is being synthesized, it lacks binding of ligand and is functionally inactive. At least one mutation in PTH1-Rc has also been detected in BOCD that causes a shift in the receptor mRNA open reading frame and thus generates truncated receptor fragments (188). Sequence analysis of all coding exons of the PTH1-Rc gene identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The

70

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CI-IAPTWR4

missense mutation produces a shift in the open reading frame, leading to a truncated protein after amino acid 364 in the second extracellular loop. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7. These are precisely the regions thought to be critical for (1) interaction of PTH1-Rc with the activation domain at the extreme N terminus of PTH and PTHrP and (2) the activation of coupled G proteins at the intracellular surface of the receptor. Functional analysis of the m u t a n t receptor in COS-7 cells and of dermal fibroblasts obtained from the patient demonstrated that the mutation was inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts increased cAMP accumulation in response to PTH or PTHrE

LIGAND-RECEPTOR BIMOLECULAR COMPLEX-CENTERED APPROACH One of the most effective ways of characterizing any ligand-acceptor system is to study the intact bioactive bimolecular complex under conditions that will not perturb their active conformation. Routinely, X-ray crystallography and NMR spectrometry are the tools of choice. These methods yield very detailed structures that have been utilized in rational drug design and have generated unprecedented leads for the development of novel therapeutic agents. Enzyme-substrate/inhibitor such as cathepsin K-inhibitor (189) and HIV protease-inhibitor (190), and soluble protein acceptor-ligand systems, such as the h u m a n growth h o r m o n e (hGH)-extracellular domain of the hGH receptor (191), erythropoietin (EPO)-EPO receptor (192), and ligand-FK506 binding protein (193) are just a few of a long list of successes demonstrating the power of studying the bimolecular complex and identifying intermolecular interfaces. Unfortunately, membrane-embedded proteins like the G protein-coupled receptors are not amenable for either NMR or X-ray analysis because of their large molecular weights and inability to form crystals. Two approaches, one ligand-centered and the other receptor-centered, have been pursued to further the understanding of ligand-PTH-Rc interactions and each has made important contributions (see preceding sections). The hormone-centered approach succeeded in mapping functional domains within the h o r m o n e for receptor-binding and activation. In some cases, structural features responsible for biologic properties have been identified down to the level of a single amino acid. However, this approach cannot be used to deduce the domains of the Rc that are in contact with the horm o n e across the interface. Furthermore, in many cases, the consequences of modifying the primary structure of

the h o r m o n e cannot be assumed to alter Rc interaction unambiguously. Although structural modifications of the h o r m o n e may alter directly the interaction with an important complementary structural feature of the Rc, some substitutions in the h o r m o n e may produce their effect on bioactivity through either local or global conformational changes within the h o r m o n e that prevent adaptation of an optimal "bioactive conformation." In essence, the hormone-centered approach is "blind" to the structure of Rc. The receptor-centered approach also has succeeded in providing valuable insights. PTH receptors with amino acid substitutions or chimeric structures (interspecies of PTH1-Rc, such as rat with opossum Rc, or interhormone receptors, such as PTH1-Rc with calcitonin or secretin Rcs) have been created, and information has been obtained regarding the importance of specific Rc domains and single amino acids necessary for Rc function. However, analysis of the consequences of modification of Rc structure alone cannot be used to deduce interacting complementary elements in the hormone. Furthermore, one usually cannot determine unambiguously whether a modification in the Rc disrupts function as a result of either a local change in an important "contact site," which affects directly the interaction with a site in the hormone, or an internal global conformational change in Rc leading to modified Rc topology, and thereby altered interaction with hormone, or both. Hence, despite the attractiveness of both lines of investigation and the importance of the contributions each makes, conclusions drawn from both the hormone-centered and the Rc-centered approaches have inherent limitations and are inferential at best. Therefore, the most appealing m e t h o d for identifying hormone-Rc interacting domains is a direct one, based on the analysis of cross-linking sites. Photoaffinity labeling has emerged as an effective methodology for studying interactions of biologic macromolecules with their ligands (194-197). It is now feasible to use a photocross-linked conjugate as a starting point for mapping "contact domains," and even "amino acid-to-amino acid contact points," between a biologically active comp o u n d and an interacting macromolecule (198-210). We and others have embarked on a challenging program to map the bimolecular interface between a large peptide h o r m o n e and a seven-transmembrane-spanning Rc. The approach, using photoaffinity scanning (PAS) to identify directly contact sites in the hPTH1-Rc responsible for h o r m o n e binding and signal transduction, relies on six parallel efforts: (1) the design and synthesis of bioactive PTH analogs that are resistant to certain kind of cleavage agents and enzymes, and that incorporate a photoreactive moiety and a radionucleide; (2) production of sufficient high quantities of functional

PTH/PTHrP/RECEPTOR INTERACTIONS /

tify at a highly localized level the structural elements critical for h o r m o n e - r e c e p t o r interaction. The mapping effort is interdisciplinary. Site-directed mutagenesis is used to generate new specific cleavage sites or to eliminate existing ones in an attempt to validate the digestion map generated from the wild-type receptor. Alternatively, mutagenesis is important to reduce the size of a cross-linked fragment in order to further delineate the contact site. Synthesized or expressed receptor sequences that include a contact site are conformationally analyzed, in the presence of micelles to mimic the m e m b r a n e milieu, to provide insight on the bioactive conformation of these domains. Finally, homology searches, computer modeling employing distance geometry, and molecular dynamics are used to merge the various inputs in an effort to generate a unified experimentally based model of the bimolecular ligand-GPCR complex. Indeed, this is an iterative process in which every new finding added to the data base results in the modification or confirmation of the emerging bimolecular model. Because of the nature of this approach it cannot yield molecular structures of the same resolution as those obtained by X-ray crystallography or NMR analysis. Nevertheless, u n d e r the current circumstances and with the technology available at h a n d it yields the best approximation of the actual ligand-receptor complex.

native and mutant hPTH-Rcs to permit cross-linking, exhaustive digestion, purification (epitope-tagged hRc), and subsequent analysis of fragments generated; (3) devising a strategy for a cascade of cleavages that identifies unambiguously hormone-binding sites within the hPTH-Rcs; (4) production of antibodies to various hRc extracellular epitopes for use in purification and analysis; (5) expressing receptor domains that contain the contact sites for conformational studies; and (6) integration of the cross-linking data with our Rc mutagenesis data, and eventually with conformational analysis and molecular modeling data to generate an experimentally based model of h o r m o n e - R c complex. Special design, and synthesis of PTH and PTHrP analogs containing photophores that are strategically and uniquely inserted along the h o r m o n e sequence permit the identification of h o r m o n e - r e c e p t o r interaction sites. These could be either interactions between an amino acid in the ligand and a contact site in the receptor, or more precisely between an amino acid in the ligand and an amino acid in the receptor, namely point-to-point interactions (Fig. 8). A radiolabeled h o r m o n e - r e c e p t o r photoconjugate thus generated is fragmented using enzymatic or chemical cleavage methods. The radiolabeled h o r m o n e or its fragment is covalently linked to a segment of the receptor containing a binding domain, and is subsequently isolated and characterized, thereby identifying small regions of horm o n e and receptor that are in contact with or in proximity to each other. By moving the photoreactive cross-linking moiety along the peptide sequence to certain discrete positions in PTH or PTHrP where bioactivity can be maintained, it will be possible to map precisely the binding sites of the receptor and to iden-

Radiolabe!ed Photoreactive

Receptor

Ligand

Ligand-Receptor Complex

71

Photoreactive Analogs The initial efforts to generate a photoreactive, radiolabeled, and biologically active analog of PTH aimed to identify the receptor as a distinct molecular entity (211-214). All of these studies used poorly character-

Ligand-Receptor Ligand-Receptor Conjugate ConjugatedFragment

2A

7

=/m i

_u

Binding

UV

3

• '

Radiolabef ~ - - Photoreactive moiety

j

FI6. 8 Schematic approach to photoaffinity scanning of PTH receptors. Photocross-linking is followed by fragmentation of the resultant radiolabeled hormone-receptor photoconjugate. Comparison of the fragmentation pattern elucidated by SDS-PAGE analysis with the theoretical restriction digestion map of the receptor identifies the putative contact site. Mass spectroscopic and microsequence analysis will identify the cross-linked residue in the receptor. (See color plates.)

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CI-IAeTWI~4

ized nitroarylazide-based photophores and reported molecular masses ranging between 28 and 95 kDa for the hormone-receptor complex. Shigeno and coworkers carried out a careful synthesis and characterization of the nitroarylazide-based photoligand and identified it to be [Nle8'18,Lysla (N~-(4-N~-2-NO2-phenyl),Tyra4] PTH(1-34)NH 2, a fully active analog in ROS 17/2.8 cells (Kd = 2.8 nM compared to 0.8 n M for the corresponding photoinactive radiolabeled PTH) (215). Using this photoaffinity ligand, they were able to identify in the same cells a plasma membrane glycoprotein corresponding to the PTH receptor that had the apparent molecular mass of 80 kDa (215,216). Nevertheless, more extensive characterization of the cross-linking site could not be carried out beyond this level with the arylazide-based photoreactive PTH analogs. Our strategy for the ligand/receptor-based approach was to introduce arylketone-based PAS methodology (27,217-220) into the field of calciotrophic hormones and their corresponding receptors (26,28,29,31, 220-222). In this section we summarize the major achievements in the design and development of benzophenone-containing PTH and PTHrP ligands, and their contribution to the mapping of the bimolecular ligand-receptor interface. The benzophenone moiety (which cross-links with >50% efficiency and has greater specificity than arylazides) was employed successfully in other systems (198-210). But in the mid-1990s the methodology was still at an early stage of development and was never applied to any of the calcium-regulating h o r m o n e / G proteincoupled Rc systems. The photogenerated triplet state of the benzophenone is capable of inserting into many types of C-H bonds, provided close proximity is achieved (223). This is in contrast to most other photoaffinity labels, e.g., azidoaryl functions, which generate highly reactive electrophilic species and therefore interact preferentially with nucleophilic groups on proteins (194). There are several advantages of benzophenones over other photophores. Relatively low-energy UV radiation is needed for photoactivation (224) and the reactive biradical is nearly nonreactive in water (225). Therefore, during cross-linking experiments, a large excess of photoaffinity label is unnecessary: efficiency of cross-linking is high because only a small amount is lost to hydrolysis. In addition, the photolabile moiety is compatible with solid-phase peptide synthesis methodology. Furthermore, synthesis, purification, and biological evaluation can be conducted in the laboratory under normal ambient light conditions. Radioiodination was chosen as the tagging method of choice because of its high specific radioactivity translating into high sensitivity of detection of the radiolabeled conjugated ligand-receptor complex and the fragments derived from it. The drawbacks are the regulatory constraints imposed on working with radioactive material

and the relatively short half-life of the radiolabeled material. Needless to say, radioiodination is not necessarily an innocuous modification. In several cases, radioiodination of otherwise bioactive benzophenone-containing PTH analogs resulted in a radiolabeled analog devoid of the binding affinity required for efficient photocross-linking. Because current technology has not optimized substitution of the benzophenone moiety with a radioiodine (226), these two features must be presented on two different amino acid moieties in the ligand. Therefore, successful PAS analysis requires maintaining the connectivity between the radiotag and the photophore throughout the controlled degradation of conjugated ligand-receptor complex. Modifications in PTH (1-34), which include Met 8 and 18 _...) Nle 8 and 18, Lysl~,26, and 27 .__) Lys13,26, and 27, and Yrp 2~ --+ 2-naphthylalanine 2~ (Nal), render the ligand resistant to the various chemical and enzymatic cleavage agents [i.e., CNBr, lysyl endopeptidase (Lys-C), and BNP-skatole, cleaving at the carboxyl side of Met, Lys, and Trp, respectively]. The premise of any photoaffinity cross-linking study is that analogs with similar pharmacologic profile share with the parent peptide hormone similar bioactive conformation and generate topochemically equivalent ligand-receptor complexes. The photoreactive benzophenone-containing analogs of PTH and PTHrP were designed specifically for PAS studies aimed at investigating the bimolecular interactions of the activation and binding domains of PTH and PTHrP with either the PTH1-Rc or the PTH2-Rc subtypes. Table 1 summarizes all bezophenone-containing analogs of PTH and PTHrP reported to date.

Identification o f Contact Sites We and others have identified contact sites for positions 1, 13, and 27 in PTH and positions 1, 2, and 23 in PTHrP using the PAS methodology (26-29,222,227). Two different photophores were used in different studies; p-benzoylphenylalanine (Bpa) (28,29,227) and Lys(N~-p-benzoylbenzoyl) (Lys(N~-pBz2)(26,27,29,222). The former has the benzophenone moiety attached to the peptide backbone through a [3-carbon while the latter is presented on a relatively long side chain removed by six atoms from the backbone. The differential positioning of the benzopheneone moiety relative to the backbone may play a limited role in selecting the cross-linking sites. Cross-Linking to Position 1 in PTH 1 Photocross-linking of [Bpa 1 ,Nle'8 1 8 ,Arg~'26'Z7,NalZ~,Tyr34]bPTH(1-34)NH2 (Bpal-PTH) to the human PTH1-Rc stably overexpressed (---400,000 Rcs/cell) in human embryonic kidney cell line 293 (HEK293/C-21)

PTH/PTHrP/R~cF.PToR INTERACTIONS / TABLE 1

Analog I

II III IV V Vl VII VIII IX X Xl Xll XlII XlV XV XVl XVll XVlII XlX XX XXl XXll XXlII XXiV XXV XXVl XXVll XXVlII XXlX XXXl XXXll XXXlII XXXlV XXXV

73

Benzophenone-containing analogs of PTH and PTHrP Analog (Ref.)

Position

[Bpa', Nle8'~8,Arg~3'26'2z,Na123,Tyr34]bPTH( 1-34)NH 2 (28) [Bpa2,Nle8'~8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa3, Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa4,Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa5, Nle8'~8,ArgO3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa6, Nle8"8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa7,NleS"8,Na123,Tyr34]bPTH(1-34)NH2 (217) [Arg2, Lys7(N'-pBz2),Tyr34]bPTH(1-34)NH 2 (217) [NleS"8,BpaZ,a-Nal'2, Na123,Tyr34]bPTH(7-34)NH2 (217) [Nle8'~8,Bpa~2,Na123,Tyr34]bPTH(1-34)NH2(217) [Nle8"8,Bpa'2,Na123,Tyr34]bPTH(7-34)NH2 (217) [NleS,,8,Lys,3(N,pBz2),Na123,Tyr34]bPTH(1_34)NH2 (217) [NleS,,8,D.Nal,2,Lys,3(N,.pBz2), Na123,Tyr34]bPTH(7_34) N H2 (217) [Nle8,,8,Lys,3(N,_p(3_l_Bz)Bz),Na123,Arg26,27,Tyr34]bPTH(1_34)NH2 (26) [Nle8,,8,Lys,3(N,.pBz2), Na123,Arg26,27,Tyr34]bPTH (1_34) N H2 (263) [Arg2, Lys '3(N'-pBz2),Tyr34]bPTH(1-34)N H2 (217) [NleS"8,Bpa23,Tyr34]bPTH(1-34)NH2 (217) [Nle8"8,D-NaI~2,Bpa23,Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,D-NaI'2,Na123,Lys26(N'-pBz2),Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,Na123,Lys26(N'-pBz2),Tyr34]bPTH(1-34)NH2 (217) [Nle8'~8,Arg'3'26,L-2-Na123,Lys27(N'-pBz2),Tyr34]bPTH(1-34)NH2 (222) [Bpa~, Ile~,Arg'l"3,Tyr36]PTHrP(1-36)NH 2 (29) [Bpa',IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa2,11e~,Arg~'~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa2, IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa3,11eS,Arg~"~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa3,11eS,Trp23,Tyr36]PTHrP(1-36)N H2 (227) [Bpa4,11e~,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa4, IleS,Trp2~,Tyr36]PTHrP(1-36)N H~ (227) [BpaS,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa5,Trp23,Tyr36]PTHrP(1-36) N H2 (227) [Bpa6,11e5,Arg'~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa6, Ile~,Trp~3,Tyr36]PTHrP(1-36)NH2 (227) [lleS,Bpa23,Tyr36]PTHrP(1-36) N H2 (30)

1 2 3 4 5 6 7 7 7 12 12 13 13 13 13 13 23 23 26 26 27 1 1 2 2 3 3 4 4 5 5 6 6 23

generates an 87-kDa photoconjugate (28). Chemical digestions by CNBr and BNPS-skatole, which cleave at the carboxyl end of Met and Trp, respectively, and enzymatic digestions by lysyl endopeptidase (Lys-C) and endoglycosidase F/N-glycosidase F (Endo-F), which cleave at the carboxyl end of Lys and deglycosylate the aspargines at the consensus glycosylated sites, respectively, generate a digestion restriction map of the photoconjugated receptor. Although the resolving power of polyacrylamide gel electrophoresis is limited, the combination of consecutive cleavages (e.g., Endo-F followed by Lys-C followed by CNBr) carried out in

reversed order (e.g., Lys-C followed by BNPS-skatole and BNPS-skatole followed by Lys-C) is extremely powerful. It generates a reproducible pattern of digestions and produces a set of fragments delimited by specific end residues and the presence or absence of glycosylation sites. Comparing the putative digestion map of the hPTH1-Rc with the actual fragments identifies 125 the sequence of the smallest I-radiolabeled 1 Bpa-PTH-PTH1-Rc conjugated fragment (---4 kDa). This fragment includes the ligand (4489 Da) modified by a moiety contributed by a Met residue belonging to

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CHAPTER 4

the receptor (28). Two Met residues, 414 and 425, present at the midregion and the extracellular end of TM6, emerged as potential contact sites for position 1 in PTH. Contact between residue 1 in PTH and M 414 requires the N terminus of PTH to protrude into the seven helical and hydrophobic transmembrane domain bundle. In contrast, contact with M 425 can be achieved while the N terminus is dipping superficially into the transmembrane domain bundle. These biochemical methods can be supplemented by molecular biology to provide additional resolving power to the PSA. Transient expression of two point-mutated hPTH1-Rcs, [M414L] and [M425L], in COS-7 cells, generated fully active receptors (28). The 125I-labeled Bpa ~PTH lost its ability to photocross-link to [M425L] but not to [M414L], suggesting that position 425 is the putative contact site for position 1 in PTH. Cross-Linking to Position 13 in PTH

Biochemical analysis of the photocross-linking product of radiolabeled [NleS'lS,Lysl~(Aff-p(3-I-Bz)Bz) ,NalZ3,ArgZ6'ZW,Tyr~4]bPTH (1-34) NH 2 [Lys 13(PBz2)PTH] with hPTH1-Rc expressed in HEK293/C-21 cells identifies a glycosylated radioactive band of---6 kDa, which is delimited by Lys-C and CNBr cleavage sites at the N and C termini. The theoretical cleavage restriction map of hPTH1-Rc reveals the minimal 125 13 radiolabeled I-Lys (PBz2)-PTH-hPTH1-Rc conjugated fragment, corresponding to hPTH1-Rc(173-189) located at the C-terminal region of the extracellular N terminus (26). Site-directed mutagenesis within the 17 amino acid residues comprising hPTH1-Rc(173-189) combined with subsequent biochemical analysis further delineates the boundaries of the contact site for 125I-Lys13(pBz2)PTH to hPTH1-Rc(182-189), an 8-amino acid sequence (263). Several single site-mutated receptors were generated, which include a new Lys-C-susceptible cleavage site. The mutant [RlSlK]hPTH1-Rc was stably expressed in HEK293 cells (---200,000 Rcs/cell) and was fully functional. Compared to the wild-type receptor, Lys-C cleavage of the 125I-Lys13(pBz~)-PTH-[RlS1K] photoconjugate produces a smaller conjugated fragment (---18 vs. --~9 kDa, respectively), corresponding to a cleavage site upstream to the N-glycosylated Asn 176. Interestingly, the only functional mutations that failed to cross-link to 125I-Lys13(pBzz)-PTH were the [RlS6K/A] mutants (263). However, [R~S6K]hPTH1-Rc stably expressed in HEK293 cells cross-links effectively to 125IBpal-PTH and displays wild-type receptor-like cyclase activity and binding affinity similar to that in HEK293/C-21 cells. These findings suggest that R 186 participates in an interaction with the ligand that either

provides a contact site for position 13 in the ligand or provides an interaction that brings the ligand into the close spatial proximity required for cross-linking within the hPTH1-Rc(182-189) contact site (263). This interaction does not appear to be essential for a productive ligand-receptor interaction because [RlS6K] is fully functional and cross-links effectively with a25I_Bpaa_PTH. Cross-Linking to Position 27 in PTH

Though the previously mentioned studies address interaction between residues in the extended activation domain of PTH comprising residues 1-13, a similar approach was directed toward the principal binding domain (sequence 24-34). We have analyzed • • • 8 18 13 26 the blmolecular Interaction between [Nle' ,Arg ' ,L-223 27 34 27 Nal ,Lys (N~-PBz2),Tyr ]bPTH(1-34)NH 2 [Lys (pBzz)PTH], modified by a benzophenone-containing photophore at position 27, and hPTH1-Rc by employing a combination of biochemical analysis of the photoconjuate and site-directed mutagenesis (222). Analysis of the 5 27 I-Lys (pBzz)-PTH-PTH1-Rc photoconjugate by CNBr/ Endo-F and BNPS-skatole/Endo-F degradation pathways produced an overlapping sequence corresponding to L232-W 298. This contact domain includes part of TM2, ECL1, and the entire TM3. Secondary digestions of the CNBr- and BNPS-skatole-derived fragments by endoproteinase Glu-C, which predominantly cleaves at the carboxyl side of Glu, converged on an overlapping 38-amino acid sequence corresponding to L261-W298, which includes part of ECL1 and the entire TM3 (222). To further delineate and validate the sequence containing the cross-linking site for position 27 in PTH, three mutated receptors were generated and transiently expressed in COS-7 cells. All three receptors, [R262K], [LZ61M] a n d [LZ61A], were expressed and displayed characteristic binding affinity and PTHstimulated adenylyl cyclase activity compared to wildtype receptor. [RZ62K] a n d [LZ61M] were designed to modify the Lys-C and CNBr cleavage pattern, respectively. The [L26~A] was introduced to eliminate a favorable insertion site at position 261. Restriction digestion 125 27 262 analysis of the ' I-Lys (pBzz)-PTH-[R K] photoconjugate delineated the contact site to hPTH1-Rc(232-262). Taken together, the minimal contact sites (sequence 261-298) and (sequence 232-262) obtained from the analysis of the wild-type and m u t a n t [RZ62K] receptors, respectively, suggest either L 261 or R 262 as the contact site for Lys27 . Treatment of the 125I-Lys27(pBz2)-PTH-[LZ61M] photoconjugate with CNBr generated a conjugated fragment similar in size to the ligand. This result suggests position 261 in the receptor to be the contact

PTH/PTHrP/RECEPTOR INTERACTIONS / site for position 27 in the ligand. This was further confirmed by the elimination of effective cross-linking of I125-LysZ7(pBzz)-PTH to the mutated receptor [L261A] i n which a reactive insertion site such as Leu is replaced by Ala, a poor insertion site for the photoactivated benzophenone-derived biradical. Position 261, the contact site for position 27 in PTH, is located near the center of ECL1 (222). The identification of L 261 in hPTH1-Rc as a contact site in f o r Lysz7 in PTH provides important information for mapping the PTH-PTH1-Rc interface. The remoteness of position 27 from positions 1 and 13 in PTH, and that of L 261 f r o m R 186 and M e t 425 in hPTH1-Rc, generates an important additional structural constraint that can be used to refine the emerging experimentally based model of the PTH-PTH1-Rc complex. Based on conformational analyses and structure-activity studies of PTH(1-34) and PTHrP(1-34), the prevailing view argues that these two hormones interact very similarly if not identically with the PTH1-Rc. In line with this assumption, radioiodinated [Bpa1,Ile5,TrpZ3,Tyr36]PTHrP (1-36) NH 2 [lZ5IBpal-PTHrP] photocross-links to M 425 in hPTH1-Rc in the same fashion as the corresponding PTH analog, lz5I-Bpal-PTH (29). Cross-Linking to Position 23 in PTHrP

Another photoreactive analog of PTHrP, [Ile5,Bpa 2~, Tyr~6]PTHrP(1-36)NH2 [Bpa2~-PTHrP], modified by a benzophenone moiety incorporated at position 23, was reported by Mannstadt and co-workers to cross-link to Y23-L4°, located at the very N-terminal end of rat PTH1Rc (30). CNBr analysis of the 125I-Bpa2~-PTHrP-rPTH1Rc photoconjugate suggests that the contact site resides at the N terminus of the receptor, rPTH1-Rc(23-63). A combination of site-directed mutagenesis (single point mutation [M63I] and the double mutants [M63I,L4°M] and [M6~I,L41M]) and CNBr cleavages further delineates the contact site to span the sequence 23-40. Earlier findings demonstrated that the two mutant rPTH1-Rcs with deletions of residues 26-60 or 31-47 transiently expressed in COS-7 cells had little or no capacity to bind 125I-labeled PTH, therefore suggesting these regions to be important for ligand binding (228). Only two of the 31 32 33 four cassette mutant receptors ([V A,F A,T A, 35 36 37 38 K34A,Ea5A] and [E A,Q A,I A,F A]) spanning the 31-47 sequence displayed diminished 125I-labeled PTH binding capacity. Finally, in an Ala-scan of the 31-38 region, mutants [Ta3A] and [Q~WA] exhibited the largest loss in binding affinity of 125I-labeled PTHrP and complete loss of binding affinity toward the antagonist 11 12 [Leu ,D-Trp ]PTHrP(7-34)NH 2. Relying primarily on mutagenesis-based analysis, Mannstadt and co-workers

75

suggest that the first 18 amino acid residues of the PTH1Rc comprise the contact site for position 23 in PTH, and T 33 a n d Q37 are functionally involved in binding of the 7-34 region in PTH rather than the 1-6 region (30). The location of contact sites for two closely spaced residues in PTH/PTHrP (23 and 13) at both ends of the extracellular amino terminus of the receptor (within 23-40 and in proximity to R 186,respectively) is consistent with the current model of the ligand-receptor binding interface. The extensive length of the putative extracellular amino terminus of PTH1-Rc (---167 residues) allows for assumption of secondary and tertiary structures by the receptor that can accommodate simultaneously the above-mentioned bimolecular interactions. Cross-Linking of Position 1 of Agonist vs. Antagonist

A very interesting observation that directly distinguishes between the nature of the bimolecular interaction of an agonist versus antagonist with PTH1-Rc was recently reported by Behar and co-workers (29). Photoconjugation of radiolabeled [Bpa2,Ile5,Arg 11'13, Tyra6]PTHrP (1-36) NH 2 [BpaZ-PTHrP], a highly potent antagonist, to hPTH1-Rc was carried out in HEK293/ C-21 cells (29). Unlike the analog [BpaZ,Nle s'18, Ar g 132627 ' , , NalZ3,Tyra4]bPTH(1-34)NH2 [BpaZ-PTH], which is a full agonist (28) and cross-links t o M 425 in PTH1-Rc in a manner similar to 125I-Bpa1-PTH o r 125IBpal-PTHrP, 125I-BpaZ-PTHrP also cross-links to a proxi' mal site within the receptor domain p415-M425 (29). These results may reflect either differences between the binding modes of agonist and antagonist or differences in the interaction between the two consecutive positions in the PTHrP(1-36) sequence and PTH1-Rc. In an attempt to distinguish between these two possibilities, we utilized the agonist analog BpaZ-PTH, which carries the same photoreactive moiety at the same position as the antagonist BpaZ-PTHrE Analysis of 125I-Bpa2-PTH photoconjugates with wild-type [MalaL] and [M4Z5L] mutated hPTH1-Rcs indicates that this ligand cross-links only to the e-methyl of Met 4z5, similar to Bpal-PTHrP and to Bpal-PTH cross-linking (28). These results, therefore, provide strong support for the hypothesis that the differences observed between 2 the cross-linking of 125I-Bpa1- and 125 I-Bpa-PTHrP may reflect different interaction modes of an agonist versus an antagonist with the PTH1-Rc. Interestingly, two additional Bpa-containing PTHrP(1-36) analogs, [Bpa 2 ,Ile 5 ,Trp 23 ,Tyr36 ]-and [Bpa 4, 5 23 36 Ile ,Trp ,Tyr ]PTHrP(1-36)NH 2, were reported to preferentially antagonize and cross-link to hPTH1-Rc and hPTH2-Rc stably expressed in LLC-PK1 cells, respectively (227). However, in homologous systems composed of hPTH1- and hPTH2-Rcs expressed in a human cellular

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/

C~TF~k4

background (HEK293/C-21 and HEK293/BP-16, respectively), Bpa2-PTH is a full agonist and Bpa4-PTH is a very weak agonist with a slightly better affinity for the hPTH2-Rc (28,227). Similar to [Bpa4,Ile5,Trp2~,Tyr~6]PTHrP (1-36) NH 2 (227), [Bpa4,Ile5,Arg 11'13,TyrS6]PTHrP(1-36)NH 2 displays poor binding affinity and negligible efficacy in HEK293/C-21 cells expressing the hPTH1-Rc (29). Although PTH2-Rc may not be the physiologic target for PTH or PTHrP, its structural resemblance to PTH1-Rc, its high binding affinity, specific cross-linking, and effective coupling to the PTH-induced intracellular signaling pathways make it an attractive target for exploring structure-function relations in the P T H / PTHrP-PTH1-Rc system. Analysis of the photoconjug ates obtained on cross-linking of '25I-BpaI-PTH and 25I-Lys'~(PBz2)-PTH to hPTH2-Rc stably expressed in HEK293 cells (HEK293/BP-16, ---160,000 Rcs/cell) revealed that both hPTH1-Rc and hPTH2-Rc use analogous sites for interaction with positions 1 and 13 (31). The PAS methodology offers the only readily available experimental approach to study directly the bimolecular ligand-GPCR interface. To practice this methodology we introduce b e n z o p h e n o n e moieties, radioiodine, and substitutions that provide resistance to specific chemical and enzymatic cleavages. These modifications are tolerated as long as the modified photoreactive ligand binds to the receptor specifically and with high affinity, and stimulates adenylyl cyclase in a PTH-like manner. The photoinsertion site of the benzophenone moiety is dictated by spatial proximity. But it is also biased toward the more reactive insertion sites within its reactivity sphere. Last but not least, the cleavages employed and the level of resolution allowed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis limit analysis of the photoconjugate. The validation of a putative contact site by site-directed mutagenesis is not necessarily benign. It generates some degree of perturbation, which we accept as long as the mutated receptor is expressed and functions similarly to the wild type. Taken together, PAS is not the perfect method, but we believe it is the best available. Future improvement in separating the photoconjugated receptor a n d / o r fragment from the nonconjugated species, and the elimination or replacement of the radioactive tag with a nonradioactive tag will be very helpful in terms of allowing access to high-resolution instrumental techniques. The currently labor-intensive PAS methodology will become less time consuming and more robust as the technology evolves.

Experimentally Based Molecular Modeling The contact sites identified in the cross-linking studies described above are only a small fraction of the large

ensemble that forms the bimolecular interface. The cumulative effect of these multisite bimolecular interactions results in specific ligand recognition, binding affinity, and eventually a conformational change in the receptor that leads to specific intracellular signal transduction. In general, not all contact sites revealed by PAS methodology will have the same functional significance. However, all these contact sites will be part of the ligand-receptor interface and are therefore indispensable targets in mapping efforts. The objective of the PAS studies is to generate a series of constraints, which will be used in mapping the bimolecular interface. To this end, merging the information generated by the PAS studies with information about the conformation of the ligand and receptor domains, as well as molecular modeling, generates an integrated approach that results in an experimentally based ligand-receptor model. This integrated approach based on experimental data is in contrast to the more c o m m o n approaches that predict conformation and molecular models solely on a theoretical basis. The model for the PTH-PTH1-Rc complex is steadily evolving as new bimolecular contact sites are identified (28,229,230). The combination of hydrophobicity profile analysis and search of the Brookhaven Protein Data Bank (PDB) employing the Basic Logic Alignment Search Tool (BLAST) can identify and refine, respectively, the location of the TM helices (231,232). The identification of the TM domains of the PTH1-Rc is in good agreement with respect to the location of peptides containing TM helical regions as determined by high-resolution NMR (233,234). The arrangements of the TM heptahelical bundle in rhodopsin and bacteriorhodopsin (235-239) were used as templates for the initial arrangement of the putative TM helical domains of the PTH-Rcs. Rotating the helices about their long axis to orient the hydrophobic m o m e n t toward the membrane environment and optimize their helix-helix, helix-core, and helixmembrane interactions generated the putative core of our receptor model (Fig. 9) (234). The modeling of the extracellular and intracellular domains responsible for binding the ligand and coupling to the G protein and other adapter molecules is not as straightforward as that of the membraneembedded core. Unlike the high structural similarity for the arrangement of the seven TM domains bundle (240), the cytoplasmic and ectopic domains of the GPCR are extensively variable and no a pr/0r/ structure is available. The loops are constrained to some extent by the TM helical domains to which they are attached. Additional constraints are imposed by the three disulfide bridges at the extracellular N terminus and the disulfide bridge connecting the first and the second ECL. All of these cysteines are highly conserved in the class II GPCRs, of which PTH1-Rc is a member.

PTH/PTHrP/RECEPTOR INTERACTIONS /

TM1 TM5

IC3 FIG. 9 Depiction of the PTH1-Rc from a molecular dynamic simulation. The transmembrane oL-helices are depicted as cylinders. The regions of the receptor that have been experimentally determined are depicted as ribbons. The regions of the receptor that have been shown to crosslink with PTH analogs, PTH1-Rc(173-181) (26,27) and M42s (28), are depicted in gray (241). (See color plates.)

Unfortunately, the pattern of disulfide bridge formation in the ectopic part of the class II GPCRs is not known and therefore cannot be used in building the receptor model. Homology search with BLAST (232) helps to assign putative elements of secondary structure to regions in the receptor that are homologous to regions of protein for which a secondary structure is available. Once a secondary structure element is identified, it can be incorporated into the model. Practically, we focus only on the identification of ahelices, which can be studied in isolation from other secondary structural elements. Indeed, sequence homology searches identify conformational preferences of the C-terminal portion of the extracellular amino terminus proximal to TM1 of PTH1- and PTH2Rcs and the third extracellular loop of PTH1-Rc (28,229). These homology searches indicate that the third ECL adopts a helical conformation that is highly p r o n o u n c e d for T435-Y443 (229), and amphiphatic helices for K172-M 189 and L129-E 139 in PTH1- and PTH2Rcs, respectively (28,229). Unfortunately, such homology searches may not always result in the assignment of distinct secondary structure to a specific receptor sequence.

77

The modeling procedure for the receptor and receptor-ligand complex is described in detail by Mierke and Pellegrini (241). Briefly, the primary structure of the PTH1-Rc is embedded into a three-phase, H z O / d e c a n e / H 2 0 (40 A each) simulation cell. A multistep simulation is carried out in which either the heptahelical bundle a n d / o r cytoplasmic and extracellular domains are allowed to move freely. PTH, in its membrane-associated conformation, is added to the receptor model, applying the ligand/receptor distance constraints elucidated from the cross-linking experiments, and additional simulations are carried out. Indeed, additional constraints obtained via sitedirected mutagenesis, and chimera studies, PTH2-Rc and other class II GPCRs can be incorporated into the development of the model. The most direct way to identify the conformational features of the cytoplasmic and ectopic domains of the GPCR is by generating these receptor fragments and examining them by NMR in a membrane-mimetic system. Adding a small portion of the corresponding TMs to the otherwise flexible receptor-derived termini or loops provides anchors that partially reproduce the native orientation of the receptor domain relative to the membrane-mimicking milieu. Another design element useful in restraining an excised loop sequence from assuming extended conformations is the covalent binding of both ends of the sequence by a linker of---12 A, the approximate distance between two consecutive TM domains (235,237). To this end, we have characterized the conformational features of the third intracellular loop (ICL) of PTH1-Rc, which was constructed as a 29-amino acid peptide with the side chains of Cys residues in positions 1 and 28 bridged by an octamethylene linker (233,234). This linker also assists in the association of the peptide termini with the membranemimicking micelle. In the presence of either SDS or dodecylphosphocholine (DPC) micelles, the peptide assumes two helical domains composed of residues L e u 4 L e u 8 and Yyr22-Leu26, and [3-turns at Glul°-Ala 13 and Glya4-Asp 17. The hydrophobic residues of the N-terminal amphiphilic helix are embedded in the hydrophobic portion of the micelle, whereas the polar side chain protrudes into the aqueous phase. The two [3-turns point away from the membrane and are exposed to the aqueous solvent. The structure of the bridged peptide in the presence of micelles is distinct from the same peptide in the absence of micelles as well as from the linear form of the peptide. A similar approach was applied to characterize the conformational features of two ectopic domains found to photocross-link to Lys13 a n d Lys27 in PTH(1-34) (28,221,222). Position 13 in PTH was found to cross-link within an 8-amino acid domain,

78

/

C~a'TER4

PTH1-Rc(182-189), located at the C-terminal portion of the N-ECD proximal to the first TM helix (28,221). The combination of homology search and molecular dynamic calculations, using a two-phase simulation cell consisting of H20 and CC14 (to mimic a membrane-water interface), suggests that the segment R179-E-R-E-V-F-D-R-L-G-M189forms an amphipathic oL-helix whose axis is parallel to the membrane surface and points away from the heptahelical bundle (28,242). 1H NMR analysis of the synthetic peptide hPTH1-Rc(168-198) in presence of micelles (to provide the membranelike environment) was carried in combination with distance geometry and molecular dynamic simulation (Fig. 10) (242). The analysis identifies a C-terminal helix, hPTH1-Rc (190-196), corresponding to the ectopic portion of the first TM helix, which was perpendicularly embedded in the micelle. Two oL-helices, (180-188) and (169-176), lie on the m e m b r a n e surface. The polar residues in the linker, E 177 and R 170, and in the middle helix, R TM, E 182, D 185, and R 186, are exposed to the solvent while the hydrophobic residues, F 173, F TM,and L 187, are projecting

toward the hydrophobic membrane (242). Based on the finding that the contact site for Lys 1~ in PTH is within residues (182-189), which includes negatively charged amino acids, coulombic interaction between these charges and the positive charge on Lys is may function as one of the ligand/receptor-specific interactions. Nevertheless, this may not be an essential interaction because analogs in which the e-amino on Lys 1~is blocked by acylation maintain high affinity and efficacy. At this point one can bring together findings obtained from the cross-linking studies of position 13 in hPTH(1-34) with the putative bioactive conformation of PTH (64), and the conformational analysis of the receptor domain containing the contact site for position 13 in order to generate the first generation of an experimentally based model of the PTH-hPTH1-Rc complex (28). Using the cross-linking data as a docking cue to position the ligand (in its putative bioactive conformation) places its C-terminal amphiphilic helix parallel to the membrane-aligned portion of the receptor-derived peptide. This allows the formation of complementary coulombic interactions between the

A 8o

G188

¸¸¸i i Cytoplasm

B

FIG. 10 Structural features and topological orientation of PTH1-Rc (168-198) located at the C-terminal region of the extracellular N terminus followed by the ectopic portion of the first TM domain (241,242). (A) Schematic representation of the experimentally determined conformation. The structure consists of three o~-helices, two of which have been determined to lie on the surface of the membrane; the third, at the top of TM1, is membrane embedded. (B) The orientation of this peptide is shown with respect to the surface of the dodecylphosphocholine micelles used in the NMR study. The hydrophobicity of the molecule is indicated (blue, polar; red, hydrophobic). The decane molecules of the water/decane simulation cell used in the structure refinement are shown in green as CPK space-filling spheres. (See color plates.)

P T H / P T H r P / R E c E P T O R INTERACTIONS /

polar residues in the helix comprising the principal binding domain of the ligand and the polar residues E 177, R 179, R TM, E 182, D 185, and R 186 in the receptorderived peptide. Interestingly, this docking procedure brings only M 425, a n d not M 414, into sufficient proximity to permit cross-linking to position 1 in 125I-Bpa1-PTH. Therefore, these observations are in complete agreement with the results obtained through cross-linking studies (Fig. 11) (28). The structural features of the first ECL in the presence of dodecylphosphocholine micelles were revealed from the high-resolution NMR study, followed by distance geometry calculations and molecular dynamic simulations of a peptide, hPTH1-Rc(241-285), comprising the loop and few residues from the ectopic portions of TM2 and TM3 of the receptor (230). This peptide contains L 26], which was found to cross-link to Lys27 in hPTH(1-34) (222). The structure of this receptor fragment includes three or-helices, (241-244), (256-264), and (275-284). The first and the last helices correspond

79

to the ectopic pordons of TM2 and TM3, respectively. The topological orientation of the helices relative to the membrane surface was examined in the presence of 5and 12-doxylstearic acids, nitroxide radical-containing molecules that serve as reporters for the localization of amino acid residues in the membrane. The amino acids corresponding to the ectopic portion of the TMs are more strongly associated with the lipid micelle and may serve as membranal anchors. All of the hydrophobic residues in the partially ordered central helical portion (terminated by the unique helix-breaking sequence p258_p_p_p261) are projecting toward the lipid surface (230). The conformational analysis of the first ECL is very helpful in gaining important insights into the bimolecular ligand-receptor interaction revealed by crosslinking studies. The benzophenone moiety o n Lys27 in 125I-KZT(N~-pBzz)-PTH cross-links to L 261 in hPTH1-Rc (222). The long, amphiphilic C-terminal helix, which i n c l u d e s Lys27, was found to lie on the surface of the

W457

Extraeellu!ar

M414 s~

Intraeellu!ar Receptor

B TM3 ~.

TM2

i~:

N-Terminus .....

~

TM7

TM5 C.Te~in~

N~Te~ni:~s PTH Ligand

FIG. 11 Model for the binding of hPTH(1-34) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains hPTH1-Rc(173-189) and hPTH1-Rc(409-437)) (A, side view; B, top view). The amphipathic oL-helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high-resolution, low-energy structure of hPTH(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of hPTH(1-34) are denoted in yellow. The C-terminal amphipatic oL-helix of hPTH(1-34) is aligned in antiparallel arrangement with the amphipatic cx-helix of the extracellular N-terminus hPTH1-Rc(173-189), contiguous with TM1 and encompassing the 17-amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue M414 and M425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc(S"°9-W"37)) are shown (28). (See color plates.)

80

/

CHAPTER4

micelle with its hydrophobic face projecting into the lipid layer (64). We therefore propose that these two helices, the C-terminal helix in PTH and the central helix in the first ECL in PTH1-Rc, interact in an antiparallel fashion allowing exposed charged residues on both helices to form numerous intermolecular interactions. Integrating these findings into the PTH-PTH1-Rc model results in the enhancement and refinement of the overall bimolecular topology by positioning the C-terminal helix of PTH between the first ECL and the C-terminal helix of the N-ECD of hPTH1-Rc. This topological organization is consistent not only with the individual bimolecular contact sites between positions 1, 13, and 27 in PTH and the respective sites in PTH1-Rc (namely, M 425, a site in the proximity of R 186, and L261), but also accommodates the contact site between position 23 in PTHrP and YZ~-L4° in PTH1-Rc (Fig. 12). R61z and co-workers constructed the PTH1-Rc and PTH2-Rc as described previously and used the membrane-bound conformation of hPTH(1-34) (64) and the contact sites identified for positions 1 and 13 in PTH (26,28,221) to dock hPTH(1-34) to the receptors (229). Using these models, they identify interresidue contacts within the seven-transmembrane helical bundle (Fig. 13) and suggest explanations for ligand specificity (46,243), site-directed mutagenesis (23,173,177,179,180), constitutively activated receptors (182,244), cross-linking outcomes (28), substitutions

N-terminus 261

C-terminus FIG. 12 Schematic representation of the binding of PTH to its G protein-coupled receptor, PTH1-Rc. The locations of the contact points in PTH1-Rc identified by photoaffinity crosslinking are indicated (SerlmM 42s, Lys13--R 186,Trp23--T33/Q37, Lys2L--L261). The structural features of the PTH and fragments of PTH1-Rc are indicated (230).

C281-C351

. , a t e , TM2

TM1 TM4

TM7 TM6 FIG. 13 Illustration of some key residue-residue contacts within the seven-transmembrane helix bundle of PTH1-Rc. These contacts provide support that the model contains the correct topology of the seven-TM helices. Reprinted with permission from Ref. 229. Copyright 1999 American Chemical Society.

within the ligand (39,245), and signal transduction. These authors also suggest some mutations, which may reverse the specificity of the PTH1- and PTH2-Rcs for their respective ligands, P T H / P T H r P and PTH (229). An interesting study reported by Shimizu and coworkers has incorporated data generated by the ligandand receptor-centered approaches in a very innovative way to design a constitutive active ligand-tethered hPTH1-Rc (Fig. 14) (246). Four concepts were established: (1) A peptide as small as PTH(1-14) can stimulate weak cAMP formation with both wild-type and N-ECD-truncated rPTH1-Rc, rANt (18). (2) Residues 1-9 in PTH (1-14) are critical for interacting with the rANt (18). (3) Position 13 in PTH photocross-links in the proximity of R ]86 in PTH1-Rc (26,27). (4) The hydrophobic residues F TM and L 187 in PTH1-Rc are functionally important for the interaction with the 3-14 portion of PTH(1-34) (172). In this ligand-tethered hPTH1-Rc, the N-ECD was truncated from E 182, juxtaposed to the TM1 (AN-ECD-hPTH1-Rc), and was extended by a Gly4 spacer (Ga-AN-ECD-hPTH1-Rc) linked to PTH(1-9) (246). Transient expression of this construct in COS-7 cells resulted in 10-fold higher basal cAMP levels compared to the control, wild-type

PTH/PTHrP/REcEPTOR INTERACTIONS /

~NH2

[A1-V-S-E-I-Q-L-M-H9 ]

PTH[ 1-9]

[AI-V-S-E-I-Q-L-M-H-NI° I

PTH[ 1-10]

[AI-V-S-E-I-Q-L-M-H-N-L 11]

PTH[1-11]

[A'-V-S-E-I-Q-L-M-H-N-R 1']

Arg11PTH[1-11]

y

H2N~77-/'~G_G_G_G

HOOC ~

81

H2N

HOOC hPTH1-Rc

HOOC -

Tether-G4-AN-ECD-

AN-ECD-hPTH1-Rc

hPTH1-Rc FIG. 14 Schematics include the wild-type hPTH1-Rc, the AN-ECD-hPTH1-Rc, and the Tether-G4-AN-ECDhPTH1-Rc. Also listed are the different N-terminal sequences derived from PTH, which are tethered to E 182 (solid diamond) via a tetraglycine (G4) spacer. All the receptor constructs retain the 23-amino acid native hPTH1-Rc signal sequence. Therefore, the putative N-terminal residue in all the receptors is y23 generated on signal peptidase cleavage. Modified from Shimizu et aL (246).

hPTH1-Rc in the same expression system. T e t h e r i n g the extended and more potent [Arg11]PTH(1-11) resulted in 50-fold higher basal c-AMP levels than those seen with the wild-type hPTH1-Rc. Interestingly, similar to the PTH(1-14) (18) in which Val 2, Ile 5, and Met 8 were the most critical residues for activation they were also the most critical ones for the constitutive activity of the [Arg11]PTH(1-11)-G4-AN-ECD-hPTH1-Rc (246). The elegance of this study is in devising a unique way to specifically "immobilize" the principal activation domain of the ligand in the proximity to the contact sites critical for receptor activation. The correspondence between the substitutions in the PTH (1-11) that increase the efficacy of the free and the tethered peptide supports the notion that both exercise the same contact points responsible for receptor activation. The high effective molarity of the tethered ligand minimizes the role of binding affinity as it is known for the free ligand, thus allowing the identification of residues within the tethered ligand essential for induction of activity. However, the accessibility to the tethered ligand-receptor system is limited to the recombinant technology and therefore to coded amino acids, and the stringent requirements for efficient expression may turn out to be major obstacles in practicing and extending this approach in the future. It

remains to be shown whether the tethered ligand-receptor system may be a source for identifying structural constraints that can contribute to the refinem e n t of the experimentally based ligand-receptor model and to rational drug design. The elimination of most of the entropic c o m p o n e n t from the ligand-receptor interaction may generate contact interactions and produce activation mechanisms that differ from those involved in the interaction with a diffusable ligand. The quality of any model, namely, its capacity to represent ligand-receptor interactions realistically and predict the nature of the interface, is based primarily on the data and procedures used in construction of the model. A model can become highly speculative and thus only remotely relevant to biology if overloaded with data derived from indirect and circumstantial conclusions. It is important to avoid overinterpretation of model and r e m e m b e r the assumptions and approximations used in its construction. Last, any extrapolation derived from the model must be tested in order to validate its predictive potential. Therefore, the evaluation of any models for complexes of PTH and PTHrP with PTH1-Rc and PTH with PTH2-Rc should follow the above-mentioned principles.

82

/

CHAPTER4

FUTURE DIRECTIONS T h e most powerful insights into the nature of h o r m o n e - r e c e p t o r interactions are emerging from direct PAS studies of h o r m o n e - r e c e p t o r photoconjugates. Future experimentally based models of the bimolecular interface will be m o r e refined and better validated. As additional contact sites are demonstrated, other constraints are generated for the model, which further refines the entire model. W h e n sufficiently advanced, this experimentally based model of the PTH-PTH1-Rc interface will b e c o m e a powerful tool for u n d e r s t a n d i n g structure-based mechanisms responsible for differences in biologic activities of h o r m o n e agonists, signaling-selective agonists, antagonists, partial agonists, a n d inverse agonists. It will also provide the means to u n d e r s t a n d aberrant mechanisms underlying pathologic mutations of PTH1-Rc leading to the clinical disorders of Jansen's metaphyseal chondrodysplasia and Blomstrand's osteochondrodysplasia. Finally, the detailed and validated model of the h o r m o n e - r e c e p t o r complex will serve as a molecular template for design of therapeutically advantageous analogs of PTH and PTHrP. O n e area of research that has grown steadily in interest over the past decade is the potential utility of PTH- or PTHrP-derived agonists for the t r e a t m e n t of osteoporosis. It is very well established that low-dose intermittent administration of several forms of P T H stimulates b o n e formation, leading to an overall anabolic effect on b o n e (247,248). Further observations have b e e n m a d e in vivo in animals and in h u m a n studies (249-255). This beneficial effect on b o n e occurs despite the well-documented action of P T H in stimulating b o n e resorption via increased osteoclast n u m b e r and activity. Nevertheless, instigating osteoblasfic bone formation without concomitant activation of osteoclasts a n d resultant b o n e resorption remains an ultimate goal for t r e a t m e n t of osteoporosis. To this end, future focus on the developm e n t of signaling-selective PTH or PTHrP analogs is one of the m o r e promising directions for analog design. Unfortunately, the chronic n a t u r e of osteoporosis implies that long-term or even life-long t r e a t m e n t is required. This poses serious c o m p l i a n c e issues due to the fact that the administration of a peptide-based d r u g such as P T H is generally limited to p a r e n t e r a l routes. In the short a n d i n t e r m e d i a t e term, we anticipate the d e v e l o p m e n t of i m p r o v e d d r u g delivery systems that will greatly e n h a n c e the t h e r a p e u t i c potential of PTHa n d PTHrP-derived agonists a n d antagonists. However, in the long term, d e v e l o p m e n t of small n o n p e p t i d e PTH-mimetic drugs is of major interest. In the past few years a growing n u m b e r of small n o n p e p t i d e peptide-mimetic agonists for GPCRs have b e e n r e p o r t e d (256-262). This raises our expectations that either

through rational d r u g design (based on the P T H - P T H 1 - R c e x p e r i m e n t a l model), or high-throughp u t screening of collections of synthetic c o m p o u n d s , natural products, a n d culture broths, new lead molecules will be discovered. These "leads" can then be optimized chemically into n o n p e p t i d e PTH-rnimetic anabolic agents. To this end, studying the s t r u c t u r e activity relations a n d the degrees of structural tolerance of P T H ( 1 - 1 4 ) is a p r o m i s i n g initiative (18-20). Given the probability of substantial progress in developing a m o d e l for the h o r m o n e - r e c e p t o r complex, in analog design, in elucidating h o r m o n a l m e c h a n i s m s of action, a n d in peptide delivery systems, P T H or PTHrP agonists are likely to find clinical utility in t r e a t m e n t of disorders of calcium a n d b o n e metabolism.

REFERENCES

1. Chorev M, Rosenblatt M. Structure-function analysis of parathyroid hormone and parathyroid hormone-related protein. In: Bilezikian JP, Levine MA, Marcus R, eds. The parathyroids. New York: Raven, 1994:139-156. 2. Chorev M, Rosenblatt M. Parathyroid hormone: Structurefunction relations and analog design. In: BilezikianJP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego, Academic Press, 1996:305-323. 3. PottsJT, Jr, Gardella TJ, Jfippner H, Kronenberg HM. Structure based design of parathyroid hormone analogs. J Endocrinol 1997;S15-$21. 4. Whitfield JF, Morley E Small bone-building fragments of parathyroid hormone: New therapeutic agents for osteoporosis. Trends Pharmacol Sci 1995;16:382-386. 5. Dempster DW, Cosman E Parisien M, Shen V, Lindsay R. Anabolic actions of parathyroid hormone on bone. Endocr Rev 1993;14:690-709. 6. Rosenblatt M. Peptide hormone antagonists that are effective in vivo: Lessons from parathyroid hormone. N Engl J Med 1986;315:1004-1013. 7. Rosenblatt M, Callahan EN, Mahaffey JE, Pont A, Potts JT, Jr. Parathyroid hormone inhibitors: Design, synthesis, and biologic evaluation of hormone analogues. J Biol Chem 1977;252:5847-5851. 8. Rosenblatt M, Chorev M, Nutt RF, Caulfield ME Horiuchi N, Clemens TL, Goldman ME, McKee RL, Caporale LH, FisherJE, LevyJJ, Reagan JE, Gay T, DeHaven E New directions for the design of parathyroid hormone antagonists. In: Massry SG, Fujita T, eds. New actions of parathyroid hormone. New York: Plenum, 1993:61-67. 9. Nussbaum SR, Rosenblatt M, Potts JT, Jr. Parathyroid hormone renal receptor interactions: Demonstration of two receptorbinding domains. J Biol Chem 1980;255:10183-10187. 10. Caulfield ME McKee RL, Goldman ME, Duong LT, Fisher JE, Gay CT, DeHaven PA, LevyJJ, Roubini E, Nutt RF, Chorev M, Rosenblatt M. The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: The receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endocrinology 1990;127:83-87. 11. Gardella TJ, Wilson AK, Keutmann HT, Oberstein R, PottsJT,Jr. Kronenberg HM, Nussbaum SR. Analysis of parathyroid hor-

P T H / P T H r P / R E c F ~ V T O R INTERACTIONS

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

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233. Mierke DE Royo M, Pellegrini M, Sun H, Chorev M. Peptide mimetic of the third cytoplasmic loop of the PTH/PTHrP receptor. J Am Chem Soc 1996;118:8998-9004. 234. Pellegrini M, Royo M, Chorev M, Mierke DE Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor. Biopolymers 1997;40: 653-666. 235. Schertler GF, Villa C, Henderson R. Projection structure of rhodopsin. Nature 1993;362: 770-772. 236. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 1990;213:899-929. 237. Schertler GF, Hargrave PA. Projection structure of frog rhodopsin in two crystal forms. Proc Natl Acad Sci USA 1995;92:11578-11582. 238. Grigorieff N, Ceska T, Downing K, Baldwin J, Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Bio11996;259:393-421. 239. Pebay Peyroula E, Rummel G, Rosenbusch JP, Landau EM. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science 1997;277: 1676-1681. 240. Baldwin JM. The probable arrangement of the helices in G protein-coupled receptors. E M B O J 1993;12:1693-1703. 241. Mierke DE Pellegrini M. Parathyroid hormone and parathyroid hormone-related protein: Model systems for the development of an osteoporosis therapy. Curr Pharm Design 1999;5: 21-36. 242. Pellegrini M, Bisello A, Rosenblatt M, Chorev M, Mierke D. Binding domain of human parathyroid hormone receptor: From conformation to function. Biochemistry 1998;37:12737-12743. 243. Behar V, Nakamoto C, Greenberg Z, Bisello A, Suva LJ, Rosenblatt M, Chorev M. Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes. Endocrinology 1996;137:4217-4224. 244. Schipani E, Jensen GS, Pincus J, Nissenson RA, Gardella TJ, Jfippner H. Constitutive activation of the adenosine 3', 5'monophosphate signaling pathway by parathyroid hormone (PTH)/PTH-related peptide receptors mutated at the two loci for Jansen's metaphyseal chondrodysplasia. Mol Endocrinol 1997;11:851-858. 245. Rosenblatt M, Goltzman D, Keutmann HT, Tregear GW, Potts JT, Jr. Chemical and biological properties of synthetic, sulfurfree analogues of parathyroid hormone. J Biol Chem 1976;251:159-164. 246. Shimizu M, Carter PH, Gardella TJ. Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand. J Biol Chem 2000;275:19456-19460. 247. Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ. Parathyroid hormone stimulates bone formation and resorption in organ culture: Evidence for a coupling mechanism. Proc Natl Acad Sci USA 1981;78:3204-3208. 248. Tam CS, Heersche JNM, Murray TM, Parsons JA Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continual administration. Endocrinology 1982;110:506-512. 249. Reeve J, Davies UM, Hesp R, Katz D. Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride. Br MedJ 1990;301:31 4-318. 250. Tada K, Yamamuro T, Okumura H, Kasai R, Takahashi H. Restoration of axial and appendicular bone volumes by hPTH(1-34) in parathyroidectomized and osteopenic rats. Bone 1990;11:163-169.

251. Hock JM, Gera I, Fonseca J, Raisz LG. Human parathyroid hormone-(1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122:2899-2904. 252. Hodsman AB, Fraher LJ. Biochemical responses to sequential human parathyroid hormone (1-38) and calcitonin in osteoporotic patients. Bone Miner 1990;9:137-152. 253. Tsai K-S, Ebeling PR, Riggs BL. Bone responsiveness to parathyroid hormone in normal and osteoporotic postmenopausal women. J Clin Endocrinol Metab 1989;69:1924-1027. 254. Slovik DM, Rosenthal DI, Doppelt SH, Potts JT, Jr, Daly MA, CampbellJA, Neer RM. Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D. J Bone Miner Res 1986;1:377-381. 255. Wronski TJ, Yen C-F, Qi H, Dann LM. Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats. Endocrinology 1993;132:823-831. 256. Kivlighn SD, Huckle WR, Zingaro GJ, Rivero RA, Lotti VJ, Chang RSL, Schorn TW, Kevin N, Johnson RG, Greenlee WJ. Discovery of L-162, 313: A nonpeptide that mimics the biological actions of angiotensin II. A m J Physio11995;268:R820-R823. 257. Aquino CJ, Armour DR, Berman JM, Birkemo LS, Carr RAE, Croom DK, Dezube M, Dougherty RW, Ervin GN, Grizzle MK, Head JE, Hirst GC, James MK, Johnson ME Miller LJ, Queen KL, Rimele TJ, Smith DN, Sugg EE. Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger."J Med Chem 1996;39:562-569. 258. Yang L, Berk SC, Rohrer SP, Mosley RT, Guo L, Underwood DJ, Arison BH, Birzin ET, Hayes ED, Mitra SW, Parmar RM, Cheng K, Wu TJ, Butler BS, Foor E Pasternak A, Pan Y, Silva M, Freidinger RM, Smith RG, Chapman K, Schaeffer JM, Patchett AA. Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2. Proc Natl Acad Sci USA 1998;95:10836-10841. 259. Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor E Mitra SW, Degrado sJ, Shu M, Klopp JM, Cai sJ, Blake A, Chan WWS, Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM. Rapid indentification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 1998;282:737-740. 260. Tian SS, Lamb P, King AG, Miller SG, Kessler L, Luengo JI, Averill L, Johnson RK, Gleason JG, Pelus LM, Dillon SB, Rosen J. A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor. Science 1998;281:257-259. 261. Hansen TK, Ankersen M, Hansen BS, Raun K, Nielsen KK, Lau J, Peschke B, Lundt BE Thogersen H, Johansen NL, Madsen K, Andersen PH. Novel orally active growth hormone secretagogues. J Med Chem 1998;41:3705-3714. 262. Zhang B, Salituro G, Szalkowski D, Li Z, Zhang Y, Royo I, Vilella D, Diez MT, Pelaez F, Ruby C, Kendall RL, Mao X, Griffin P, CalaycayJ, Zierath JR, HeckJV, Smith RG, Moller DE. Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science 1999;284:974-977. 263. Adams A, Bisello A, Chorev M, Rosenblatt M, Suva L. Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone. Mol Endocrinol 1998; 12:1673-1683. 264. Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, Rodda CP, Ebeling PR, Hudson PJ, Zajac JD, Martin TJ. Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA 1987 ;84:5048-5052.

P T H / P T H r P / R E C E P T O R INTERACTIONS 265. Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI. A parathyroid hormone-related protein implicated in malignant hypercalcemia: Cloning and expression. Science 1987;237:893-896.

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266. Horiuchi N, Caulfield ME Fisher JE, Goldman ME, McKee RL, Reagan JE, LevyJJ, Nutt RF, Rodan SB, Schofield TL, Clemens TL, Rosenblatt M. Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 1987;238:1566-1568.

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Receptors for Parathyroid Hormone and Parathyroid Hormone-Related Protein Signaling and Regulation ROBERT A. NISSENSON Endocrine Unit, San Francisco VA Medical Centeg, and Departments of Medicine and Physiology, University of California, San Francisco, California 94121

INTRODUCTION The endocrine effects of parathyroid hormone (PTH) and the paracrine actions of PTH-related protein (PTHrP) are initiated by the same intrinsic plasma membrane receptor (1,2). Several years ago, the sequence of the PTH/PTHrP receptor cDNA was obtained (3), and it was evident that the protein had a predicted topology similar to that of other known G protein-coupled receptors (GPCRs). In particular, the receptor is predicted to contain seven membrane-spanning helices, with a long amino-terminal extracellular domain, three extracellular loops, three intracellular loops, and a large carboxyterminal cytoplasmic tail (Fig. 1). Despite containing seven membrane-spanning segments, the PTH/PTHrP receptor does not share a number of the specific sequence motifs present in the largest subfamily of GPCRs (the class I family, which includes receptors for a diverse group of ligands ranging from photons to polypeptide hormones). Instead, the PTH/PTHrP receptor is a member of a second GPCR subfamily (class II) that includes receptors for calcitonin, glucagon, and a number of other polypeptide ligands (4). The evolutionary relationship between members of the class II GPCR subfamily is evident from the similarity of the intron/exon boundaries of their cognate genes, as well as the presence of a variety of conserved protein sequence motifs, particularly in their transmembrane domains (4,5). Moreover, these receptors generally utilize G s (coupling to adenylyl cyclase) and Gq (coupling to phospholipase C) for generating intracellular signals. Members of the class II GPCR subfamily presumably share a common The Parathyroids, Second Edition

NHz

COOH

FIG. 1 Structural representation of the three-dimensional topology of the PTH/PTHrP receptor, based on the model developed for other GPCRs. The large, glycosylated aminoterminal domain of the receptor is on the extracellular side of the membrane, and the large carboxyl-terminal tail is cytoplasmic. The seven transmembrane helices interact with one another and line a central polar cavity in the receptor.

basic mechanism of G protein activation, but have evolved determinants of specificity that permit binding and activation by only the appropriate peptide ligands. 93

Copyright © 2001 John P. Bilezikian, Robert Marcus, and Michael A. Levine.

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The molecular basis of ligand specificity and interaction with the P T H / P T H r P receptor is discussed elsewhere in this volume. What follows is a summary of the current understanding of molecular events that underlie activation of signal transduction following the binding of PTH or PTHrP to the P T H / P T H r P receptor; the mechanisms by which the P T H / P T H r P receptor is regulated following receptor activation, and diseases that are associated with abnormalities in P T H / P T H r P receptor expression and function.

SIGNAL TRANSDUCTION

PTH/PTHrP RECEPTOR

BY

THE

The G Protein-Coupled Receptor Superfamily Even before determination of the P T H / P T H r P receptor sequence, functional studies suggested that the P T H / P T H r P receptor was a member of the GPCR superfamily. For example, GTP and its analogs were found to regulate the affinity of PTH for the receptor, and to potentiate PTH-induced stimulation of adenylyl cyclase (6--11). This prediction was confirmed with the cloning of the cDNA encoding the P T H / P T H r P receptor (3), which revealed a predicted protein sequence containing seven putative membrane-spanning domains, a topology characteristic of members of the G protein-coupled receptor superfamily (12,13). There are well over 1000 known GPCRs, all of which appear to mediate agonistdependent G protein activation through a c o m m o n basic molecular mechanism (Fig. 2) (14,15). In brief, in GDP

Inactive

G protein

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GDP-(~y

GTP

GTPa+ ~y ~nGS ~L'~ GDP-(~

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FIG. 2 G protein activation by a GPCR. The rate-limiting step in G protein activation-dissociation of GDP from the G protein o~subunit is catalyzed by the agonist-activated receptor (HR*). This permits GTP to bind to the o~ subunit, resulting in subunit dissociation. Both the GTP-bound oL subunit and the 137 subunit dimer are capable of activating effectors such as adenylyl cyclase (AC) and phospholipase C (PLC), sometimes synergistically. Termination of signaling requires the hydrolysis of GTP to GDP by the intrinsic GTPase activity of the oL subunit. For some G proteins, the rate of GTP hydrolysis is enhanced by the action of a regulator of G protein signaling (RGS protein). The GDP-oL subunit complex then binds the 137 subunit dimer, regenerating the inactive heterotrimeric G protein.

the resting, inactive state, G proteins are plasma membrane-associated heterotrimers consisting of or, [3, and ~/subunits. The heterotrimeric form is stabilized by the binding of GDP to the guanylyl nucleotide binding site on the e~ subunit. Agonist binding to its GPCR induces the receptor to interact with the heterotrimeric G protein, producing a conformational change that results in the release of GDP from the e~ subunit. This allows GTP (which is more abundant than GDP in the cell) to bind to the oL subunit. Binding of GTP in turn induces a structural change in the G protein that results in G protein activation, i.e., dissociation of the e~ subunit-GTP complex from the [3y complex (the latter are tightly associated under all physiologic conditions). The free e~ subunit-GTP complex is able to regulate activity of specific effector systems (e.g., adenylyl cyclase, phospholipase C) that can produce a variety of second messengers. It has become clear that the [3y complex can also participate in regulation of effector activity, often (but not always) synergistically with the GTP-bound 0L subunit. Termination of signaling is effected by the intrinsic GTPase activity of the oLsubunit, resulting in the generation of a GDP--a subunit complex that rapidly reassociates with [3~/. In some cases, GTPase activity can be accelerated by the activity of a "regulator of G protein signaling" (RGS) protein (16). The inactive heterotrimeric G protein is now regenerated and poised to respond to another round of receptor activation. There are over 20 genes encoding G protein oL subunits, as well as multiple genes for [3 and ~/ subunits (17,18). Although there is evidence that the [3~/ complex participates in the specificity of G proteins for receptors and effectors, it is the oLsubunit that plays the p r e d o m i n a n t role in determining specificity. In the case of the P T H / P T H r P receptor, the major G proteins that can be activated are G s and Gq, which contain the e~ subunits % and C~q, respectively. Activation of G s leads to increased adenylyl cyclase activity, resulting in increased cellular levels of cyclic AMP and activation of Protein Kinase A (PKA). Activation of Gq results in stimulation of phospholipase C-[3 (PLC-[3) resulting in mobilization of intracellular calcium and activation of PKC. The relative activation of these pathways in a given cellular context presumably depends on the relative abundance of receptors as well as G s a n d Gq, and the relative affinity of the agonist-occupied receptor for these G proteins. In the case of the P T H / P T H r P receptor, signaling via G s appears to be preferred, probably due to greater affinity of the receptor for G s v e r s u s Gq. Thus, signaling by the P T H / P T H r P receptor through the cyclic AMP pathway can be detected at levels of PTH that occupy only a minute fraction of cellular receptors, which in part accounts for the ability to use urinary excretion of n e p h r o g e n o u s cyclic AMP as an in vivo bioassay for circulating levels of PTH (19) that are

RECEPTOR SIGNALING AND REGULATION

100--

---

Adenylyi cyclase

80--

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60--

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40--

,,.

m

20--

0"-0

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11

10

9

8

7

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FIG. 3 Relative potency of PTH in activating two signal transduction pathways in UMR-106 osteoblastic cells. In this system, PTH is at least an order of magnitude more potent in stimulating the adenylyl cyclase pathway versus the phospholipase C pathway (measured indirectly by the increase in cytosolic calcium). This result probably reflects a greater affinity of the activated PTH/PTHrP receptor for Gs as compared to Gq. Redrawn from Ref. 20; M Babich, H Choi, RM Johnson, KL King, GE Alford, RA Nissenson; Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways. Endocrinology, Vol. 129, pp. 1463-1470, 1991. © The Endocrine Society.

in the picomolar range. Preference of the P T H / P T H r P receptor for the cyclic AMP signaling pathway is also suggested by studies on PTH target cells in vitro, where activation of adenylyl cyclase occurs at m u c h lower concentrations of added PTH than does activation of phospholipase C (Fig. 3) (20).

Transmembrane Signaling by the P T H / P T H r P Receptor To perform their physiological functions properly, the activation of GPCRs must be tightly regulated by the

BASAL III

VI

His

Inactive

+PTH III

95

binding of receptor agonists. In the absence of agonists, the p r e d o m i n a n t receptor conformation is one that does not interact productively with the G protein on the cytoplasmic surface of the plasma membrane. Binding of an agonist to extracellular a n d / o r transmembrane domains of the receptor stabilizes a receptor conformation that favors interaction with and activation of the target G protein. One of the most intriguing questions in the GPCR field is how binding of an agonist to sites in the extracellular and transmembrane regions of a GPCR alters the structure of the cytoplasmic domain in a way that promotes G protein activation. In the case of well-studied GPCRs such as rhodopsin, the inactive and active receptor conformations can be distinguished by differences in the interactions between residues in the transmembrane helical domains (21,22). Alterations in the relative orientation of the transmembrane domains that are induced by agonist binding result in changes in the three-dimensional structure of the intracellular loops. This exposes key amino acids that participate in the activation of the cognate G protein(s) on the cytoplasmic face of the plasma membrane. In the case of the visual receptor rhodopsin, there is evidence that one of the critical conformational changes that accompanies receptor activation is the relative m o v e m e n t of the cytoplasmic ends of transmembrane helices 3 and 6 away from one a n o t h e r (23,24). In addition, specific interactions between amino acids in t r a n s m e m b r a n e domains 2 and 7 are i m p o r t a n t in stabilizing the active conformation of the receptor (22). Available evidence suggests that the f u n d a m e n t a l conformational shift that occurs on agonist binding to GPCRs m a y be conserved across the receptor superfamily. To examine this issue for the P T H / P T H r P receptor, a study was carried out to d e t e r m i n e whether preventing the relative m o v e m e n t of t r a n s m e m b r a n e domains 3 and 6 would inhibit PTH-induced receptor activation (Fig. 4). This study took advantage of the ability of zinc ions to complex with histidine residues in proteins. The P T H / P T H r P receptor contains a

.... Cai ++ 0

/

+PTH VI

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Active

III

VI

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\z/

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FIG. 4 Activation of the PTH/PTHrP receptor may require the relative movement of the cytoplasmic ends of transmembrane domains 3 and 6. A mutated receptor in which histidine residues are present in the cytoplasmic end of these two transmembrane domains is fully functional in the present of PTH. However, the addition of zinc to coordinate the histidines, thereby constraining the relative movement of transmembrane helices 3 and 6, inhibits receptor activation by PTH. See the text for further details.

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histidine at the cytoplasmic end of transmembrane domain 3, and a second histidine residue was inserted at the cytoplasmic end of transmembrane domain 6 by mutagenesis. The modified P T H / P T H r P receptor was fully functional with respect to supporting PTHsimulated adenylyl cyclase, but activation of the G protein G s by PTH was blocked by the addition of zinc ions. Inhibition of receptor activation by zinc required the presence of both histidine residues, indicating that the mechanism of zinc inhibition involved coordination of the histidines by zinc ions. This coordination apparently prevented the movement away from one another of the cytoplasmic ends of transmembrane domains 3 and 6 of the P T H / P T H r P receptor. Mutagenesis studies have also demonstrated a probable interaction between transmembrane domains 2 and 7 in the activated P T H / P T H r P receptor (25), similar to what has been reported for rhodopsin. Much of what we know about the structural basis for activation of the P T H / P T H r P receptor has come from studies of receptor mutations. A variety of receptor mutations have been shown to result in diminished agonist-dependent receptor activation with retention of ligand binding. This might be due to effects of mutations on the agonist-induced conformational switch in the receptor that is required for activation. Indeed, replacement of any of three amino acids (serine, arginine, serine) along a polar face of transmembrane domain 2 of the P T H / P T H r P receptor result in diminished responsiveness to PTH (26) (Fig. 5). These mutations may disrupt the interaction of transmembrane domains 2 and 7, and indeed mutation of a glutamine residue in transmembrane domain 7 likewise diminished signaling through both the adenylyl cyclase and phospholipase C pathways (25). Mutation of specific cytoplasmic sequences in the receptor can also disrupt PTH-induced signaling by the P T H / P T H r P receptor, presumably by directly interfering with receptor-G protein interactions. The critical amino acids for agonist-

AC PLC

COOH PLC

stimulated activation of G, and Gq appear to lie in the second and third cytoplasmic loops of the receptor. A variety of mutations in the second cytoplasmic loop produce a reduction in phospholipase C activation, without major loss of adenylyl cyclase activation, indicating that this region is particularly crucial for efficient receptor coupling to Gq (27). A lysine residue in the third cytoplasmic domain (near the cytoplasmic end of transmembrane helix 5) was found to be essential for signaling both to G s and Gq, whereas mutations of nearby amino acids resulted in selective reduction in the signaling to either G s or Gq (28). Studies of a synthetic peptide mimetic of the third cytoplasmic loop indicate that these critical amino acids are within a domain capable of forming an oL-helix in a nonpolar environment (29). This is reminiscent of other GPCRs, whereby positively charged helical domains in the cytoplasmic loops are essential for G protein activation. Taken together, these results demonstrate that multiple sites in the cytoplasmic domain of the P T H / P T H r P receptor are involved in the activation of G proteins. There appear to be some sites that are important generically for G protein activation, presumably contacting structural features that are shared by G s and Gq. Other sites in the receptor are involved in the selective activation of either of these G proteins. It is clear that more information is needed concerning the threedimensional structure of the cytoplasmic domain of the P T H / P T H r P receptor in order to establish the molecular basis for the initiation of signal transduction. The precise role of the cytoplasmic tail of the P T H / P T H r P receptor in signaling is not entirely clear. In one study, truncation of the cytoplasmic tail of the rat P T H / P T H r P receptor was found to enhance PTH-stimulated adenylyl cyclase, but not phospholipase C activity (30). This finding is consistent with previous reports that the P T H / P T H r P receptor is able to couple to a third G protein (Gi) that inhibits activation of adenylyl cyclase (31), and that this may involve deter-

FIG. 5 Location in the PTH/PTHrP receptor of specific amino acids that are essential for agonist-stimulated signal transduction. Mutations shown to inhibit either adenylyl cyclase (AC) activation or phospholipase C (PLC) activation, or activation of both pathways, are specified. Note that these mutations are found in transmembrane helices 2 and 7, as well as in the second and third cytoplasmic loops. As discussed in the text, these receptor domains are important for G protein activation by many members of the GPCR superfamily.

RECEPTOR SIGNALING AND REGULATION

minants in the receptor's cytoplasmic tail (32). It is not yet clear whether the P T H / P T H r P receptor is able to activate G i in PTH-responsive bone and kidney cells. In a second study, truncation of the cytoplasmic tail of the opossum P T H / P T H r P receptor had no effect on the adenylyl cyclase response to PTH (33), indicating that there may be species-specific differences in P T H / P T H r P receptor-G protein coupling. The cytoplasmic tail of the P T H / P T H r P receptor also contains determinants of cell surface targeting and expression of the receptor (33). It is possible that some of the observed effects of receptor truncation on signaling in response to PTH are due to altered receptor targeting a n d / o r expression rather than to altered signal transduction.

REGULATION OF THE PTH/PTHrP RECEPTOR As with other GPCRs, signaling by the P T H / P T H r P receptor is tightly regulated by both homologous and heterologous mechanisms. Homologous regulation occurs in response to agonist binding whereas heterologous regulation occurs in response to factors acting though different pathways. Regulation can be manifest at multiple levels, including suppression of the ability of the agonist-occupied receptor to promote activation of cognate G proteins (desensitization) and physical removal of the receptor from the cell surface into an intracellular compartment (internalization/sequestration). Long-term regulation of receptor signaling is accomplished by agonist-induced changes in steadystate levels of expression of receptors, due to increased receptor catabolism following receptor internalization (down-regulation) and to changes in de n o v o receptor synthesis. Homologous regulation commonly involves all of these mechanisms, whereas heterologous regulation most often occurs through changes in steady-state levels of receptor expression.

PTH Receptor Phosphorylation and Desensitization Homologous regulation of P T H / P T H r P receptor signaling has been extensively d o c u m e n t e d . Treatment of cultured bone and kidney cells with PTH generally dampens the adenylyl cyclase and phospholipase C responses to a second addition of the hormone (34-43). Generally, desensitization of the PTH response occurs rapidly, within minutes of initial exposure to PTH, suggesting that the P T H / P T H r P receptor has become acutely uncoupled from its cognate G proteins. The mechanisms underlying acute desensitization have been explored in depth for GPCRs

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such as rhodopsin and [3-adrenergic receptors (44-46). A major mechanism underlying acute desensitization of these receptors is phosphorylation of the cytoplasmic domain of the receptor by a GPCR kinase (GRK) (46,47). Activation of rhodopsin or the [3-adrenergic receptor alters the conformation of the cytoplasmic domain in a way that favors the binding of a GRK to the receptor. There are at least six distinct GRK genes, encoding GRK1, specifically a rhodopsin kinase, and GRKs 2, 3, and 5, which are capable of phosphorylating other GPCRs (48). Once bound, the GRK phosphorylates serine a n d / o r threonine residues in the cytoplasmic tail or (less commonly) the third cytoplasmic loop of the receptor. Phosphorylation of the receptor by a GRK promotes the binding of arrestin proteins, which physically uncouple the receptor from its cognate G protein(s) (49) and also facilitate the entry of the receptor into clathrin-coated pits, thereby promoting receptor internalization (50). There is increasing evidence that similar mechanisms apply to the regulation of P T H / P T H r P receptor signaling. The P T H / P T H r P receptor is subject to phosphorylation in response to agonist binding (51,52), and this appears to occur largely if not exclusively on serine residues in the proximal portion of the cytoplasmic tail (52-54). Available evidence suggests that GRKs are largely responsible for agonist-stimulated phosphorylation of the P T H / P T H r P receptor. Thus, GRK2 is expressed in a variety of osteoblastic cell lines (55), and this GRK, and to a lesser extent GRKs 3 and 5, have been shown to phosphorylate the P T H / P T H r P receptor in isolated membranes (56). Moreover, the recombinant cytoplasmic tail of the P T H / P T H r P receptor is a substrate for phosphorylation by GRK2 (53). Overexpression of GRK2 in cells promotes the phosphorylation of the P T H / P T H r P receptor (54) and inhibits P T H / P T H r P receptor signaling (56). Interestingly, the latter effect was also seen with a C-terminally truncated form of the P T H / P T H r P receptor lacking the sites of phosphorylation by GRKs (51). This finding raises the interesting possibility that recruitment of GRKs to the receptor in response to agonist binding might suppress signal transduction by a mechanism distinct from receptor phosphorylation (e.g., steric interference with G protein activation). In further support for a role of GRKs in regulating PTH action, stable expression of a dominant-negative form of GRK2 in SaOS-2 cells was found to suppress PTH-induced desensitization of P T H / P T H r P receptor signaling (57). In the case of the [3-adrenergic receptor, agonistinduced phosphorylation can result from activation of a second messenger-dependent kinase (PKA) as well as from GRKs (58). These kinases phosphorylate different sites in the cytoplasmic domain of the [3-adrenergic

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receptor, but each can promote desensitization of receptor signaling. At least two second messengerd e p e n d e n t kinases are activated in response to P T H / P T H r P signaling, PKA and PKC. Studies with the recombinant cytoplasmic tail of the P T H / P T H r P receptor and purified kinases indicate that this domain is potentially a substrate of both of these kinases, and that the sites of phosphorylation are different from the sites phosphorylated by GRK2 (53). Moreover, exposure of cells expressing P T H / P T H r P receptors to either forskolin (to activate PKA) or phorbol esters (to activate PKC) resulted in increased receptor phosphorylation. However, studies using inhibitors of these kinases have given equivocal results. In h u m a n embyonic kidney (HEK293) cells expressing the opossum P T H / P T H r P receptor, inhibitors of PKA and PKC had little effect on PTH-induced receptor phosphorylation (51), suggesting that GRKs are responsible for receptor phosphorylation in that system. However, staurosporine (at a dose than inhibits both PKA and PKC, but not GRKs) was found to inhibit partially PTH-induced phosphorylation of the rat P T H / P T H r P receptor in COS-7, LLC-PK1, and ROS 17/2.8 cells (52). Thus, the precise role of PKA and PKC in the agonistinduced phosphorylation and desensitization of the P T H / P T H r P receptor remains to be fully defined.

Endocytosis and Down-Regulation of the PTH/PTHrP Receptor Chronic exposure of target cells to high levels of PTH or PTHrP results in a decrease in the number of cellular P T H / P T H r P receptors (down-regulation), and a corresponding reduction in the maximal signaling response to the hormone (42,59-62). This has been demonstrated in a large n u m b e r of studies in vitro, but receptor down-regulation may also have pathophysio-

459EVQ (-)

/

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logic relevance. For instance, vitamin D deficiency can be associated with target cell resistance to PTH (63-65). In animal studies, this resistance can be reversed by parathyroidectomy, suggesting that it is the secondary hyperparathyroidism that is responsible for target cell resistance (66). Infusion of PTH to levels typical of severe secondary hyperparathyoidism produces down-regulation of P T H / P T H r P receptors and a reduction in the adenylyl cyclase response to PTH (59). In chronic renal failure, factors other than hyperparathyroidism may also contribute to reduced target cell expression of P T H / P T H r P receptors (67). It is also possible that down-regulation of P T H / P T H r P receptors is one of the factors that contributes to the decreased anabolic response of the skeleton to high level continuous administration of PTH, as compared to intermittent treatment. For these reasons, there has been considerable interest in better defining the cellular and molecular bases for agonist-induced down-regulation of the P T H / P T H r P receptor. The initial step in down-regulation of P T H / P T H r P receptors appears to be agonist-induced accumulation of the receptor in plasma membrane clathrin-coated pits (68,69). These pits are endocytic organelles that pinch off from the plasma membrane, thus becoming endocytic vesicles. Once internalized, P T H / P T H r P receptors can be recycled to the plasma membrane, or can presumably progress further down the endocytic pathway to the lysosomes for degradation. The molecular mechanisms underlying the agonist-induced internalization of the P T H / P T H r P receptor are not entirely clear. The role of specific structural features of the receptor in the endocytic process has been investigated by mutagenesis (Fig. 6). The results of early studies demonstrated that a truncated P T H / P T H r P receptor lacking all but 16 amino acids in the cytoplasmic tail was capable of signaling but displayed only about 50% of the

FIG. 6 Amino acids in the PTH/PTHrP receptor that are important for regulating receptor endocytosis, identified by targeted mutagenesis. The positive endocytic signals include a tyrosine-based signal in the cytoplasmic tail, a lysine residue in the third cytoplasmic loop (also important for signal transduction), and an asparagine residue in the third transmembrane helix. A negative endocytic signal in the juxtamembrane region of the cytoplasmic tail is also indicated. The amino acids corresponding to endocytic signals are conserved among various species of PTH/PTHrP receptors. The position numbers are for the opossum PTH/ PTHrP receptor. See the text for more details.

RECEPTOR SIGNALING AND REGULATION

normal rate of agonist-stimulated receptor internalization (69). For some other GPCRs, receptor phosphorylation has been shown to promote internalization at least in part by allowing the binding of arrestins proteins and their subsequent interaction with clathrin (50). The truncated PTH/PTHrP receptor lacked the sites of GRK-mediated phosphorylation, and it was therefore logical to hypothesize that this was the basis for the reduced endocytosis. However, a PTH/PTHrP receptor mutated to eliminate selectively the sites of phosphorylation (leaving the bulk of the cytoplasmic tail intact) was not impaired in its ability to be internalized (54). Progressive truncation of the cytoplasmic tail of the P T H / P T H r P receptor revealed that mutation of a stretch of sequence containing the amino acids Tyr-GlyPro-Met resulted in impaired receptor endocytosis (69). This sequence fits the consensus sequence of endocytic motifs that have been demonstrated in the cytoplasmic domain of a large number of plasma membrane proteins (70). It appears that this sequence mediates the interaction of these proteins with the AP-2 protein complex of the clathrin-coated pit (71), and this is likely to be the major endocytic signal in the cytoplasmic tail of the P T H / P T H r P receptor. Interestingly, this same study identified a potential negative endocytic signal (Glu-Val-Gln) at the junction between the seventh transmembrane segment and the cytoplasmic tail. Mutation of this sequence resulted in enhancement of agoinst-dependent receptor internalization. Although the mechanism underlying the actions of negative endocytic signals is unclear, they may prevent internalization by interacting with proteins that are excluded from clathrin-coated pits. Despite these results, there is evidence that the GRK/arrestin system participates in P T H / P T H r P receptor internalization and down-regulation. Expression of a dominant-negative form of GRK2 resulted in diminished PTH-induced down-regulation of the P T H / P T H r P receptor in SaOS-2 cells (57). Whether this effect resulted from blockage of receptor phosphorylation is not clear, particularly in light of the finding (discussed earlier) that GRK2 can exert phosphorylation-independent actions on P T H / P T H r P receptor function. It has been demonstrated that activation of the P T H / P T H r P receptor resulted in translocation of arrestin from the cytoplasm to the cell membrane, with subsequent colocalization of the receptor and arrestin in intracellular vesicles (72). This elegant result suggests that arrestins play a role in facilitating receptor endocytosis, as has been seen with a number of other GPCRs. Whether agonist-induced phosphorylation of the receptor is required for this effect of arrestin remains to be established.

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99

Regulation of PTH/PTHrP Receptor Gene Expression Long-term regulation of PTH/PTHrP receptor levels can occur through changes in the expression of the receptor gene. Exposure to PTH is reported to decrease levels of PTH/PTHrP receptor mRNA in osteoblasts by a mechanism involving the cyclic AMP pathway (73,74). This may be due to direct regulation of transcriptional activity of the PTH/PTHrP receptor gene by transcription factors phosphorylated by PKA, but the details of this pathway have yet to be elucidated. Homologous control of PTH/PTHrP receptor expression appears to be target cell specific in that PTH reportedly does not reduce expression of the PTH/PTHrP receptor gene in the kidneys of rats with secondary hyperparathyroidism (67,75). Heterologous factors are also reported to regulate levels of PTH/PTHrP receptor expression in bone and kidney. The cytokine TGF-[3 up-regulates the expression of the PTH/PTHrP receptor in osteoblastic osteosarcoma cells (76), although the opposite effect is reported in primary cultures of fetal rat osteoblasts (77) and in OK cells (78). Dexamethasone treatment produces an increase in expression of the PTH/PTHrP receptor in osteoblastic cells, but not in kidney cells (79,80), whereas 1,25(OH) 2 vitamin D down-regulates expression of the PTH/PTHrP receptor gene (81). Many of these studies have been carried out in cultured bone and kidney cells in vitro, and further work is needed to establish the physiologic relevance of the changes in PTH/PTHrP receptor gene expression.

GENETIC DISORDERS OF THE PTH/PTHrP RECEPTOR H u m a n genetic diseases are associated with both loss-of-function and gain-of-function mutations in the P T H / P T H r P receptor (Fig. 7) (82). Homozygous loss of expression of functional P T H / P T H r P receptors is responsible for the rare familial disorder Blomstrand lethal chondrodysplasia (82-85). Blomstrand infants have abnormalities reflecting the lack of PTHrPdirected signaling during endochondral bone development. They display short-limbed dwarfism with increased bone density, accelerated skeletal maturation, and reduced numbers of proliferating growth plate chondrocytes. Two mutant P T H / P T H r P receptor alleles have been identified in such patients, both containing point mutations. One is in the coding region, and encodes a leucine residue rather than the normal proline at position 132 in the receptor's N-terminal extracellular domain (84,85). The other mutation produces a splice variant that encodes a

100

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CHAPTER5

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P T H / P T H r P receptor lacking 11 amino acids in the transmembrane helix (83). These mutations probably alter the conformation of the receptor in a way that precludes effective ligand binding. As discussed previously, activation of GPCRs ordinarily requires agonist binding to stabilize the active receptor conformation. However, a large number of mutations have been identified that allow partial stabilization of the active conformation of GPCRs in the absence of ligand. Such ligand-independent ("constitutive") activation of GPCRs is frequently associated with functional dysregulation and overt disease. Mutated P T H / P T H r P receptors with constitutive activity have been identified in patients with Jansen's metaphyseal chondrodysplasia. This is a rare, dominantly inherited disorder that is characterized by severe growth plate abnormalities, including a delay in chondrocyte differentiation, increased bone resorption, hypercalcemia, and hypophosphatemia (86--89). Circulating levels of PTH and PTHrP are normal or low in these individuals. Three mutated alleles of the P T H / P T H r P receptor have been identified in Jansen's chondrodysplasia. These encode receptors with single amino acid transversions toward the cytoplasmic end of transmembrane helix 2, 6, or 7 (90-92). Each of these mutant receptors displays constitutive activity for the adenylyl cyclase pathway. That is, these receptors promote activation of adenylyl cyclase even in the absence of PTH or PTHrE Constitutive signaling in developing cartilage presumably mimics the effect of excessive PTHrP, thus producing the growth plate phenotype, whereas constitutive signaling in bone and kidney reproduces the effect of excess PTH, resulting in the abnormalities in bone turnover and mineral homeostasis. It is of interest that these mutant receptors do not produce constitutive activation of phospholipase C when expressed in cultured cells, although it is possible that this pathway is activated in vivo. The structural basis for the constitutive signaling by these mutant P T H / P T H r P receptors is not established.

FIG. 7 Human PTH/PTHrP receptor mutations identified in patients with Blomstrand (B) and Jansen's (J) chondrodysplasias. Blomstrand mutations result in a loss of receptor function, whereas Jansen's mutations result in receptors that display ligand-independent (constitutive) activity.

As noted previously, the cytoplasmic ends of transmembrane domains 3 and 6 must separate for agonistinduced signaling to occur. The threonine at position 410 in transmembrane domain 6 may be crucial for preventing the separation of these regions in the absence of agonist. Mutation of this threonine to any of several amino acids, including proline, as seen in some patients with Jansen's chondrodysplasia, would allow separation of these domains and thus signal transduction in the absence of agonist binding. As discussed earlier, there is evidence that transmembrane domains 2 and 7 interact during the course of receptor activation. It is possible that the mutations in these regions in Jansen's patients (histidine to arginine at position 223 and isoleucine to arginine at position 458) stabilize this interaction even in the absence of agonist. Most antagonist analogs of PTH and PTHrP are neutral competitive antagonists. That is, they bind to the P T H / P T H r P receptor (and thereby competitively inhibit agonist binding), but they do not stabilize a particular receptor conformation. However, a few analogs have been shown to function as inverse agonists with respect to constitutively active P T H / P T H r P receptors (93). The binding of these analogs stabilizes the mutated receptor in an inactive conformation and thereby suppress its constitutive activity. It is conceivable that such inverse agonists will prove to be useful for treating individuals with Jansen's metaphyseal chondrodysplasia.

SUMMARY There has been great progress in understanding the molecular basis of activation of the G protein-coupled receptor for PTH and PTHrE Ligand binding to defined sites in the extracellular and transmembrane domains facilitates a conformational change in the receptor that appears to include the movement of the cytoplasmic ends of transmembrane helices 3 and

RECEPTOR SIGNALING AND REGULATION

6 away from one another. Specific cytoplasmic amino acids, particularly in the second and third cytoplasmic loops, have been found to mediate the activation of the cognate G proteins G s a n d Gq, resulting in activation of adenylyl cyclase and phospholipase C. Once activated, PTH/PTHrP receptors are subject to regulatory phosphorylation on serine residues in the cytoplasmic tail. Members of the GRK family, particularly GRK2, appear to be primarily responsible for phosphorylation, with a lesser role for PKC. Phosphorylation of the receptor followed by the binding of arrestin may participate in both desensitization and endocytosis of the P T H / P T H r P receptor, but this has yet to be demonstrated unequivocally. The receptor also contains other determinants of endocytosis (both positive and negative), and these are likely to regulate PTH/PTHrP receptor down-regulation during chronic exposure to agonists. Naturally occuring loss-of-function and gain-of-function mutations in the human PTH/PTHrP receptor have been identified, and these are associated with Blomstrand lethal chondrodysplasia and Jansen's metaphyseal chondrodysplasia, respectively. Further progress in understanding the structural basis of PTH/PTHrP receptor function will continue to provide insights into the control of cellular function by these essential regulatory polypeptides.

6.

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12.

13. 14.

15.

ACKNOWLEDGMENTS

16. 17.

I am grateful to Margaret Bencsik for skillful assistance in the preparation of this manuscript. Portions of the work discussed here were supported by NIH Grant DK35323 and by the Medical Research Service of the Department of Veterans' Affairs.

18. 19. 20.

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78. Law F, Bonjour JP, Rizzoli R. Transforming growth factor-beta: A down-regulator of the parathyroid hormone-related protein receptor in renal epithelial cells. Endocrinology 1994;134:2037-2043. 79. Urefia P, Iida-Klein A, Kong XE Jfippner H, Kronenberg HM, Abou-Samra AB, Segre GV. Regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid by glucocorticoids and PTH in ROS 17/2.8 and OK cells. Endocrinology 1994;134:451-456. 80. Yaghoobian J, Drfieke TB. Regulation of the transcription of parathyroid-hormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells. Nephrol Dial Transplant 1998;13:580-586. 81. Wald H, Dranitzki-Elhalel M, Backenroth R, Popovtzer MM. Evidence for interference of vitamin D with PTH/PTHrP receptor expression in opossum kidney cells. Pfluegers Arch EurJPhysiol

1998;436:289-294.

82. Nissenson RA. Parathyroid hormone (PTH)/PTHrP receptor mutations in human chondrodysplasia [editorial; comment]. Endocrinology 1998;139:4753-4755. 83. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, Le Merrer M, Silve C. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 84. Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365-3368. 85. Karaplis AC, He B, Nguyen MT, Young ID, Semeraro D, Ozawa H, Amizuka N. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia [see comments]. Endocrinology 1998;139:5255-5258. 86. Jansen M. Uber atypische chondrodystrophie (achondroplasie) und uber eine noch nicht beschriebene angeborene wachstumsstarung des knochensystems: Metaphysare dysostosis. Z Orthop Chir 1934;61:253-286. 87. Ozonoff MB. Metaphyseal dysostosis of Jansen. Radiology 1969;93:1047-1050. 88. Charrow J, Poznanski AK. The Jansen type of metaphyseal chondrodysplasia: Confirmation of dominant inheritance and review of radiographic manifestations in the newborn and adult. Am J Med Genet 1984;18:321-327. 89. Kruse K, Schfitz C. Calcium metabolism in the Jansen type of metaphyseal dysplasia. EurJPediatr 1993;152:912-915. 90. Schipani E, Kruse K, Jfippner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 1995;268:98-100. 91. Schipani E, Langman CB, Parfitt AM,Jensen GS, Kikuchi S, Kooh SW, Cole WG, Jfippner H. Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen's metaphyseal chondrodysplasia [see comments]. N EnglJ Med 1996;335: 708-714. 92. Schipani E, Langman C, Hunzelman J, Le Merrer M, Loke KY, Dillon MJ, Silve C, Jfippner H. A novel parathyroid hormone (PTH)/PTH-related peptide receptor mutation in Jansen's metaphyseal chondrodysplasia. J Clin Endocrinol Metab

1999;84:3052-3057.

93. Gardella TJ, Luck MD, Jensen GS, Schipani E, Potts JT, Jr, Jfippner H. Inverse agonism of amino-terminally truncated parathyroid hormone (PTH) and PTH-related peptide (PTHrP) analogs revealed with constitutively active mutant PTH/PTHrP receptors. Endocrinology 1996;137:3936-3941.

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CHAPTER 6

Nuclear Actions of PTHrP

ANDREW C. KARAPLIS AND M. T. AUDREY NGUYEN Division of Endocrinology, Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research, McGill University, Montrial, Quebec, Canada H3T 1E2

INTRODUCTION

midregion fragment (amino acid residues 37-86) that is proposed to play a role in placental calcium transport (41); and a carboxyl-terminal fragment [PTHrP(107-139)] that is reported to inhibit osteoclastic bone resorption (13), stimulate osteoblast growth (12), and induce calcium transients in hippocampal neurons (17). As can be inferred from this discussion, PTHrP should be regarded as a prototypical polyhormone that encompasses several distinct, functional regions that mediate unique and independent biological processes under highly specific circumstances (54). Nevertheless, for the most part, the nature of these circumstances remains an open question.

Parathyroid hormone-related protein (PTHrP) was initially identified as the humoral factor responsible for hypercalcemia in malignancy. It is now recognized, however, that its role in calcium regulation represents only a fraction of the wide spectrum of its physiologic actions (41). Indeed, PTHrP controls a diverse range of developmental and homeostatic functions in a wide variety of tissues by acting primarily at the local or cellular level (68). The mature PTHrP sequence is preceded by a 36amino acid prepro segment, in which the first 20-30 amino acids likely represent a signal sequence critical in directing the nascent peptide from the cytosolic compartment to the rough endoplasmic reticulum (ER). It is presumed that the signal peptide is cleaved cotranslationally in the ER by signal peptidase, likely within the - 1 5 to - 5 region, although the exact site has not been rigorously proved. The presence of a prosequence is even less well characterized, although circumstantial evidence and parallels drawn from knowledge of PTH processing overwhelmingly support its existence (9). Cleavage of the propeptide either in the Golgi apparatus or in secretory granules yields the mature PTHrP form with alanine at position + 1. The PTHrP protein is generally thought to comprise several biologically active domains (Fig. 1A). These include the amino-terminal 1-36 peptide, which binds and activates the type 1 PTH cell surface receptor (PTHR1 or P T H / P T H r P receptor) and thereby influences cellular proliferation and differentiation in cartilage (35), bone (4), breast (85), and skin (16); a The Parathyroids, Second Edition

THE PTHrP N U C L E A R / N U C L E O L A R LOCALIZATION SEQUENCE The portion of the mature protein spanning amino acids 87-106 comprises two clusters of basic amino acids (88-91 and 102-106) that have been previously viewed as putative endoproteolytic processing sites (Fig. 1B). This sequence also bears structural homology to a nuclear localization sequence (NLS). The two best defined classes of nuclear import signals are the monopartite and bipartite NLSs, such as PKKKRKV found in the SV40 large T antigen (34), and the nucleoplasmin sequence KR(PAATKKAGQA)KKKK (72), that consists of two basic domains separated by 10 intervening "spacer" amino acids (indicated in parentheses), respectively. This observation formed the basis for the early studies that set out to investigate whether 105

Copyright © 2001 J o h n R Bilezikian, Robert Marcus, and Michael A. Levine.

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FIG. 1 PTHrP as a polyhormone. (A) PTHrP cDNA encodes a prepropeptide and mature forms of 139, 141, and 173 amino acids. The three isoforms are identical for the first 139 amino acids, which is the portion depicted here. Proposed biologically active domains within the protein are shown. SP, Signal peptide; P, propeptide. (B) The PTHrP bipartite nuclear localization sequence. This segment encompasses amino acids 88-106 of the mature form (indicated in the single-letter amino acid code) and comprises two basic clusters separated by 10 intervening "spacer" amino acids, resembling the Xenopus laevis nucleoplasmin nuclear localization sequence (72). Furthermore, this region conforms to the structural requirements for a nucleolar localization sequence, as described in key regulatory proteins of human retroviruses (HTLV-1 Rex, and HIV-1 Tat and Rev), consisting of an "arginine hinge" (KRK, in blue) and an adjacent Q inserted between two putative nuclear localization sequences (22). (C) Subcellular distribution of PTHrP in transfected COS-7 cells. Plasmid constructs encoding PTHrP forms having either an intact coding region, deletions within the coding region, or fused in frame to the Escherichia coli lacZ gene were expressed in COS-7 cells and the subcellular localization of the recombinant protein was determined by indirect immunofluorescence. SP, Secretory pattern; N, nucleolar, C, cytoplasmic. Size of lettering on the right-hand side is indicative of the levels of PTHrP immunoreactivity in the various subcellular compartments (24). (See color plates.)

the NLS in PTHrP is indeed functional (24). When preproPTHrP cDNA was transiently expressed in COS-7 cells, the subcellular localization of the protein, as determined by indirect immunofluorescence, was consistent with that of a secretory protein (Fig. 1C). Moreover, in these randomly cycling cell populations, approximately 10% of transfected cells also displayed nucleolar PTHrP staining, suggesting that the putative NLS could target PTHrP to the cell nucleus/nucleolus. When plasmid expressing an engineered mature form of PTHrP (i.e., lacking the prepro sequence) was transfected in COS-7 cells, this mature form, in striking contrast to the predominantly cytoplasmic accumulation of the expressed preproPTHrP eDNA, was targeted almost exclusively to the nucleus, where it was distributed in a nucleolar pattern. Removal of the putative NLS resulted in purely cytoplasmic staining. A similar deletion from the preproPTHrP eDNA construct elicited an exclusively secretory pattern for the expressed protein. These findings suggested that, in the absence of the prepro sequence, PTHrP is preferentially directed to the nuclear compartment and that deletion of the NLS effectively abolishes intranuclear localization of the recombinant protein. Interestingly, fusion of the PTHrP NLS to the cytoplasmic protein [3-galactosidase can target the fusion protein to the nucleus, consistent with the PTHrP NLS sequence being sufficient to target PTHrP to the nucleus. In addition to the foregoing studies, endogenous native PTHrP has also been shown, both in vitro as well as in situ, to localize to the nucleolus (Fig. 2). In osteoblasts, immunoelectron microscopy detected endogenous PTHrP over the dense fibrillar component of nucleoli, a subnucleolar structure and major site for transcription of rRNA genes (24). Nuclear/nucleolar PTHrP immunoreactivity has subsequently been described in keratinocytes (47), vascular smooth muscle (55), breast cancer (10), malignant melanoma (87), astrocytoma (77), and glial and neuronal cells (A. C. Karaplis and M. T. Audrey Nguyen, personal observation, 2000). It is becoming apparent, therefore, that nuclear translocation of PTHrP constitutes a means by which this peptide growth factor could modulate cell function via an intracrine mechanism of action. This unconventional and rather contentious view of PTHrP action is not unique to this protein. Over the past two decades, other molecules that bind to cell surface receptors, such as insulin (21); growth hormone (52); prolactin (71); somatostatin (59); nerve growth factor (26); fibroblast growth factors (FGFs) such as bFGF (7,8), aFGF (28), and FGF3 (36,37); plateletderived growth factor (PDGF) (53); angiogenin (27,60); and insulin-like growth factor-binding proteins (IGFBP)-3/IGFBP-5 (75,76) have been proposed to

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influence cellular function in a dual m a n n e r [for reviews, see (23,32)], first, by binding to cell surface receptors in their classic e n d o c r i n e / p a r a c r i n e mechanism of action, and second, by targeting of the protein to the nucleus a n d / o r nucleolus of the cell in an intracrine signaling pathway. Little is known about the events that govern the timing and extent of nuclear transport of these peptides or their nuclear actions in normal cell biology. In this review, we highlight the present knowledge of the PTHrP nuclear actions, concentrating on four specific issues: (1) How does PTHrP, a secreted protein, gain access to the cytosol? (2) Once in the cytosol, how is PTHrP transport to the cell nucleus regulated? (3) How does n u c l e a r / n u c l e o l a r PTHrP modulate cell functions? (4) What are the cellular consequences of nuclear signaling by PTHrP?

HOW DOES PTHrP, A SECRETED PROTEIN, G A I N A C C E S S TO THE C Y T O S O L ? In attempting to understand how PTHrP, a secreted pepdde, gains access to the cytosol for subsequent nuclear targeting, three distinct pathways should be considered (Fig. 3). First, depicted by pathway A in Fig. 3, PTHrP could be internalized after secretion. This may be a receptor d e p e n d e n t or i n d e p e n d e n t process. What evidence exists that secreted PTHrP utilizes this route to access the cell cytosol? Intuitively, one would assume that binding to the type 1 PTH receptor is the primary mode for PTHrP internalization. In concordance with this assumption, Lam et al. (45) have demonstrated that PTHrP(1-108), when added to culture medium, can be taken up specifically by receptor-expressing

FIG. 3 Potential pathways (A, B, and C) utilized by PTHrP to gain access to the cytosol. In pathway A, secreted PTHrP undergoes internalization at the cell surface in a "receptor"dependent manner. Endocytosis could be mediated by the type 1 PTH receptor (PTHR1) or a binding protein that is distinct and recognizes either the N-terminal domain or other regions of PTHrP (R). In pathway B, PTHrP, after entering the ER lumen, "dislocates" back to the cytosol via the Sec61p translocon, a key component of the mammalian cotranslational protein translocation system, which functions as a twoway channel shuttling proteins both into the ER and back to the cytosol. Ubiquination of preproPTHrP may serve as the signal for retrograde transport of the peptide. In pathway C, initiation of translation in PTHrP mRNA downstream from the initiator methionine generates a protein with a shorter signal peptide. Such a protein would fail to be targeted for secretion and remain in the cytosol for subsequent nuclear import. Experimental evidence supporting each of these pathways is illustrated in Fig. 4. (See color plates.)

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UMR-106.01 cells. This process is blocked by an excess of either PTHrP(1-108) or PTHrP(1-34), indicating that the endocytosis is mediated by the type 1 PTH receptor (Fig. 4A). Further support for type 1 PTH receptor-mediated uptake has come from studies showing receptor immunoreactivity in the nucleus of osteoblast-like cells at the time of DNA synthesis and mitosis (84). This, however, does not preclude involvement of the distinct amino-terminal PTHrP receptor, previously described in keratinocytes and squamous cell lines (67), or the novel splice variant of the type 1 receptor that lacks the signal peptide and displays low levels

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of cell surface expression (33), in the reuptake pathway. In contrast to the above reports, another study found nuclear import of the protein in both type 1 PTH receptor-positive and-negative cells, thus suggesting that a core motif within the PTHrP NLS is responsible for endocytosis and nuclear targeting of the protein (2). This argues for the existence of an as yet-unidentified cell surface receptor, distinct from the type 1 PTH receptor, that mediates ligand internalization. Second, PTHrP could be diverted away from the secretory route by retrograde translocation from the ER lumen to the cytosol (Fig. 3, pathway B). This novel

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,~.-preproPTHrP ions act in distinct but sometimes c o m p l e m e n t a r y ways to regulate these various biologic processes. T h e near constancy of C a 2+ o ensures the availability of Ca 2+ ions for their extracellular roles, such as serving as a cofactor for adhesion molecules, clotting factors, and o t h e r proteins and regulating cardiac contraction and n e u r o n a l excitability (1). F u r t h e r m o r e , salts of calcium and p h o s p h a t e form the mineral phase of bone, thereby providing both a rigid framework that affords protection of vital structures and enables locomotion a n d other bodily movements as well as serving as a large reservoir of these ions for times of n e e d (2). The cytosolic free calcium c o n c e n t r a t i o n (CaZi+), in contrast, is m a i n t a i n e d at a basal level of---100 n M but increases 10-fold or m o r e on cellular activation by extracellular signals acting on their respective cell surface r e c e p t o r s - - o w i n g to influx of C ao2+ a n d / o r release o f C a 2+ f r o m its i n t r a c e l l u l a r stores (3). C a 2+i plays pivotal roles in controlling cellular processes as diverse as muscular contraction, cellular motility, differentiation a n d proliferation, secretion of h o r m o n e s and o t h e r factors, and apoptosis (4). Because all intracellular C a 2+ ultimately originates from that p r e s e n t in the extracellular fluids (ECFs), the availability of a constant source The Parathyroids, Second Edition

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mammals and other free-living terrestrial organisms. This chapter also addresses these newly emerging relationships between Ca2o+ homeostasis and other physiologic regulatory mechanisms.

C L O N I N G OF THE CALCIUMSENSING R E C E P T O R Only a decade ago, the idea that there was a specific 2+ Ca o -sensing "receptor" was held by only a few investigators and was based largely on a body of indirect evi2+ dence in a very limited number of Cao-sensing cells, such as bovine parathyroid cells (1,6-8). The lack of direct evidence that this putative receptor actually existed necessitated undertaking a molecular cloning approach that utilized a bioassay detecting 2+ Ca o -sensing activitymnamely, expression cloning in Xenopus laevis oocytes. Racke et al. (9) and Shoback and co-workers (10) independently showed that X. laevis oocytes become responsive to Ca 2+ o -sensing receptor agonists after being injected with messenger RNA (mRNA) extracted from bovine parathyroid glands. Subsequently, Brown et al. used a similar assay to screen a bovine parathyroid cDNA library and isolated a single full-length, functional clone of the Ca 2+ o -sensing receptor (11). It was then possible to use traditional, hybridizaion-based methodologies to isolate cDNAs encoding CaRs from human parathyroid (12) and kidney (13), rat kidney (14), brain (viz. striatum) (15) and C cell (16), rabbit kidney (17), and chicken parathyroid (18) [for review, see (5)]. All are very similar in their predicted structures and are thought to represent the various tissue and species homologs of the same ancestral gene [for review, see (5)].

PREDICTED S T R U C T U R E A N D BIOCHEMICAL PROPERTIES OF THE CALCIUM-SENSING R E C E P T O R Predicted Structure o f the CaSR and Its H o m o l o g y to Other GPCRs The topology of the human parathyroid CaSR prot e i n ~ p r e d i c t e d from the nucleotide sequence of its cDNA--is shown in Fig. 1. It exhibits three principal structural domains. These are, respectively, its (1) large, 600-amino acid amino-terminal extracellular domain (ECD), (2) "serpentine" motif of seven membranespanning domains characteristic of the superfamily of G protein-coupled receptors (GPCRs), and (3) substantial carboxyl (C)-terminal tail of some 200 amino acids. Over the past decade, several different subfamilies of GPCRs have been identified that share the large

ECD exhibited by the CaSR as well as a modest (20-30%) degree of amino acid identity within their transmembrane domains (TMDs). These structurally related GPCRs are called the family C receptors (19) and contain three separate groups--the metabotropic glutamate receptors (mGluRs) (group I), the CaSR and a family of putative p h e r o m o n e receptors (group II), and the G protein-coupled receptors for y-amino butyric acid (GABA) or GABAB receptors (group III). The mGluRs are G protein-coupled receptors for glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS) (20), whereas the GABAB receptors are the GPCRs for GABA, the central nervous system's principal inhibitory neurotransmitter (21,22). The putative pheromone receptors (VRs) within the group II GPCRs are found exclusively in neurons of the rat vomeronasal organ (VNO) that express the guanine nucleotide regulatory (G) protein, G% (23). The VNO is a small sensory organ regulating instinctual behavior via input from environmental pheromones (23). Additional GPCRs closely related to the CaSR a n d / o r VRs, which are, respectively, taste and putative odorant receptors have been identified in mammals (24) and fish (25). They may represent evolutionary precursors of the pheromone receptors identified in rats, and they also exhibit the characteristic topology of the family C GPCRs. Thus all of the family C GPCRs seem to have as ligands small molecules that serve as environmental cues (i.e., pheromones) or are extracellular messengers within the CNS (e.g., glutamate or GABA) or systemic extracellular fluid (ECF) (viz. Ca2o+).As described below, the CaSR (and probably the other family C GPCRs as well) bind their respective ligands within their ECDsmin contrast to many other GPCRs, whose small ligands (e.g., epinephrine or dopamine) bind within the respective receptors' TMDs a n d / o r extracellular loops (ECLs). The "sensing" function of the ECD of the family C GPCRs likely has its origin in a class of extracellular binding proteins in bacteria (26)rathe periplasmic binding proteins (PBPs), which are receptors for a variety of small ligands, including ions (although these ions apparently do not include Ca2o+) and amino acids (27). The PBPs regulate bacterial chemotaxis toward these environmental substances and can participate in their cellular uptake by coupling the binding of a given ligand to its respective PBP to subsequent transport of that ligand by an associated transport system (27). Thus the family C GPCRs can be thought of as fusion proteins comprising an extracellular ligand-binding, "sensing" motif (the ECD) and a signal-transducing (e.g., serpentine) motif that couples the sensing process to intracellular regulators of various cellular functions (i.e., G proteins and their associated effector

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elements). Interestingly, these functions include the same ones regulated by the PBPs, namely, chemotaxis [e.g., of monocytes toward high levels of Cao2+ (28) ] and cellular transport [i.e., of C a 2+ o by CaSR-regulated, Ca 2+permeable channels (29)]. Furthermore, as described later (see Possible Additional Ca 2+ Sensors), the CaSR binds not only Ca2o+ but also several other ligands, including various amino acids ( 3 0 ) - - f u r t h e r supporting its functional and evolutionary relationships to the other members of the family C GPCRs and, ultimately, to the PBPs.

Biochemical Properties of the CaSR Studies utilizing chimeric receptors that comprise the ECD of the CaSR coupled to the TMDs and C tail of the mGluRs (and vice versa) have shown that C a 2+ o binds to the CaSR ECD (31). Studies have suggested that

residues within the CaSR ECD (e.g., Ser-147 and Ser170) may participate in the binding of C ao2+• These residues correspond to key amino acids thought to be involved in the binding of glutamate and GABA to the mGluRs and GABA B receptors, respectively (32). Given that the CaSR likely binds several calcium ions, however, because of its apparent "positive cooperativity" and the resultant steep slope of the curve describing the activation of the receptor by its various polycationic agonists (e.g., C a 2+ o and Mg2o+) (1), further work is n e e d e d to define the nature of these Ca2+o binding site(s). Of interest in this regard, the CaSR resides on the cell surface principally as dimers (33,34) that are linked by disulfide bonds within its ECD involving cysteines at amino acid positions 129 and 131 (35). Furthermore, functional interactions occur between the m o n o m e r i c subunits of these CaSR dimers, because two individually inactive CaSRs harboring mutations

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within different domains (e.g., the ECD and C tail) can recover substantial biological activity when they form heterodimers after being cotransfected into h u m a n embryonic kidney (HEK293) cells (36). That is, even though the individual CaSRs are inactive, they can "complement" one another's deficiencies to form a partially active heterodimer, presumably via intermolecular interactions. It is possible, therefore, that some of the receptor's apparent positive cooperativitymwhich is crucial to ensure that the CaSR responds over the narrow range of Ca2o+ that regulates, for example, PTH secretion (see later)mresults from the presence of 2+ • Ca o -binding sites on both ECDs of the dimer a n d / o r even at the site(s) where the two ECDs within the dimer interact with one another. Ultimately, solving the three-dimensional structure of the CaSR ECD by X-ray crystallography will shed a great deal of light on how the CaSR binds C a 2+ o and its other polycationic ligands, including the locations, numbers, and interactions, if 2+ • any, between these Ca o -binding sites. The ECD of the cell surface form of the CaSR is extensively N-glycosylated with complex carbohydrates (37), and eight of the predicted N-glycosylation sites within the h u m a n CaSR ECD are efficiently glycosylated (38). Disruption of four to five of these sites reduces cell surface expression of the receptor by 50-90%. Thus, glycosylation of at least three of the sites is essential for cell surface expression, although glycosylation per se is not critical for CaSR binding of Ca~o+ and its subsequent activation of signal transduction (38). Within its intracellular loops and C-terminal tail, the h u m a n CaSR has five predicted protein kinase C (PKC) and two predicted protein kinase A (PKA) phosphorylation sites (12,39). The functional importance of the PKA phosphorylation sites is unknown. Activation of PKC inhibits CaSR-induced stimulation of phospholipase C (PLC), and studies using site-directed mutagenesis have revealed that phosphorylation of a single PKC site within the CaSR C-terminal (C) tail (Thr-888) accounts for most of the PKC-mediated modulation of the receptor's function (39). PKC-evoked phosphorylation of the CaSR C tail, therefore, may provide a mechanism for negative feedback control of its coupling to PLC, whereby PLC-mediated activation of protein kinase C m b y phosphorylating the receptor at Thr888mlimits further activation of this pathway.

I N T R A c E L L u L A R S I G N A L I N G BY T H E CaSR CaSR agonists activate phospholipases C, A 2 (PLA2), and D (PLD) in bovine parathyroid cells as well as in HEK293 cells stably transfected with the CaSR (40).

These actions are most likely mediated by the CaSR in both cell types, because high C a 2+ o no longer produces them in nontransfected HEK293 cells, which do not express an endogenous CaSR, or in parathyroid cells maintained in culture for 3-4 days, in which the level of CaSR expression diminishes by about 80% (41,42). In most cells, CaSR-elicited activation of PLC involves the pertussis toxin-insensitive G protein, Gq (43), although in some it occurs via pertussis toxin-sensitive pathways, likely involving one or more isoforms of the G protein, G i (44). In bovine parathyroid and CaSR-transfected HEK293 cells, activation of PLA 2 and PLD involve PKCd e p e n d e n t pathways that are activated by the CaSR, presumably via PLC (40). The high Ca 2+ • • o -elicited, transient rise in C a 2+ i in bovine parathyroid cells and CaSR-transfected HEK293 cells probably results from the activation of PLC (40) and the resultant IP~-mediated release of Ca 2+ from its intracellular stores (37). High Ca2o+ likewise evokes sustained increases in CaZi+ in both CaSR-transfected HEK293 cells (37) and bovine parathyroid cells (1) through incompletely defined CaZo+ influx pathways. We have shown using the patch-clamp technique that 2+ the CaSR enhances the opening of a Ca o -permeable, nonselective cation channel (NCC) in CaSR-transfected HEK cells (45). A similar NCC is present in bovine parathyroid cells and is activated by high Ca2o+, presumably through a CaSR-dependent pathway (29), suggesting that it could contribute to the high Cao2+-elicited, • sustained elevation in C ai2+ in these cells (46). High C a 2+ o markedly inhibits agonist-stimulated cAMP accumulation in bovine parathyroid cells (47). This action is thought to involve direct inhibition of adenylate cylcase via one or more isoforms of G i, because it is pertussis toxin sensitive (47). In other cells, however, high Ca 2+ o -evoked, CaSR-mediated inhibition of cAMP accumulation can involve indirect pathways, such as suppression of the activity of a 2+. Ca i-lnhibitable isoform of adenylate cyclase by the associated rise in CaZi+ (48). The CaSR also stimulates mitogen-activated protein kinase (MAPK) activity in several cell types, including rat-1 fibroblasts (49), ovarian surface cells (50), and CaSR-transfected but not nontransfected HEK293 cells (50a). As has been described with other GPCRs, the CaSR activates MAPK via PKC- and tyrosine kinase-dependent pathways involving c-Src-like cytoplasmic forms of the latter enzyme, which are downstream of Gq a n d / o r G i, respectively (49,50). That is, in CaSR-transfected HEK293 cells, PKC inhibitors reduce high Ca o2+_evoked, CaSR-mediated activation of MAPK by about 50%, and the remaining activation of the enzyme can be largely abolished by the further addition of pertussis toxin a n d / o r tyrosine kinase inhibitors (50a).

o RECEPTORS/SIGNALING FOR C a 2+

THE CALCIUM-SENSING R E C E P T O R GENE A N D ITS R E G U L A T I O N Very little is currently known about the structure of the CaSR gene and the factors that control its expression. The h u m a n CaSR gene resides on the long arm of chromosome 3 as d o c u m e n t e d by linkage analysis (51) and in band 3q13.3-21 as determined by fluorescent in situ hybridization (52), whereas the rat and mouse CaSR genes, respectively, reside on chromosomes 11 and 16 (52). The h u m a n CaSR gene has seven e x o n s m the first encodes upstream untranslated regulatory regions, the next five code for various portions of the ECD, and the last encodes the r e m a i n d e r of the CaSRmfrom its first TMD to the C terminus (53). Understanding the upstream regulatory regions of the CaSR gene will be of substantial interest, because expression of the CaSR can change in a variety of circumstancesmsome of which are described below. Several factors are associated with increased expression of the CaSR gene. Both high Ca2+o and 1,25(OH)zD ~ can up-regulate expression of the CaSR gene in certain cell types [e.g., adrenocorticotropin h o r m o n e (ACTH)-secreting, pituitary-derived AtT-20 cells (44) and in both rat kidney and parathyroid (54), respectively]. Interleukin-l[3 modestly raises the level of CaSR mRNA in bovine parathyroid gland fragments, which could contribute to the associated decrease in PTH secretion (55). In the kidney there is substantial up-regulation of the CaSR during the peri- and postnatal periods, and the resultant higher level of CaSR expression persists t h r o u g h o u t adulthood (56). There is also a developmental increase in CaSR expression in the brain, but in contrast to that occurring in the kidney, the increase in the brain takes place about 1 week postnatally (57). Furthermore, the increased CaSR expression in the brain is transient, and it decreases severalfold about 2 weeks later, reaching a lower level that remains stable thereafter (57). The biologic significance of these developmental changes in CaSR expression are unknown. Conversely, CaSR expression decreases in several circumstances. Calf parathyroid cells show a rapid and marked (80-85%) decrease in CaSR expression after they are put in culture (41,42), which likely is a major factor contributing to the concomitant reduction in high 2+ Ca o -evoked suppression of PTH release. The level of expression of CaSR in the kidney is also decreased in chronic renal insufficiency induced in the rat by subtotal nephrectomy (58). This change in the level of CaSR expression could potentially contribute to the hypocalciuria that occurs in the setting of renal insufficiency, because reduced renal CaSR expression a n d / o r activity are associated with increased tubular reabsorption

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131

of C a 2+ (59). Because, as noted above, 1,25(OH)2D ~ increases renal CaSR expression (54), the decrease in the receptor's expression with impaired renal function could result, in part, from the associated reduction in circulating 1,25(OH)zD ~ levels (2). The detailed mechanisms underlying these changes in the expression of the CaSR gene, however, including the relative contributions of changes in gene transcription and posttranscriptional mechanisms, require additional investigation.

ROLES OF THE CALCIUM-SENSING R E C E P T O R IN TISSUES M A I N T A I N I N G Ca2o+ HOMEOSTASIS Parathyroid The parathyroid glands of humans (60), rats (61), mice (62), rabbits (17), and chickens (18) express a b u n d a n t CaSR mRNA and protein. Several lines of evidence support the CaSR's role as the key mediator of the inhibitory action of elevated C a 2+ o on PTH secretion, although the principal intracellular signaling mechanisms through which it exerts this action remain uncertain [for review, see (63)]. As described above, the reduction in CaSR expression in cultured parathyroid cells is associated with loss of inhibition of PTH secretion by high Ca o2+ (41). Furthermore, humans with familial hypocalciuric hypercalcemia (FHH), who are heterozygous for naturally occurring, inactivating mutations of the CaSR gene (59), or mice heterozygous for targeted disruption of this gene (62) show modest right-shifts in their relationships between C a 2+ o and inhibition of PTH secretion, indicative of "resistance" to Ca2o+. Humans and mice homozygous for such defects (59,62), in turn, show m u c h more severe i m p a i r m e n t of high Ca 2+. o -induced suppression of PTH release, docu m e n t i n g that this parathyroid "C a 2+ o resistance" is inversely related to the n u m b e r of normally functioning CaSR alleles. Thus mice with "knockout" of the CaSR gene as well as the naturally occurring CaSR knockout in humans prove the central role of the CaSR 2+ in Ca o -regulated PTH secretion. Another feature of parathyroid function that is likely CaSR regulated is PTH gene expression. Garrett et al. (64) showed in preliminary studies that the calcimimetic CaSR activator, NPS R-568, which activates the receptor by an allosteric mechanism involving an increase in the receptor's apparent affinity for Ca2o+, decreases the level of PTH mRNA in bovine parathyroid cells. Finally, the CaSR appears to inhibit parathyroid cellular proliferation tonically, because humans homozygous for inactivating CaSR mutations (59) or mice homozygous for targeted disruption of the CaSR gene (62) show m a r k e d

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parathyroid cellular hyperplasia. Moreover, treatment of rats with experimentally induced renal impairment with NPS R-568 prevents the parathyroid hyperplasia that otherwise occurs in this setting (65), providing additional evidence that CaSR activation inhibits parathyroid cellular proliferation.

C Cells In contrast to PTH release, CT secretion is stimulated by elevating Ca2o+, similar to the more classic, positive relationship between Ca 2+ and exocytosis in most other hormone-secreting cells (1,2). This was one of 2+ several lines of indirect evidence that the Ca o -sensing mechanism in C cells might differ fundamentally from that in parathyroid cells. Recent studies, however, have shown that rat, human, and rabbit C cells express CaSR mRNA and protein (16,17,66). Moreover, cloning of the CaSR gene from a rat C cell tumor line revealed that it was identical to that expressed in rat kidney (16). It is currently thought, therefore, that the CaSR is the mediator of the stimulatory action of high Ca2o+ on CT secretion, although studies have not been p e r f o r m e d in humans or mice with knockout of the CaSR gene, for example, to establish this point definitively. Tamir and co-workers have provided evidence that the following sequence of events underlies CaSR-stimulated CT secretion (67). High C a 2+ o initially activates phosphatidylcholine-specific PLC, which provides a source of diacylglycerol for PKC-induced activation of an NCC. The latter allows cellular uptake of Na + and Ca 2÷, thereby producing cellular depolarization and resultant activation of voltage-gated, principally L-type, C a 2+ channels, elevating CaZi + a n d stimulating exocytosis of 5-hydroxytryptamine- and CT-containing secretory vesicles. Kidney In the adult rat kidney, the CaSR is expressed in almost all segments of the nephron, with the highest levels at the basolateral m e m b r a n e of the cortical thick ascending limb (CTAL) (68), which reabsorbs divalent minerals in a regulated m a n n e r (69,70). The CaSR is also expressed basolaterally in the distal convoluted tubule (DCT), where the tubular reabsorption of CaZ+~like that in C T A L ~ i s known to be stimulated by PTH. Additional sites of expression of the receptor include the base of the brush border of the proximal tubule (68), the basolateral m e m b r a n e of the cells of the medullary thick ascending limb (MTAL) (68), and the luminal surface of the inner medullary collecting duct (IMCD) (71). None of these sites is directly involved in renal Ca2o+ handling, although CaSRs in these n e p h r o n segments may regulate the handling of

other solutes a n d / o r water. For instance, in the proximal tubule, it is conceivable that the CaSR mediates the direct phosphaturic action of raising Ca2o+ (72), which might contribute to the reduction in serum phosphate levels in patients with hypoparathyroidism treated with vitamin D and calcium supplementation (2). As will be described in more detail below, the CaSR in CTAL, in addition to regulating reabsorption of Ca 2+ and Mg 2+, also modulates renal handling of Na +, K +, and C1- (73). Finally, as is discussed later, the CaSR in the IMCD is thought to mediate the well-known action of high GaZeo to inhibit vasopressin-stimulated water reabsorption (71,74), which contributes to the defective urinary concentrating capacity in hypercalcemic patients (5,59). The localization of the CaSR in the basolateral membrane of the CTAL suggests that it could mediate the previously demonstrated inhibitory action of elevated peritubular but not luminal Ca2o+ on Ca 2+ and Mg 2+ reabsorption in perfused tubules from this n e p h r o n segment (70). Figure 2 illustrates schematically how the CaSR likely acts at a cellular level to inhibit PTHstimulated reabsorption of divalent cations in the CTAL. As shown in detail in Fig. 2, it acts in a "lasixlike" m a n n e r to inhibit the overall activity of the Na/K/2C1 cotransporter that generates the lumen positive potential normally driving the passive paracellular reabsorption of about 50% of NaC1 and nearly all of the C a 2+ and Mg 2+ in this n e p h r o n segment (75). Interestingly, persons with F H H have a markedly reduced capacity to up-regulate their urinary excretion of C a 2+ in response to hypercalcemiameven after total parathyroidectomy (76). Therefore, there is a PTH-independent, overly avid reabsorption of C a 2+ in F H H that likely results, at least in part, from a reduced n u m b e r of normally functioning CaSRs in the CTAL, thereby rendering the tubule "resistant" to Ca2o+ and limiting the capacity of elevated levels of Ca2o+ to reduce tubular reabsorption in this n e p h r o n segment (59). Furthermore, in normal persons the hypercalciuric action of hypercalcemia results from at least two distinct CaSR-mediated actionsm(1) inhibition of PTH secretion and (2) direct suppression of tubular reabsorption of C a 2+ in the CTAL. It is not currently known if the CaSR modulates PTH-stimulated Ca '~+ reabsorption in the DCT.

Intestine The intestine is an important participant in the maintenance of Ca2o+ homeostasis owing to its capacity for regulated absorption of dietary Ca2o+ in response to active metabolites of vitamin D (1,2). The d u o d e n u m is the major site for 1,25-dihydroxyvitamin D3-dependent intestinal Ca 2+ absorption, involving a process of active transport that most likely includes the vitamin D-dependent CaZ+-binding protein, calbindin. In con-

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trast, the j e j u n u m and ileum not only absorb lesser amounts of C a 2+ but also s e c r e t e Ca 2+, which may enable chelation of fatty acids and bile acids, thereby forming insoluble "calcium soaps" that mitigate potential damage to colonic epithelial cells from soluble, unchelated fatty acids and bile acids. Even though the major function of the colon in fluid and electrolyte metabolism is to absorb water and Na +, it nonetheless absorbs significant amounts of Ca 2+ by both vitamin D-dependent and -independent routes, particularly in its proximal segments (77). The CaSR is expressed in all segments of rat intestine and at the highest levels on the basal aspect of the absorptive cells of the small intestinal villi and the crypt cells of the small intestine a n d colon, and in the enteric nervous system (78). Does the CaSR in any of these locations participate in systemic Ca 2+ o homeostasis? The CaSR in the enteric nervous system, which controls gastrointestinal secretomotor functions, could conceivably mediate the known actions of high and low Ca2o+ (e.g., in hyper- and hypocalcemic individuals) to reduce and increase GI motility, respectively. Such an action on intestinal motility, however, even if CaSR-mediated, has no known relevance to systemic C a 2+ o homeostasis. Available evidence, however, is consistent with a role for the CaSR in regulating intestinal C a 2+ absorption. Hypercalcemia inhibits the absorption of dietary Ca 2+ (79). Furthermore, direct measurement of the level of Ca2o+ in the interstitial fluid beneath the small intestinal absorptive epithelial cells has revealed that Ca2o+ increases by nearly two fold when luminal C a 2+ o is elevated to the levels known to be reached following • . intake of C a 2 + -containing foods (80). This level of

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FIG. 2 Possible mechanisms by which the CaSR controls intracellular second messengers and ionic transport in CTAL. Hormones elevating cAMP (e.g., PTH) enhance paracellular Ca 2÷ and Mg 2÷ reabsorption by stimulating the NaK2CI cotransporter and an apical K ÷ channel and by increasing Vt. The CaSR, likewise on the basolateral membrane, stimulates PLA 2 (2), increasing free arachidonic acid, which is metabolized by the P450 pathway to an inhibitor of the apical K ÷ channel (4) and, perhaps, the contransporter (3). Both actions decrease overall cotransporter activity, reduce Vt, and, therefore, diminish paracellular divalent cation transport. The CaSR also inhibits adenylate cyclase (1) and, therefore, hormone-stimulated divalent cation reabsorption. Reprinted from B o n e , Vol. 20; Brown EM, Hebert SC. Calcium-receptor regulated parathyroid and renal function; pp. 303-309. Copyright 1997, with permission from Elsevier Science.

Ca2o+ would be more than sufficient to activate CaSRs resident on the basal aspect of the small intestinal absorptive cells. It is possible, therefore, that a homeostatically relevant, negative feedback control of intestinal C a 2+ absorption takes place via local increases in C a 2+ o occurring as a result of the absorptive process per se or increases in the systemic level of Ca2o+. It is not currently known whether the CaSR regulates the secretion of C a 2+ o and other solutes by the small intestinal a n d / o r colonic crypts, although hypercalcemia has been found to stimulate intestinal calcium secretion in some studies (79):

Bone and Cartilage The levels of C a 2+ o within the bony microenvironm e n t are likely to vary substantially owing to the regulated turnover of the skeleton through osteoclastic resorption of small portions of bone followed by their replacement by bone-forming osteoblasts~a process totally replacing the h u m a n skeleton about every 10 years (2). Indeed, the levels o f Ca2o+ beneath an actively resorbing osteoclast can be as high as 8-40 m M (81). Furthermore, Ca2o+ exerts a variety of actions on the functions of bone cells in vitro that may serve physiologically useful functions, although it has not yet been proved that these same actions take place in vivo. For instance, high Ca2+o enhances osteoblastic functions that could promote their recruitment to sites of future bone formation, such as chemotaxis and proliferation, a n d / o r p r o m o t e their differentiation to mature osteoblasts [for review, see (82,83)]. In addition, high Ca2o+ inhibits both the formation (84) and activity (85) of osteoclasts in vitro. Thus C a 2+ o exerts effects on

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osteoblasts and osteoclasts a n d / o r their precursors that are homeostatically appropriate. For instance, increasing Ca2+owould promote net movement of calcium into the skeleton by enhancing bone formation and inhibiting its breakdown in the short and longer term. Moreover, locally high levels of Ca o2+generated by osteoclasts at sites of bone resorption could potentially contribute to the "coupling" of bone resorption to the subsequent replacement of the missing bone by osteoblasts, by amplifying and recruiting the resident pool of preosteoblasts to such sites and promoting their differentiation to mature osteoblasts (82,83). As described below, however, the molecular nature of the 2+ Ca o -sensing mechanisms in bone cells remains uncertain, although the CaSR is expressed in at least some bone cells, and therefore, could potentially participate in this process. A substantial body of indirect evidence accumulated prior to and around the time of the cloning of the 2+ CaSR suggested that the Ca o -sensing mechanisms in osteoblasts and osteoclasts differed in their pharmacologic and certain other properties from those of the CaSR [for reviews, see (82,85)]. In addition, some investigators have failed to detect the expression of CaSRs in osteoblast- (86) and osteoclast-like cells (87). Other studies, however, have provided strong evidence that the CaSR is expressed by a variety of cells that originate from the bone and bone marrow, including various hematopoietic precursors (88), at least some osteoblast-like and osteoclast-like cell models in vitro, and cells of both lineages in situ within sections of bone [for review, see (83)]. The ST-2 stromal cell line (89), which is derived from the same mesenchymal stem cells that serve as progenitors for osteoblasts, expresses CaSR mRNA and protein, as do several osteoblast-like cell lines, including SaOS-2, MC-3T3-E1, UMR-106, and MG-63 cells (90-92). Moreover, Chang et al. have shown expression of both CaSR mRNA and protein in most osteoblasts in sections of murine, rat, and bovine bone (92). With regard to cells of the osteoclast lineage, most h u m a n peripheral blood monocytes, which are known to contain osteoclast precursors, express a b u n d a n t CaSR mRNA and protein (93). Preosteoclastlike cells formed in vitro also expresses the CaSR (84), as do osteoclasts isolated from rabbit bone (94). In murine, rat, and bovine bone sections, however, only a minority of multinucleated osteoclasts expressed CaSR mRNA and protein (92). Further studies are needed, therefore, to clarify whether only osteoclast precursors and not mature osteoclasts express the CaSR. Moreover, additional work is n e e d e d in which the activity of the CaSR in bone cells a n d / o r their precursors is blocked using genetic a n d / o r pharmacologic approaches in order to clarify whether the CaSR that has been found to be expressed in at least

some cells of both lineages is the actual mediator of some or even all the effects of high Ca2o+ on these cells. One study, which failed to detect the CaSR in transformed osteoblast-like cells derived from either wildtype mice or those with knockout of the CaSR, found that these cells still exhibited responses to Ca2o+ and 2+ A1~+, suggesting the presence of another Ca o -sensing receptormas has been postulated by the same workers in earlier studies (95). Although the cartilage-forming chondrocytes do not directly participate in systemic C a 2+ o homeostasis, they play a key role in the growth of the skeleton by providing a cartilaginous model of the future skeleton that is gradually replaced by bone. Moreover, the cartilaginous growth plate represents a site where longitudinal growth takes place until the skeleton is fully mature at the end of puberty. The availability of C a 2+ is known to be important to ensure proper growth and differentiation of cartilage cells and resultant skeletal growth in vivo (96,97). In addition, altering Ca2o+ modulates the differentiation a n d / o r other functions of cells of the cartilage lineage (98,99). The latter arise from the same mesenchymal stem cell, giving rise to osteoblasts, adipocytes, smooth muscle cells, and fibroblasts (100,101). It is of interest, therefore, that the rat cartilage cell line, RCJ3.1C5.18, expresses CaSR mRNA and protein (102). Furthermore, various types of cartilage cells within intact bone also express CaSR mRNA and protein, including the hypertrophic chondrocytes within the growth plate that participate importantly in long bone growth (92). Therefore, the CaSR could potentially mediate some or all of the previously demonstrated direct actions of Ca2o+ on chondrocytes and cartilage growth. Indeed, raising the level of Ca2o+ exerts several direct actions on RCJ3.1C5.18 cells, including dose dependently decreasing the levels of the mRNAs encoding a major proteoglycan in cartilage, aggrecan, as well as the ~x1 chains of types II and X collagen and alkaline phosphatase (102). Moreover, treating the cells for 48-72 hours with a CaSR antisense oligonucleotide lowered the level of the CaSR protein significantly and p r o m o t e d increased expression of aggrecan mRNA (102), consistent with a mediatory role of the CaSR in the regulation of this gene. These results indicate, therefore, that (1) Ca2o+ regulates the expression of several biologically important genes in this chondrocytic cell line, (2) cartilage cells express the CaSR, and (3) the receptor mediates some or all of these actions of Ca2o+ in the RCJ3.1C5.18 cartilage cell model. Thus the CaSR potentially not only regulates bone turnover a n d / o r the coupling of bone resorption to bone formation through effects on bone cells a n d / o r their precursors, but may also regulate skeletal growth through its actions on chondrocytes.

RECEPTORS/SIGNALING FOR Ca2o+ /

THE CALCIUM-SENSING RECEPTOR AND T H E I N T E G R A T I O N OF CALCIUM A N D WATER M E T A B O L I S M Hypercalcemic patients not infrequently have abnormally decreased urinary concentrating capacity and, occasionally, frank nephrogenic diabetes insipidus (103,104). The presence of the CaSR in several n e p h r o n segments participating in the urinary concentrating mechanism (68,71) has provided novel insights into the likely mechanism(s) underlying the long-recognized but poorly understood inhibitory effects of high C a 2+ o on this parameter of renal function. As noted above, high Ca2o+ levels, probably by activating CaSRs present on their apical membranes, reversibly inhibit vasopressin-elicited water flow by about 35-40% in the IMCD (71). The CaSR has been shown to be present within the same apical endosomes containing the vasopressin-regulated water channel, aquaporin-2 (71). This observation suggests that the CaSR could potentially reduce vasopressin-stimulated water flow in the IMCD by either stimulating the endocytosis or inhibiting the exocytosis of these endosomes out of or into the apical plasma m e m b r a n e , respectively (71). Moreover, inducing chronic hypercalcemia in rats by treatment with vitamin D decreases aquaporin-2 expression (74), which would further diminish vasopressinstimulated water flow in the terminal collecting duct. In addition to the mechanisms just described, high Ca o -elicited, CaSR-mediated inhibition of NaC1 reabsorption in the MTAL (105,106), by diminishing the magnitude of the medullary c o u n t e r c u r r e n t gradient, would be expected to reduce further the maximal urinary concentrating power of hypercalcemic persons (Fig. 3). What is the evidence that these various actions of high Ca2o+ are mediated via the CaSR? Interestingly, individuals with inactivating mutations of the CaSR concentrate their urine normally despite their hypercalcemia (107), probably because they are "resistant" to the usual inhibitory actions of C a 2+ o on the urinary concentrating mechanism. In contrast to persons with F H H or mice with targeted disruption of the CaSR gene, families have been defined in which activating mutations of the CaSR produce a form of autosomal d o m i n a n t hypocalcemia (59). Because parathyroid and kidney are "overresponsive" to the usual actions of C a 2+ o in such families, the Ca2+o homeostatic system is "reset" to maintain stable hypocalcemia in association with relative hypercalciuria (e.g., affected persons have hypercalciuric hypocalcemia). In contrast to persons with inactivating CaSR mutations, those with activating mutations can develop symptoms of diminished urinary concentrating capacity even at normal levels of C a 2+ o when treated with vitamin D and calcium supplementation, 2+

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probably because their renal CaSRs involved in urinary concentration are overly sensitive to Ca2o+ (108). Is there any physiologic relevance to the defective renal handling of water in hypercalcemic patients? We have previously postulated that it affords a mechanism for integrating the renal handling of divalent cations, particularly C a 2+, and water, thereby allowing appropriate "trade-offs" in how these aspects of renal function are regulated u n d e r specific physiologic conditions (73). For instance, when a systemic C a 2+ load must be disposed of, a CaSR-mediated increase in urinary C a 2+ excretion ensues owing to reduced tubular reabsorption of C a 2+ in the CTAL. The resultant increase in the luminal levels of Ca2o+ in the IMCD, especially in a dehydrated person, might predispose to forming Ca2+-containing renal stones if it were not for the associated high Ca 2+. o -induced, presumably CaSRmediated, inhibition of maximal urinary concentration. In addition, a b u n d a n t CaSRs are expressed in the subfornical organ (SFO) ( 1 0 9 ) m a n important thirst center (110)mwhich may provide an additional layer of integration of C a 2+ o and water homeostasis. That is, a Ca2o+ -induced, CaSR-mediated increase in drinking owing to activation of CaSRs in the SFO could prevent dehydration that might otherwise result from loss of free water in the kidney because of concomitant inhibition of the urinary concentrating mechanism (Fig. 3). Finally, prior studies have docum e n t e d the existence of a specific "calcium appetite" in rats (111) that could provide a mechanism for a physiologically appropriate modulation of the intake of calcium-containing food during hypo- and hypercalcemia. Some reduction in the intake of Ca2o+containing foods would presumably also result from activation of CaSRs in the area postrema of the b r a i n m a "nausea center" (109)mowing to the resultant anorexia/nausea. We postulate, therefore, that multiple layers of CaSR-mediated integration and coordination participate in the regulation of water and calcium metabolism, serving to optimize the capacity of terrestrial organisms to adapt to their intermittent access to dietary calcium and water (73). The modulation of vasopressin-mediated water flow in the IMCD can be thought of as an example of"local" C a 2+ o homeostasis, whereby Ca2o+ within a restricted microenvironment outside of the general ECF is only allowed to rise to a certain maximal level (5). Of interest, whereas alterations in Ca2o+ made by the system governing systemic C a 2+ o homeostasis are usually accomplished principally by adjusting the m o v e m e n t of C a 2+ into or out of the ECF (e.g., by intestine, kidney, or bone) (1), the adjustment of Ca2o+ in the IMCD takes place via alterations in the movement of water. It seems possible, however, that one function of the increased thirst in hypercalcemic patients,

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DecreasedCa2÷ • Reabsorption, Increased Ca2+Excretion J • •

Decreased Urinary Concentration

icreased Urinal Ca2÷Excretion Without Risk of Stones Decreased Gut Motility, Increased H20 Absorption FIG. 3 Possible mechanisms that interrelate systemic Ca2o+ and water homeostasis (see text for further details). (a) Renal mechanisms through which the CaSR decreases maximal urinary concentrating ability. Reproduced with permission from Brown EM, Hebert SC. Novel insights into the physiology and pathophysiology of Ca 2+ homeostasis from the cloning of an extracellular Ca2+-sensing receptor. Regulatory Peptide Letter 1997;VI1:43-47. (b) Activating CaSRs in the SFO could enhance water intake, and mitigate loss of free water that otherwise would result from diminished urinary concentration. Reproduced with permission from Brown EM, Harris HW, Jr, Vassilev P, Hebert SC. The Biology of the extracellular Ca 2+sensing receptor. In: Bilezikian JP, Raiscz LG, Rodan GA, eds. Principles of bone biology, San Diego:Academic Press.

in a d d i t i o n to p r o v i d i n g m o r e free water to r e d u c e the level of C a 2+ o in the IMCD, is to dilute C a 2+ o in t h e ECF as well. T h e r e f o r e , p e r h a p s the i n t e g r a t i o n

o f calcium a n d water m e t a b o l i s m is n o t limited to regu l a t i n g the level of Ca2o+ within the u r i n e b u t also that in b l o o d .

RECEPTORS/SIGNALINGFORC a 2+ o / OTHER CALCIUM-SENSING RECEPTOR A G O N I S T S A N D M O D U L A T O R S - - T H E CaSR AS A N I N T E G R A T O R O F P H Y S I O L O G I C S I G N A L S A N D AS A " N U T R I E N T R E C E P T O R " Agonists and Activators o f the CaSR O t h e r T h a n C a 2+ o A variety of divalent cations (Sr2o+), trivalent cations (e.g., Gd~o+), and even organic polycations [i.e., neomycin and spermine (112) ] are effective agonists of the CaSR, probably interacting with the receptor's ECD [for review, see (5)]. Only a few of these, however, are likely to be present within biologic fluids at concentrations that would activate the receptor (112). In addition to Ca2o+, Mg2o+ and spermine are two such polycationic agonists of the CaSR. It is likely that in certain microenvironments, such as in the gastrointestinal tract and CNS, the levels of spermine are sufficiently high to activate the CaSR even at levels of C a 2+ o that would not by themselves do so (5,112). Indeed, all polycationic CaSR agonists potentiate one anothers' actions on the receptor, so that only small increases in any given agonist (i.e., spermine) may be n e e d e d to activate the CaSR in the presence of a sufficient level of another agonist that is present in the m i c r o e n v i r o n m e n t (e.g., Ca2o+) (5). Some support for the role of the CaSR in "setting" Mg2o+ comes from the observation that persons with inactivating mutations of the CaSR tend to have mild 2+ whereas those with activating mutaincreases in M go, tions can have reductions in Mg2o+(59). The potency of M go2+ is about twofold less than that of Ca 2+ o on a molar basis in activating the CaSR' (11,17). Because circulating levels of Mg2o+ are, if anything, lower than those for Ca2o+ (2) , one might wonder how M go2+ could regulate its own homeostasis via changes in PTH secretion. It appears m o r e likely that M go2+ acts via the CaSR in the CTAL to regulate its own level in the ECF, because Mg2o+ is reabsorbed to a lesser extent than other solutes in proximal n e p h r o n segments, resulting in 1.6- to 1.8-fold increases in Mg2o+ in the tubular fluid of the CTAL (70). The latter levels should be sufficiently high to inhibit Mg2o+ reabsorption in this n e p h r o n segment, because raising not only Ca2o+ but also Mg2o+ inhibits the reabsorption of both divalent cations in perfused CTAL (70). Another ionic factor that modulates the effects of Ca2o+ and other polycationic agonists on the CaSR is a change in ionic strength per se (e.g., via alterations in the concentration of NaC1) (113). Increases in ionic strength reduce, and decreases in ionic strength enhance, the sensitivity of the CaSR to activation by C a 2+ o . The impact of changes in ionic strength on the CaSR may be particularly relevant in specific microenvironments, such as in the urinary or gastroin-

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testinal tracts, where this p a r a m e t e r can vary over a wide range (113).

P o s s i b l e R o l e o f the CaSR as a "Nutrient-Sensing" Receptor Calcimimetics are prototypical "modulators" of the CaSR that activate the receptor only in the presence of C a 2+ o, as opposed to the polycationic agonists of the CaSR (e.g., Gdo3+),which can activate it even in the total absence of Ca O2+ (114) ° Recent studies have identified another class of endogenous modulators of the CaSRmnamely, various amino acids (30). Although individual amino acids only activate the receptor in the presence of ~>1 m M Ca2o+ and are of relatively low potency (e.g., acting in the range of 0.1-1 m M o r higher), a mixture of amino acids emulating that present in h u m a n serum u n d e r fasting conditions substantially shifts the receptor's sensitivity to C a o2+. Moreover, changes in the levels of the amino acid mixture above and below this fasting level have readily detectable effects on the function of the CaSR (30). Although the implications of the direct effects of amino acids on the CaSR are far from clear, they may help to explain several long-standing observations linking protein and C a 2+ o metabolism and suggest future avenues for research into the possible role of the CaSR as a "nutrient-sensing" receptor. For instance, highprotein diets substantially increase urinary calcium excretion (115). Although this effect has traditionally been ascribed to buffering of the acidic products of protein metabolism by bone and direct calciuric actions of the acid load (116), perhaps direct activation of CaRs in the CTAL contributes as well. Conversely, a reduction in dietary protein has been shown to increase serum PTH levels up to twofold in normal women (117). Could it be that the latter effect results from decreased inhibition of PTH secretion owing to reduced circulating levels of amino acids and that the reduced intake of dietary prot e i n that is a standard part of the therapy of patients with chronic renal insufficiency (2) contributes to the secondary hyperparathyroidism in the latter setting? Viewing the CaSR as not only a "homeostatic" receptor for C a 2+ o but as a nutrient receptor, sensing not only C a 2+ o and M go2+but also amino acids, may enable the formulation and testing of novel hypotheses directed at understanding, for instance, the link between the needs of the organism for both protein and mineral ions during growth. Skeletal growth in childhood involves laying down both a protein and a mineral phase in bone as well as growth of soft tissuesmall of which contain varying mixtures of mineral ions and protein. Indeed, in the GI tract, the presence of an amino acid receptor regulating secretion of gastrin, gastric acid, and cholecystokinin has been postulated, and the pharmacology for

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the effects of various a m i n o acids on these parameters is remarkably similar to that for the effects of the same amino acids on the CaSR (118-120). CaSRs in the gastrointestinal system could represent a particularly suitable target for the sensing of the intake of protein and mineral ions, which are, of course, generally ingested together (i.e., in milk). F u r t h e r studies are needed, therefore, to d e t e r m i n e w h e t h e r the CaSR, in fact, represents the putative a m i n o acid receptor, and w h e t h e r recognition by this receptor of amino acids in the context of C a 2+ o and M go2+ within the gastrointestinal tract and elsewhere represents the molecular basis for a physiologically relevant link between protein and mineral ion metabolism.

POSSIBLE ADDITIONAL

Ca2o+ S E N S O R S

As n o t e d above and described in detail elsewhere (82,85), there may be C a 2+ o sensors in addition to the CaSR in osteoblasts and osteoclasts. F u r t h e r m o r e , additional work has revealed that some mGluRs can sense Ca2o+ in addition to r e s p o n d i n g to glutamate as their principal physiologic agonist, although the physiologic relevance of this Ca2o+ sensing is not yet clear. Kubo et al. (121) d e m o n s t r a t e d that mGluRs 1, 3, and 5 sense Ca2o+ over a range of a b o u t 0.1 to 10 mM, whereas mGluR2 is considerably less responsive to changes in C a 2+ o . M1 three of the mGluRs that sense Ca2o+ have identical serines and threonines, respectively, at amino acid positions equivalent to residues 165 and 188 in m G l u R l a (32). These two residues play key roles in the binding of glutamate to the ECDs of the mGluRs (26). In contrast, t h o u g h mGluRs la, 3, and 5 have a serine at a position equivalent to residue 166 in m G l u R l a , mGluR2 has an aspartate in this position (121). Changing this serine to an aspartate in mGluRs la, 3, and 5 considerably reduces their capacities to sense Ca2o+, but replacing the aspartate in mGluR2 with a ser2+ ine increases its a p p a r e n t affinity for Ca o i n t o a level similar to those of mGluRs 1, 2, and 5 (121). Therefore, the serines at a m i n o acid position 166 in m G l u R l a and the equivalent positions in mGluRs 3 and 5 apparently play key roles in the capacities of these receptors to sense Ca2o+. No d o u b t f u r t h e r studies will reveal the capacity of additional cell surface proteins to sense Ca2o+ and, perhaps, o t h e r ions, probably not only GPCRs but also o t h e r integral m e m b r a n e proteins capable of m o d u l a t i n g cellular function (e.g., ion channels).

SUMMARY In conclusion, the discovery of the CaSR has provided a molecular m e c h a n i s m mediating many of the

known actions of Ca2o+ on the functions of cells and tissues involved in systemic Ca 2+ o homeostasis. Much remains to be learned, however, a b o u t the functions of the CaSR in these tissues as well as in n u m e r o u s other CaSR-expressing cell types that are not directly involved in systemic mineral ion homeostasis. In the latter, the receptor probably serves diverse roles, making it a versatile regulator of a wide variety of cellular functions such that it could serve as an i m p o r t a n t therapeutic target. F u r t h e r m o r e , the capacity of the CaSR to integrate and coordinate several types of signals may enable it to serve as a central homeostatic regulator, not only of mineral ion homeostasis but also of processes related to water, protein, and n u t r i e n t metabolism m o r e generally.

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RECEPTORS/SIGNALING FOR C a 2+ o / 81. Silver IA, Murrils RJ, Etherington DJ. Microlectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 1988;175:266-276. 82. Quarles LD. Cation-sensing receptors in bone: A novel paradigm for regulating bone remodeling? J Bone Miner Res 1997;12:1971-1974. 83. Yamaguchi T, Chattopadhyay N, Brown EM. G Protein-coupled extracellular Ca 2+ (Ca2+ o )-sensing receptor (CAR)" Roles in cell signaling and control of diverse cellular functions. Adv Pharmacol 1999;47:209-253. 84. Kanatani M, Sugimoto T, Kanzawa M, Yano S, Chihara I~ High Extracellular calcium inhibits osteoclast-like cell formation by directly acting on the calcium-sensing receptor existing in osteoclast precursor cells. Biochem Biophys Res Commun 1999;261:144-148. 85. Zaidi M, Adebanjo OA, Moonga BS, Sun L, Huang CL. Emerging insights into the role of calcium ions in osteoclast regulation. J Bone Miner Res 1999;14:669-674. 86. Pi M, Hinson TK, Quarles L. Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines. JBone Miner Res 1999;14:1310-1319. 87. Seuwen K, Boddeke HG, Migliaccio S, Perez M, Taranta A, Teti A. A novel calcium sensor stimulating inositol phosphate formation and [Ca2+]i signaling expressed by GCT23 osteoclastlike cells. Proc Assoc Am Physicians 1999;111:70-81. 88. House MG, Kohlmeier L, Chattopadhyay N, Kifor O, Yamaguchi T, Leboff MS, Glowacki J, Brown EM. Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. J Bone Miner Res 1997;12:1959-1970. 89. Yamaguchi T, Chattopadhyay N, Kifor O, Brown EM. Extracellular calcium (Ca~o~)-sensing 2+ receptor in a murine bone marrow-derived stromal cell line (ST2): Potential mediator of the actions of Ca~o 2+~ on the function of ST2 cells. Endocrinology 1998; 139:3561-3568. 90. Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR, Jr, Sugimoto T, Brown EM. Mouse osteoblastic cell line (MC3T3El) expresses extracellular calcium (Ca2o+)-sensingreceptor and its agonists stimulate chemotaxis and prolif-eration of MC3T3E1 cells. JBone Miner Res 1998;13:1530-1538. 91. Yamaguchi T, Kifor O, Chattopadhyay N, Brown EM. Expression of extracellular calcium (Ca2+o)-Sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2. Biochem Biophys Res Commun 1998;243:753-757. 92. Chang W, Tu C, Chen T-H, Komuves L, Oda Y, Pratt S, Miller S, Shoback D. Expression and signal transduction of calciumsensing receptors in cartilage and bone. Endocrinology 1999;140:5883-5893. 93. Yamaguchi T, Olozak I, Chattopadhyay N, Butters RR, Kifor O, Scadden DT, Brown EM. Expression of extracellular calcium (Ca 2+ o )-sensing receptor in human peripheral blood monocytes. Biochem Biophys Res Commun 1998;246:501-506. 94. Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M, Izumi N, Kawashima H, Ozawa H, Ikeda K, Kameda A, Hakeda Y, Kumegawa M. Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem Biophys Res Commun 1998;245:419-422. 95. Quarles DL, Hartle II JE, Siddhanti SR, Guo R, Hinson TK. A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor. J Bone Miner Res 1997;12:393-402. 96. Jacenko O, Tuan RS. Chondrogenic potential of chick embryonic calvaria: I. Low calcium permits cartilage differentiation. Dev Dyn 1995;202:13-26. 97. Reginato AM, Tuan RS, Ono T, Jimenez SA, Jacenko O. Effects of calcium deficiency on chondrocyte hypertrophy and type X

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collagen expression in chick embryonic sternum. Dev Dyn 1993;198:284-295. 98. Bonen DK, Schmid TM. Elevated extracellular calcium concentrations induce type X collagen synthesis in chondrocyte cultures. J Cell Bio11991;115:1171-1178. 99. Wong M, Tuan RS. Interactive cellular modulation of chondrogenic differentiation in vitro by subpopulations of chick embryonic calvarial cells. Dev Bio11995;167:130-147. 100. Dennis JE, Merriam A, Awadallah A, Yoo JU, Johnstone B, Caplan M. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res 1999;14:700-709. 101. Boyan BD, Caplan M, Heckman JD, Lennon DE Ehler W, Schwartz Z. Osteochondral progenitor cells in acute and chronic canine nonunions. J Orthop Res 1999;17:246-255. 102. Chang W, Tu C, Bajra R, Komuves L, Miller S, Strewler G, Shoback D. Calcium sensing in cultured chondrogenic RCJ3.1 C5.18 cells. Endocrinology 1999;140:1911-1919. 103. Gilljj, Bartter E On the impairment of renal concentrating ability in prolonged hypercalcemia and hypercalciuria in man. J Clin Invest 1961;40:716-722. 104. Suki WN, Eknoyan G, Rector FC, Jr, Seldin DW. The renal diluting and concentrating mechanism in hypercalcemia. Nephron 1969;6:50-61. 105. Wang W, Lu M, Balazy M, Hebert SC. Phospholipase A2 is involved in mediating the effect of extracellular Ca 2+ on apical K+ channels in rat TAL. AmJPhysio11997;273:F421-F429. 106. Wang WH, Lu M, Hebert SC. Cytochrome P-450 metabolites mediate extracellular Ca~Z+Linduced inhibition of apical K+ channels in the TAL. AmJPhysio11996;271:C103-Cl11. 107. Marx SJ, Attie ME Stock JL, Spiegel AM, Levine MA. Maximal urine-concentrating ability: Familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. J Clin Endocrinol Metab 1981;52:736-740. 108. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N Engl J Med 1996;335:1115-1122. 109. Rogers KV, Dunn CK, Hebert SC, Brown EM. Localization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization. Brain Res 1997;744:47-56. 110. Simpson JB, Routenberg A. Subfornical organ lesions reduce intravenous angiotensin-induced drinking. Brain Res 1975;88: 154-161. 111. Tordoff MG. Voluntary intake of calcium and other minerals by rats. Am J Physiol 1994;167:R470-R475. 112. Quinn sJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, Brown E. The CaZ+-sensing receptor: A target for polyamines. Am J Physiol 1997;273:C 1315-C1323. 113. Quinn sj, Kifor O, Trivedi S, Diaz R, Vassilev P, Brown E. Sodium and ionic strength sensing by the calcium receptor. JBiol Chem 1998;273:19579-19586. 114. Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG, Balandrin ME Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 1998;95:4040-4045. 115. Insogna KL, Broadus AE. Nephrolithiasis. In: Broadus AE, A FL, Felig P, BaxterJD, eds. Endocrinology and metabolism, 2nd Ed. New York:McGraw-Hill, 1987:1500-1577. 116. Lemann JR, Litzgow JR, Lennon EJ. The effect of chronic acid loads in normal man: Further evidence for the participation of bone mineral in the defense against metaboic acidosis. J Clin Invest 1966;45:1608-1614. 117. Kerstetter JE, Caseria DM, Mitnick ME, Ellison AF, Gay LF, Liskov TA, Carpenter TO, Insogna KL. Increased circulating concentrations of parathyroid hormone in healthy, young

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women consuming a protein-restricted diet. Am J Clin Nutr 1997;66:1188-1196. 118. McArthur KE, Isenberg JI, Hogan DL, Dreier SJ. Intravenous infusion of L-isomers of phenylalanine and tryptophan stimulate gastric acid secretion at physiologic plasma concentrations in normal subjects and after parietal cell vagotomy. J Clin Invest 1983;71:1254-1262. 119. Mangel AW, Prpic V, Wong H, Basavappa S, Hurst LJ, Scott L, Garman RL, Hayes JS, Sharara AI, Snow ND, et al. Phenylalanine-

stimulated secretion of cholecystokinin is calcium dependent. Am J Physio11995;268:G90-G94. 120. Taylor IL, Byrne WJ, Christie DL, Ament ME, Walsh JH. Effect of individual L-amino acids on gastric acid secretion and serum gastrin and pancreatic polypeptide release in humans. Gastroenterology 1982;83:273-278. 121. Kubo Y, Miyashita T, Murata Y. Structural basis for a CaZ+-sensing function of the metabotropic glutamate receptors. Science 1998;279:1722-1725.

CI-IAPI:F R9 Immunoassays for P T H and PTHrP Clinical Applications

L.J. DEFTOS Department of Medicine, University of California, San Diego, and San Diego VA Medical Cent~ LaJolla, California 92161

INTRODUCTION

ficity for the target peptides a n d / o r polypeptides. Either the antibodies or the peptide standard, depending on assay format, are labeled so that they can be quantified by one of a variety of detection systems. The most widely available detection systems use radioisotopes, colorimetry, or chemiluminescence. Most immunoassays for PTH and PTHrP can detect n a n o g r a m (ng) to picogram (pg) concentrations of the hormone. In the case of PTH, the circulating concentrations can be readily measured in both health and disease. But normal ranges for serum PTHrP are not yet well established, and the question of the circulation of PTHrP in normal individuals remains open. This chapter reviews the development of the wide variety of PTH immunoassays and the increasing n u m b e r of PTHrP assays that are currently available, commonly with Food and Drug Administration (FDA) approval, for clinical application in the United States. T h o u g h the detailed background in relevant physiology for a full understanding of PTH and PTHrP assays is provided in other chapters of this book, this chapter provides a synopsis of the biosynthesis, secretion, and metabolism of PTH and PTHrP that will focus on the rationale that has been used for assay development. This background synopsis is followed by a more detailed discussion of the clinical application of PTH immunoassays to specific disorders of calcium and skeletal metabolism. Developments in assay theory and practice are also discussed. The m e a s u r e m e n t of circulating levels of PTHrP has only approximated the accurate and precision available for PTH assays. However, the m e a s u r e m e n t of PTHrP is assuming increasing

This chapter discusses the development and clinical application of assays for circulating levels of parathyroid h o r m o n e (PTH) and parathyroid hormone-related protein (PTHrP). The accurate and precise measurement of circulating levels of PTH has revolutionized the clinical management of patients with calcium and skeletal disorders, and assays for PTHrP are beginning to have comparable clinical impact for the patient with hypercalcemia, especially hypercalcemia due to cancer. Sensitive and specific assays for PTH and PTHrP aid in the differential diagnosis of hypercalcemia, hypocalcemia, and a variety of other calcium and skeletal diseases. In addition to their usefulness in clinical management, PTH and PTHrP serum assays have helped to elucidate the pathophysiology of many disorders of calcium and skeletal metabolism. (In this chapter, the term "serum" will be generally used for convenience to connote measurements of circulating levels of PTH and PTHrP, because, in general, serum measurements are widely used and there are few substantial differences between serum and plasma measurements. However, in some instances there are differences, and when the distinction is important, it will be addressed.) The measurements of circulating serum or plasma concentrations of PTH and PTHrP are primarily based on immunoassay procedures that recognize different peptides of the linear sequence of the native molecules. Although bioassays are available for research purposes, they are not used clinically. These immunoassay procedures utilize one or two antibodies with defined speciThe Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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clinical importance. Thus, this chapter also reviews the expanding information on the application of PTHrP immunoassays to clinical diagnosis and management. In contrast to PTH, assays for PTHrP are in their early stages of development. Furthermore, they are complicated by the more complex biosynthesis, secretion, and metabolism of this polypeptide. New and improved research assay systems for PTH and PTHrP are continually developed and touted. However, it takes substantial clinical evaluation to establish the diagnostic value of an immunoassay procedure for clinical application and ultimate FDA approval. This chapter thus focuses on describing the general principles that underlie the development and application of immunoassays for PTH and PTHrP so that the clinician can chose the most appropriate assay.

PTH BIOSYNTHESIS, SECRETION, AND METABOLISM Overview PTH is an 84-amino acid polypeptide secreted by the parathyroid glands, primarily in the chief cells of the gland (1-9). Like other peptide hormones, PTH is originally synthesized as a larger precursor molecule; the PTH precursor, named preproparathyroid hormone, is 115 residues long (2). This intracellular species of PTH is processed, metabolized, and secreted primarily as the native 84-amino acid hormone, although some intracellular metabolism also occurs. On secretion, native PTH is metabolized to amino-terminal and carboxyterminal fragments by the liver, kidney, and other peripheral sites (6). The amino terminus of native PTH, approximately the first 27 to 34 amino acids, contains the classic biologic activity of the hormone. It binds to a PTH cell surface receptor that also recognizes PTHrP (5,6). This receptor had been named the P T H / P T H r P receptor, but is now known as the PTH1 receptor (Chapter 2). Other fragments of PTH are generally considered to be inactive. Measurement of biologically active PTH species would appear to have more clinical relevance than measurement of inert fragments of the hormone, but this is where theory and practice diverge (4,8). This divergence will be illuminated by considering some aspects of the biosynthesis, secretion, and metabolism of PTH that provide insights into the rationale for development and the application to clinical studies of the various PTH immunoassays that are available. Selected aspects of PTH biology are briefly reviewed throughout this chapter. More details about the cellular and molecular physiology of the h o r m o n e can be found elsewhere in this volume (see Chapters 13-17).

Biosynthesis of PTH The biosynthesis of PTH is controlled by its relatively simple gene located on the short arm of chromosome 11 at band 11p15 (3,6). The gene contains three exons separated by two introns, with the first exon conmining most of the 5' prime noncoding sequence, the second exon coding most of the prepro sequence, and the third exon coding the mature PTH sequence (2). Several factors can regulate PTH gene transcription or mRNA stability, with calcium and 1,25-dihydroxyvitamin D being the most important (3). Low ambient calcium stimulates gene transcription, and high ambient calcium suppresses gene transcription, but to a lesser extent. Exposure to the active vitamin D metabolite, 1,25-dihydroxyvitamin D, suppresses gene transcription (1,6). Preliminary studies of estrogen and ambient phosphorus and magnesium reveal that they can also regulate gene transcription (6). The regulation of PTH gene transcription is discussed in more detail in Chapter 2. The product of the h u m a n PTH gene is a 115-amino acid preproPTH containing the 84-residue native molecule and a 29-residue prepro moiety that undergoes several intracellular cleavage steps as it passes through cellular trafficking (1,6). As the signal sequence of the nascent peptide emerges from the ribosome, it is directed by a signal recognition particle to the endoplasmic reticulum (ER), where in the lumen a signal peptidase cleaves the 25-amino acid pre sequence, releasing the intermediate proPTH form (2). ProPTH then travels to and through the Golgi apparatus, where the six-amino acid pro sequence is cleaved by furin or a furinlike molecule, leaving the mature 1-84 native PTH. The mature molecule is then concentrated into secretory vesicles for secretion in response to ambient calcium and other regulators (2,6). In addition to the mature PTH molecule, there is evidence for the presence of other intracellular forms of the hormone. A m i n o - a n d carboxy-terminal peptides, especially the latter, are derived from intracellular processing a n d / o r metabolism of PTH, and they can be secreted as well (2,8). In fact, truncation of the carboxy terminus of PTH impairs its secretion (2). These observations about the intracellular journey of PTH have led to the assignment of a functional role for different PTH peptide regions (1,2), and as will be seen later, for the corresponding sequences in PTHrE The pre region directs the nascent molecule to the ER; the pro region is required for its introduction into a Golgi pathway and the carboxy-terminal region of the mature hormone is required for transportation through the secretory pathway that leads to secretory vesicles and, ultimately, to hormone secretion.

PTH AND PTHrP IMMUNOASSAYS /

Intracellular Metabolism o f P T H PTH is metabolized in the parathyroid gland to carboxy- and amino-terminal forms (Fig. 1). Most of the information regarding the intracellular metabolism of PTH comes from animal studies and in vitro studies of h u m a n hyperplastic and adenomatous parathyroid glands (2-8). It is notable that the intracellular and secreted carboxy-terminal forms of PTH are identical at the amino terminus to the carboxy-terminal fragments of the molecule generated by peripheral metabolism of PTH, and have the corresponding structures (discussed later) (4,8). In general, generation of both the cellular and the peripheral carboxyterminal fragments of PTHrP involves cleavages within P T H ( 3 3 - 43) (8,9). The intracellular degradation of the h o r m o n e appears to be regulated (2,8,9). When secretion is stimulated by low ambient calcium, most of the h o r m o n e is in the form of the native molecule (6,9). By contrast, when secretion is suppressed by high ambient calcium, most of the secreted h o r m o n e consists of fragments. Curiously, phorbol ester-stimulated

A. PG Biosynthesis B. PG Processing/Metabolism 1. Signal Peptidase 2. Furin or Furin-like Enzyme(s) 3. Proteases

pre-pro [I-N~III.i~i........................................ ct pro [,N

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I////////////////71 E. Renal Clearance

secretion also results in an increased a m o u n t of PTHrP fragments, regardless of the ambient calcium concentration (2,9). T h o u g h a signal peptidase is responsible for cleavage of the pre sequence of preproPTH, the enzymes responsible for subsequent intracellular cleavage of PTH have not been conclusively identified (2). However, it is likely that furin or a furinlike molecule affects the cleavage of the pro sequence of proPTH. Furin, a subtilisin-like enzyme located within the Golgi cisternae of essentially all mammalian cells, cleaves proteins and peptides at the basic residues sites flanking pro sequences (6). The p r o h o r m o n e convertases (PCs) found in secretory granules, a m o n g them PC1 and PC2, are also candidates for intracellular processing of PTH (9). There is also evidence that cathepsins affect the intracellular metabolism of PTH. There is little evidence for the secretion of PTH precursors, as there is for other h o r m o n e s such as insulin and adrenocorticotropic h o r m o n e (ACTH) (2,6,9). The result of this intracellular metabolism of PTH is the intracellular development, with potential for secretion, of several species of the hormone, including intact, midregion, carboxyl, and amino-terminal forms (4,8,9). As discussed later, each of the species has the potential of being detected by immunoassay procedures based on antibodies that recognize their included PTH epitopes. But it is important to recall that only aminoterminal forms of defined length can exert biologic activity by activating the PTH receptor (5).

Secretion o f P T H

el (b)

t7 ..........................................e t (c)

D. Peripheral (Hepatic and Renal) Metabolism

145

(d)

(e)

All Circulating Forms [(a) - (e)]

FIG. 1 Biosynthesis, secretion, and metabolism of PTH by the parathyroid gland (PG) and in the periphery. Schematic representation of the cellular biology of PTH and the resulting molecular heterogeneity of circulating forms that are detected by immunoassays. The peptides (a-e) represent the circulating forms of immunoassayable PTH, including (a) native PTH, (b) amino (N)-terminal and carboxy (C)-terminal PTH, and (d) midregion (M) PTH peptides; (c) the recently postulated amino-terminal deleted PTH peptide; and (e) the uncharacterized PTH peptides that result from further metabolism and/or degradation of all other PTH forms. Circulating PTH is a complex, immunochemically heterogeneous mixture of peptides a-e, with fragments predominating. See text for full discussion.

The secretion of PTH is regulated mainly by serum calcium concentration (1,2). In a homoeostatically appropriate response, increased extracellular calcium suppresses PTH secretion, and decreased extracellular calcium stimulates PTH secretion. The primary effect of increased extracellular calcium is to inhibit the secretion of preformed PTH from secretory granules by blocking their fusion with the cell m e m b r a n e (6). This contrasts to most other cells, wherein stimulation of exocytosis is inhibited by the depletion of calcium. Thus, the inverse relationship between ambient calcium and PTH secretion contrasts to the effect of calcium on the secretion of other hormones, including calcitonin, the biological antagonist of PTH (1,6). The relationship between serum calcium and PTH is sigmoidal, with the steepest portion of the curve corresponding to the normal range of serum calcium (9). It is also likely that these two signals correspondingly regulate the growth and proliferation of parathyroid gland cells and thus exert long-term effects on horm o n e secretion (6). The major secretory effects are on native PTH rather than on PTH fragments. In fact,

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carboxy-terminal truncation of the native molecule impairs its secretion, because this end of the molecule seems necessary for guiding PTH through the secretory apparatus (9). Ambient calcium mediates its effects on PTH secretion through the calcium sensor of the parathyroid gland, as discussed in detail in Chapter 8. The secretion of PTH can also be regulated to a lesser extent by ambient magnesium and by catecholamines (4,8). The effect of magnesium on PTH secretion is quantitatively similar to that of ambient calcium but physiologically less important. However, severe magnesium deficiency and hypomagnesemia can inhibit the secretion as well as the biologic activity of PTH (2). After secretion, native PTH is rapidly cleared from the circulation with a half-life of a few minutes (9,10). By contrast, PTH fragments are cleared with a half-life of several hours (10). So these PTH fragments, especially carboxyterminal fragments, accumulate and are readily measurable in the circulation when renal function is impaired (4). In contrast, amino-terminal fragments of PTH are difficult to demonstrate in blood (4,8). The secretion of PTH in humans is episodic (pulsatile) and rhythmic, although there is controversy about the nature of the patterns in health and disease (4,8). In studies of normal subjects, some investigators have reported hourly pulses of secretion that last for minutes, whereas others have described broad peaks that last for hours (4,7,10). In circadian studies, both nocturnal and biphasic peaks have been reported (4). Abnormal patterns of PTH secretion have been reported in several disease states, including osteoporosis and primary hyperparathyroidism (8). During induced perturbations of serum calcium, there can be hysteresis in the relationship between calcium perturbation and the recovery period (1,4,7). Most studies of patterns of PTH secretion have been conducted in small numbers of subjects, and it is difficult to draw firm conclusions about the results (4,8,9). Dynamic tests have been used, respectively, to stimulate and suppress serum PTH in order to assess the secretory status of the parathyroid gland. T h o u g h such studies have helped to define the relationship between the regulatory parameters and PTH secretion, the involved maneuvers are not suitable for clinical practice. For practical clinical purposes, the effects of patterns of basal and regulated PTH secretion on diagnostic studies can be circumvented by collecting blood samples for analysis at a consistent time, preferably in the morning after an overnight fast (9,10).

Peripheral Metabolism of PTH After the glandular PTH forms, which arise from biosynthesis and intracellular metabolism, are secreted into blood, they are also metabolized at several peripheral sites into peptide fragments (Fig. 1). The liver is

the most important metabolic site for PTH, with the kidney and skeleton following (2). Binding of PTH to specific receptors in target tissues does not contribute substantially to the metabolism of the h o r m o n e (5). As was the case for studies of intracellular metabolism, most of the information regarding peripheral metabolism of PTH comes from animal studies and in vitro h u m a n studies using both labeled and unlabeled native PTH (4,8). In their aggregate, these studies reveal that PTH is peripherally processed between residues 33 and 34, 36 and 37, 40 and 41, and 42 and 43, resulting in the corresponding amino- and carboxy-terminal peptides. Most of this metabolism seems to occur in the liver in Kuppfer cells (8). After passage through the liver, the PTH fragments are routed to the kidney, where they are cleared by glomerular filtration along with the relatively smaller amounts of circulating intact PTH (4). As will be seen later, the renal,clearance of PTH and its fragments plays an important role in regulating the concentrations and types of circulating PTHrP forms. This effect becomes especially important in renal failure, where it can confound immunoassay measurements. It is not clear if the peripheral metabolism of PTH is regulated by ambient calcium, as appears to be the case for the glandular metabolism of PTH (4,8). A homeostatically appropriate process would be for hypercalcemia to promote the peripheral degradation of PTH; however, studies of this p h e n o m e n o n have not been conclusive. In summary, as a result of the biosynthesis, secretion, and metabolism of PTH, the circulation contains several forms of the molecule (Fig. 1). This immunochemical heterogeneity of circulating PTH was discovered and d o c u m e n t e d by Berson and Yalow, the first developers of PTH immunoassay, who went on to win the Nobel Prize (11). The forms that comprise this immunochemically heterogenous collection of PTH species include primarily native PTH (1-84) and midregion and carboxy-terminal PTH fragments (6). Overall, 10-20% of circulating PTH immunoreactivity comprises the intact hormone, with the remainder being a heterogeneous collection of peptide fragments corresponding to the middle and carboxy regions of the molecule. Evidence for other circulating PTH species is inconclusive, although some studies indicate the presence in the circulation of the PTH amino terminus (5). It is important to reemphasize that only the amino terminus of PTH can bind to the PTH receptor and mediate its classic biologic effects that result in hypercalcemia. So only amino-terminal-containing PTH forms have the potential for biologic activity mediated through the PTH receptor. However, it should be kept in mind that each of the circulating forms of PTH, regardless of biologic activity, contain within them peptide sequences that can be recognized by a variety of antibodies (4,8).

PTH AND PTHrP IMMUNOASSAYS / The half-life of the relatively low concentrations of intact h o r m o n e and its amino terminal fragments can be measured in minutes, whereas the higher concentrations of the biologically inactive mid- and carboxyterminal peptides have half-lives of hours (8,11). As will be seen later, antibodies directed at the diverse bioactive as well as nonbioactive epitopes included within PTH(1-84) have been used to develop immunoassays for the h o r m o n e . In general, intact and non-aminoterminal fragments of the molecule circulate in blood. However, though intact forms of the molecule containing parts of the amino terminus can be measured in blood, amino-terminal fragments, themselves, are difficult to demonstrate. Certain technical considerations can affect immunoassay p e r f o r m a n c e (4,8,9-11). Although originally considered to be a labile h o r m o n e , in vitro losses of PTH immunoreactivity are less than 10% when whole blood or serum is left at room temperature for 4 hours. However, in vitro degradation may be increased in patients with pancreatitis and the resulting high levels of circulating proteases. In most circumstances, a reasonable delay in the separation and freezing of serum should not have a major effect on assay measurement. Nevertheless, blood samples should be processed within a reasonably short period of time, 30-60 minutes, to minimize h o r m o n e degradation. Some assay procedures utilize collecting tubes that contain enzyme inhibitors. And in some assay systems, there are differences between serum and plasma measurements (9-11). In any case, the ordering physician should comply with the sample-collecting instructions of the testing laboratory. Correct interpretation of PTH assay results requires a simultaneous serum calcium measurement, because, as detailed later, the relationship between the two can distinguish between a primary and secondary secretory disorder of the parathyroid glands (10). Total serum calcium will suffice for most purposes. However, because almost half of serum calcium is b o u n d to serum albumin, a correction is sometimes n e e d e d for abnormal, usually low, concentrations of albumin. T h o u g h there are relatively complex formulas for such a correction, a change of 1 g / d l in serum albumin will generally produced a corresponding change of 0.8 m / d l in measured total serum calcium. Measurements of ionized calcium can be used to circumvent the effects of serum albumin concentrations. However, such tests are not routinely available. Abnormalities in serum g a m m a globulin concentrations do not usually affect total serum calcium measurements. However, if they are markedly increased, as they can be in certain dysgammmaglobulinemias (e.g., Waldenstrom's), serum g a m m a globulin concentrations can also artifactually increase total serum calcium measurement. Spurious increases in serum calcium can be also caused by venousstasis-induced

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changes in local albumin and pH at the time of blood collection, as can the essentially obsolete use of calcium-containing cork-stopped blood collection tubes (4,8,10). The clinician should be aware of these possibilities when there is discordance between serum measurements and the clinical status of the patient.

PTH IMMUNOASSAYS Introduction The development of accurate and precise i m m u n o assays for circulating PTH has evolved from a discouraging beginning to a promising present (4,8). Early PTH immunoassays were based on uncharacterized antibodies and impure standards. Crude extracts of animal parathyroid glands were used for production of antibodies, and heterologous and impure preparations of PTH were used as standards. The clinical value of early immunoassays for PTH was suspect. It took the revolutionary discovery of Berson and Yalow of the i m m u n o c h e m i c a l heterogeneity of circulating PTH to lead to the correction of the clinically contentious course that PTH immunoassays had taken in the 1960s (11). Following the Nobel prize-winning contributions of these two pioneers, PTH immunoassays began to evolve in a rational manner. Immunoassays were developed based on defined PTH peptides and characterized antibodies, both polyclonal and monoclonal (11,13). Even polyclonal antibodies could be purified by peptide-specific immunoaffinity c h r o m a t o g r a p h y (12,13). Immunoassays subsequent to the first crude assay systems could thus be directed at specific forms of the h o r m o n e (4). The molecular targets of PTH assays could be predicted by basic studies that elucidated the complex nature of PTH in the circulation (8). The most prevalent forms of PTH in serum would be fragments of the molecule that contained midregion and carboxyterminal epitopes, whose molecular d o m i n a n c e was e n h a n c e d by relatively long half-lives (4,11). By contrast, assays directed at the amino terminus were not likely to be clinically successful because of the small amounts of this PTH form in the circulation and it short half-life. Intact PTH species, which revealed several epitopes, occupied an intermediary position, but were later to b e c o m e targets for two-site assays (11-14). Following the development of clinically useful midregion and carboxy-terminal PTH solution immunoassays based on an antibody to the desired epitope, solid-phase, two-site immunoassays were developed that were based on the simultaneous use of two antibodies of differing specificity for the PTH molecule (4,8,15,16). The specificity of the antibodies used in these two-site formats defined the PTH species

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being detected. These two-site assays were accompanied by the supplanting of radioiodine in radioimmunoassays (RIAs) by nonisotopic methods for detection, such as the use of colorimetry or chemiluminescence (17-20). These new technologies provided the newer immunoassays with descriptive acronymic titles, such as enzyme-linked immunosorbent assays (ELISAs), immuno-radiometric assays (IRMAs), and immunochemiluminometric or -fluorometric assays (ICMAs and IMFAs, respectively). The development of novel detection systems and improvements to assay automation continues (19-21).

PTH Assay Formats Overview

Detection systems aside, there are two general formats for PTH immunoassays, one-epitope-site solution immunoassays and two-epitope-site solid-phase immunoassays (4,16,22,23). Solution immunoassays, developed first, are based on the comparison of the displacement from an antibody, usually polyclonal, of labeled, usually radioiodinated, PTH or PTH peptide representing the PTH present in a blood sample and the PTH standard. As increasing quantities of the PTH in the unknown sample and PTH standard are added to the radiolabeled PTH-antibody reaction, there is a progressive competition by the sample and standard PTH for antibody binding by the radiolabeled PTH tracer. This competition by each known amount of PTH standard produces a progressive displacement of radiolabeled PTH tracer from the antibody in a manner that can be used to generate a standard curve. The unknown amount of PTH in the unknown sample has the same effect. By comparing the displacement produced by the PTH in the unknown sample to the known PTH standard, the amount of PTH in the sample can be calculated. The sensitivity of the assay can be enhanced by sequential (nonequilibrium) rather than simultaneous (equilibrium) addition of the unlabeled PTH standard (and sample) and labeled PTH tracer (16).

Solution Immunoassaysfor t ~ H In the earliest of PTH solution immunoassays, commonly referred to as radioimmunoassays, the standards were usually impure and often heterologous, and the antibodies were not well characterized, often being generated against only partially purified preparations of glandular PTH (4,8). Many technical advances followed these early PTH solution immunoassays. Standard preparations of PTH became progressively more pure and homologous, and PTH peptides were introduced as standards. Antibodies were raised against

fully characterized forms of PTH and PTH peptides, thus allowing the development of antibodies with welldefined specificities for PTH and its peptides. Although most antibodies used in solution immunoassays were polyclonal, occasional high-affinity monoclonal antibodies were identified (4,8,16). The combination of synthetic peptide standards and antibodies of known specificity allowed the development of PTH immunoassays that could measure defined regions of the molecule (11,12). Thus, immunoassays could be developed to detect specifically the circulating forms of PTH that contained different PTH epitopes, including amino-, mid-, and carboxyterminal PTH species (8,21-23). Although each assay so designed would measure intact PTH, it preferentially measures with appropriate specificity and respectively increased sensitivity the middle and carboxy-terminal regions of PTH that circulate at severalfold higher concentrations than intact PTH (23-31). Despite their limitations, these solution immunoassays proved clinically useful, and most of them are still in use (28-35). They specifically identify a large majority of patients with primary hyperparathyroidism and patients with secondary hyperparathyroidism in the absence of renal failure. Thus, both commercial and research immunoassays for PTH are still currently available that can detect midregions of the molecule using, for example, PTH(44-68) as standard and the tryrosinated form of the same peptide as tracer and for antibody generation, and carboxy-terminal regions of PTH using, for example, corresponding reagents for PTH (68-75) (4,8,28,33-38). The clinical value of solution immunoassays for nonamino-terminal PTH forms is also due to the fact that they can measure the relatively low concentrations of PTH that circulate in the serum of normal individuals, thus providing a basis for comparison with disease states, and because these assays, especially midmolecule assays, are notably sensitive (32,35). In addition to the inherent sensitivity of such assays, their cognate nonamino-terminal forms of PTH are predominant in the circulation because of their metabolic characteristics, discussed earlier. Furthermore, these assays can detect the small amount of circulating intact hormone conmining their epitopes (34-40). In contrast, assays designed to measure the amino terminus cannot readily detect this form in normal subjects because it circulates at such low concentrations and has a short half-life, although some exceptions have been reported (40-48). Despite their continuing clinical utility, radioimmunoassays for the midregion and carboxy regions of PTH do have their limitations, especially the latter (33,38,42). Both of these fragments, especially carboxyterminal fragments, accumulate disproportionately in

PTH AND PTHrP IMMUNOASSAYS / the patient with renal disease (4,8,45). Thus, it is difficult to assess accurately parathyroid gland secretory status using carboxy-terminal assays in such patients. However, it must be kept in mind that in renal disease there is an accumulation of all of the circulating forms of PTH, including all fragments and the intact hormone, too (4,8,47). As will be discussed in detail later, assessing parathyroid gland secretory activity remains a major problem in renal disease, especially in chronic renal failure.

Solid-Phase Immunoassays The two-site assay is based on two antibodies with different recognition sites for an antigen, in this case PTH (49,50). Although the principle of such assays, generally referred to as immunometric assays, had been recognized for years, the onset of monoclonal antibody production enhanced the development of the reagents requisite for these assays and their research and clinical application (6,19,20). Reactivity in two-site assay systems is dependent on the separate recognition of two antigenic sites in PTH by a pair of antibodies respectively directed against them. One antibody is attached to a solid matrix, usually beads or microtiter wells, and the other antibody is radioiodinated (or otherwise labeled) and in solution. The antigen, PTH, binds to the antibody on the solid phase (e.g., beads) according to the antigenic recognition site of that antibody. The labeled antibody binds the PTH antigen bound to the immobilized antibody according to its (the labeled antibody) different recognition site. The radioactivity remaining after washing the solid matrix is thus proportional to the amount of antigen having both antigenic determinants. The two-site assay has many advantages (15,26,27). It can directly measure specific and defined forms of PTH; the kinetics of the two-site assay permit an extraordinary increase in assay sensitivity, even with relatively low-affinity antibodies; and this assay system is remarkably free of nonspecific protein artifacts that have continually plagued immunoassays. This latter advantage is especially important, because two-site procedures can minimize the problems associated with immunoassay in protein-rich biologic fluids such as blood (15,26-29). The antibodies used in two-site immunoassays are usually generated against specific peptides of PTH (21). Monoclonal antibodies are favored over polyclonal antibodies for two-site immunoassays. Monoclonal antibodies of exquisite specificity can be developed in the relatively large amounts needed for the solid-phase component of two-site assays (16,19). Furthermore, monoclonal antibodies can be more readily purified for labeling. A disadvantage of m o n o -

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clonal antibodies is that they generally do not have the affinity of polyclonal antibodies (4,16). However, this property is not so important for the noncompetitive kinetics of two-site assays as it is for the kinetics and competition of solution immunoassays (8,16,21,49,50). Another approach for developing epitope-specific antibodies is to purify them from polyclonal antiserum using peptide-specific affinity chromatography (16,51). Epitope-specific polyclonal antibodies can then be used as described above for the two-site assay format; mixed monoclonal-polyclonal systems are also suitable (4,8,51). Using these general approaches and their variations, assay systems have been developed for peptides that span the linear sequence of PTH. Especially popular are two-site systems using amino- and carboxyterminal PTH antibodies in concert in order to detect in tact native PTH (9,15,52). Solid-phase immunoassays also have other technical advantages over solution immunoassays (4,8,15,21). They can be completed in hours rather than in the days usually needed for solution immunoassays. They are relatively free of the nonspecific serum effects that plague (solution immunoassays, because serum is not present during the critical incubation stage of the procedure. And they can be performed with a general technical ease that provides more accuracy and precision. Intact F I ' H

Immunoassays

Assays for intact PTH(1-84) have become the holy grail of the PTH immunoassay field, even though classic solution immunoassays, especially midmolecule assays, have sufficient accuracy and precision for most clinical indications (15,23,25,28,52-57). These assays use an antibody directed against the amino terminus of PTH in tandem with an antibody directed against the carboxy-terminal regions of PTH in order to detect circulating levels of intact PTH(1-84), a (synthetic peptide version of which is used as standard (28,29). Though intact assays generally provide excellent discrimination between parathyroid disease and nonparathyroid disease, these) assays still demonstrate some overlap in their normal range with disease states (22,23). However, even in these situations, the diagnosis can usually be clarified by considering the PTH concentration together with the serum calcium concentration (4). Thus, even if the serum PTH concentration is not absolutely different from the normal range, it is inappropriately different from the normal range in hyperparathyroid disease states (8,30). So the patient with hypercalcemia due to primary hyperparathyroidism will have a serum intact PTH that is absolutely elevated above the normal range or close to the top of the normal range, and, by contrast, the patient with nonparathyroid hypercalcemia will have a

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serum intact PTH that is close to or below the normal assay limit. These considerations, of course, apply to other PTH assays. Intact PTH assays have been widely used in renal failure to assess parathyroid gland secretory status, as will be discussed in more detail subsequently (22,23). The rationale behind this application is the well-known accumulation of PTH fragments that occurs in renal failure. Because these fragments do not necessarily reflect the secretory activity of the parathyroid gland and because they are generally biologically inactive, their m e a s u r e m e n t does not give a true assessment of parathyroid gland function (4,38,57). These fragments should theoretically escape detection in a two-site assay that is designed to measure only the intact molecule, based on antibodies to the amino-terminal and carboxy-terminal regions of PTH. However, studies demonstrate that this theoretical advantage of intact PTH immunoassays may not always apply (23,57). In the first place, like PTH fragments, intact PTH also accumulates in renal failure, although less so (22,23). However, more critical, currently available intact PTH assays seem also to measure certain PTH fragments, especially in renal failure (23). This is probably due to the fact that antibodies used for presumably intact PTH immunoassays are not, respectively, directed at the far carboxy and far amino termini of the native molecule (22,23,57). Thus, nonintact fragments that are truncated at termini the PTH will react in some putatively intact PTH immunoassays (22,23). Other studies suggest that PTH(7-84) is such a fragment (22,57). This PTH species accumulates in renal failure and may even be secreted by both normal and abnormal parathyroid glands (4,8,58-60). Further confounding clinical assessment, PTH(7-84) may act as an antagonist a n d / o r weak agonist to PTH at its receptor. Assays based on antibodies to the extreme amino- and carboxy-terminal regions of intact PTH may not be so c o n f o u n d e d and may thus be more clinically useful, especially in renal disease (4,8,22,23,60-63).

SPECIFIC CLINICAL APPLICATION

OF PTH IMMUNOASSAYS

Serum PTH immunoassays are invaluable in the differential diagnosis of hypercalcemia and hypocalcemia (1,4,8,10). In general, all well-characterized PTH immunoassays can serve this function, even though studies have focused on assays of the intact PTH (4,8). Most PTH immunoassays segregate patients with hypercalcemia and hypocalcemia into two respective categories, parathyroid disease and nonparathyroid disease (4,8,16,21). In the patient with hypercalcemia, an elevated serum PTH usually means primary hyperparathy-

roidism, and low or undetectable serum PTH usually means nonparathyroid disease. In the patient with hypocalcemia, an elevated serum PTH usually means secondary hyperparathyroidism, and a low or undetectable serum PTH usually means hypoparathyroidism. Direct comparisons of PTH fragment and intact immunoassays in the differential diagnosis of hypercalcemia show comparable clinical discrimination (4,8,38,45,47,53). In fact, midregion assays seem to identify the patient with primary hyperparathyroidism with more discrimination compared to the intact assay (4,10,35). For example, in one study of 36 patients with surgically proved primary hyperparathyroidism, a midmolecule assay recorded PTH values greater than twice normal in 28 patients, whereas an intact assay was similarly elevated in only 17 patients (42). However, in patients with nonparathyroid hypercalcemia such as malignancy, serum intact PTH is more completely suppressed than is serum midregion PTH, making that differential diagnosis easier (21,27). Furthermore, intact assays are less c o n f o u n d e d than PTH fragment assays, especially those of carboxy-terminal fragments, by the accumulation of biologically inert PTH fragments that takes place especially in renal failure, as detailed subsequently (25,28). And, as discussed elsewhere, intact assays have technical advantages over solution immunoassays (21 ). The improved quality of all contemporary PTH immunoassays makes them invaluable in the differential diagnosis of hypercalcemia (10,30). With many assays available, a practical approach to the differential diagnosis of calcium disorders is to use the PTH immunoassay that is readily available and interpretable to the physician. With most assays, the correct diagnosis will be obtained, although there is still the rare false, positive or false-negative result (55). In this respect, it should be kept in mind that some patients with primary hyperparathyroidism, especially those in the early course of the disease, may have serum PTH levels that fluctuate close to the normal range (10,16). In such case, multiple PTH measurements may be necessary to establish the correct diagnosis. For even more complicated cases, m e a s u r e m e n t by more than one PTH assay can help to resolve the differential diagnosis.

Primary Hyperparathyroidism Primary hyperparathyroidism (PHPT) is the most c o m m o n endocrine cause of hypercalcemia, and the most c o m m o n cause of PHPT is a single adenoma of the parathyroid glands (6,10,30). There are about 4 million cases of PHPT worldwide, so the disease has substantial impact (10,30). Rather than presenting to the physician with symptoms, as was the case in the last millennium, the patient with PHPT is now more likely to be asymptomatic and is seen by a physician because

PTH AND PTHrP IMMUNOASSAYS / of an elevated serum calcium detected by a multichannel screening (21,30). Parenthetically, it should be noted, however, that multichannel screening is becoming more limited by health maintenance organizations. When there are clinical manifestations of PHPT, they involve the skeleton, kidney, gastrointestinal tract, and central nervous system (64). In hospitals, PHPT is second only to malignancy as a cause of hypercalcemia; in the outpatient setting, PHPT is the most c o m m o n cause. Whereas malignancy dominates the clinical picture when it causes hypercalcemia, in PHPT the physician in the clinic is often faced with the challenge of establishing the cause of an elevated calcium that was detected in a multichannel screening rather than via a specific request, even though the advent of managed care has reduced the use of such screening tests (10,64). The availability of precise and accurate serum PTH assays has made the diagnosis of hyperparathyroidism relatively easy to establish, even u n d e r these circumstances (21). In fact, this is a most c o m m o n contemporary application of PTH immunoassays (21,64). PHPT is discussed in more detail in Chapters 18-23. PHPT usually can be distinguished from the other c o m m o n cause of hypercalcemia, malignancy, by a careful history and routine testing (10,21,30,64). In many patients with malignancy, the hypercalcemia is due to the production by the cancer of parathyroid hormonerelated protein (PTHrP) (10,65). As detailed later, immunoassays have been developed for this oncoprotein (65). T h o u g h their clinical application is discussed subsequently, it can be noted here that PTHrP does not cross-react in PTH assays. An elevated serum calcium and decreased fasting serum phosphorus support the diagnosis of PHPT. (To be diagnostically useful, the serum p h o s p h o r o u s must be fasting; otherwise, it may be perturbed by prandial excursions.) An elevated serum PTH establishes the diagnosis. By contrast, in hypercalcemia of malignancy, the serum phosphorus does not have a consistent pattern, and the serum PTH is usually suppressed by the hypercalcemia (10,30). Thus, when PHPT seems likely, the evaluation can often be completed by confirmation of the suspected diagnosis with an elevated serum PTH. The vast majority of patients with PHPT will have an elevated serum PTH in essentially all established immunoassay systems. The rare patient with PHPT due to parathyroid gland cancer will have exceptionally high serum PTH levels (Chapter 41) (10,66). Up to 95% of patients with PHPT will have elevated values in both intact and midregion assays, with a slightly higher percentage in the latter (33). Hypercalcemia and increased serum PTH are the signal manifestations of PHPT, although levels of both can fluctuate close to the u p p e r limits of normal early in the disease as it waxes and wanes (10,64,65). More than one m e a s u r e m e n t of serum calcium and sometimes PTH should be made for

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a combination of the technical and biologic reasons discussed earlier. Because PHPT waxes and wanes, especially during its early course, a single m e a s u r e m e n t might miss the period of increase. Serum PTH is now most commonly measured by a two-site assay designed to detect the intact molecule (4,21). This is the most widely used test, even though well-characterized midmolecule assays are at least equally accurate and precise in diagnosis (8,33). Amino- and carboxy-terminal terminal tests are not widely used because of the limitations discussed earlier (46-48). The u p p e r limit of normal for intact PTH is about 50-65 p g / m l . However, after the age of about 45 years, even intact PTH seems to increase in normal subjects, perhaps due to decline in renal function (64). But in the younger hypercalcemic patient, an intact PTH above the mid-40 p g / m l level should cause suspicion for PHPT (10). There are converse considerations with regard to serum calcium measurements (30). Most laboratories place the u p p e r limit of normal at 10.2-10.5 m g / d l . However, serum calcium slowly declines in the aging individual (10). Thus, after approximately age 50, a serum calcium above 10 m g / d l should also be regarded with appropriate clinical suspicion. The relationship between serum calcium and PTH m e a s u r e m e n t should also be considered. Because even in PHPT calcium feeds back on the secretion of PTH, a serum PTH approaching the u p p e r limits of normal may be considered as increased in the face of a serum calcium above normal (10,30). PTH immunoassays have been applied during parathyroidectomy in order to assess the success of the surgery (67,68). For this application, rapid assay formats have been developed so that results of the assay can be known before the surgical procedure ends (67). This becomes practical, because the half-life of some circulating species of PTH can be measured in minutes, especially intact PTH and amino-terminal fragments (4,8). In addition to enhancing the success of the surgery, some studies have suggested that the time of surgery can be decreased by intraoperative PTH assay (68).

Nonparathyroid Causes of Hypercalcemia The many nonparathyroid causes of hypercalcemia are discussed in Chapters 41-43. They include hyperthyroidism and hypoadrenalism; the diagnostically elusive familial hypocalciuric hypercalcemia (FHH); vitamin D, vitamin A, and lithium intoxication; thiazide diuretics; and several granulomatous disorders, notably sarcoidosis (10,69). However, the most important nonparathyroid cause is the hypercalcemia of malignancy, where overproduction of PTHrP, to be discussed subsequently, is a c o m m o n etiological culprit (65,69,70). It can be reemphasized here that there

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is essentially no cross-reactivity of PTHrP in PTH immunoassays (65). In reference to PTH assays, these nonparathyroid diseases are characterized by suppressed PTH secretion and low or absent serum concentrations (21,70). Thus, in these patients, the detection of a low or absent serum concentration of PTH by any of the reliable assay procedures discussed confirms the absence of parathyroid disease and points to a nonparathyroid cause for the hypercalcemia (65,67). F H H may be an exception to this rule in that a small percentage of patients can have increased serum PTH concentrations, although most have PTH levels that are normal but not suppressed by the hypercalcemia (65,70). Similarly, some patients with lithium-or thiazide-induced hypercalcemia can have increased concentrations of serum PTH (70). In all diagnostic situations, and especially in patients with nonparathyroid h o r m o n e hypercalcemia, it is important that the PTH measurement be made before calciumlowering therapy is instituted. Otherwise, the induced decrease in serum calcium can cause an increase in serum PTH, sometimes up to the hyperparathyroid range, and thus confound the diagnosis (10,70).

Hypocalcemia and Secondary Hyperparathyroidism Renal failure and hypoparathyroidism are the most c o m m o n etiologies of hypocalcemia, with hypoalbuminemia being a c o m m o n artifactual cause (71,74). Renal failure is by far most frequent a m o n g the causes of hypocalcemia, with the other causes accounting for only a small minority of cases (72,73). The etiologies of hypocalcemia can be generally classified as nonparathyroid and parathyroid, and the parathyroid diseases associated with hypocalcemia can be further classified as primary hypoparathyroidism and secondary hyperparathyroidism. The primary parathyroid causes of hypocalcemia are due to inadequate secretion of PTH (73,74). This type of primary hypoparathyroidism can follow neck surgery, including thyroidectomy and parathyroidectomy; it can occur as an isolated parathyroid disease; or it can be part of an a u t o i m m u n e endocrine deficiency syndrome that variably involves, a m o n g other tissues, the adrenal, thyroid, and pancreas (74). In renal failure, hypocalcemia develops because of abnormalities in phosphorous and vitamin D metabolism. The hypocalcemia leads to the development of secondary hyperparathyroidism. These disorders are detailed in Chapters 39 and 47. There are other less c o m m o n causes of secondary hyperparathyroidism, such as osteomalacia and rickets and other vitamin D and calcium disorders (discussed in Chapters 39 and 47. In addition to these chronic causes of hypocalcemia, acute failure of normal cal-

cium homeostasis can also cause hypocalcemia (73,74). Hyperphosphatemia that results from phosphate administration, rhabdomyolysis, or tumor lysis can produce severe hypocalcemia, especially in renal insufficiency. In acute pancreatitis, sequestration of calcium by saponification with fatty acids causes hypocalcemia. Rapid or excessive skeletal mineralization can cause hypocalcemia, as in the "hungry bone" syndrome and in osteoblastic metastases (10,30). These are discussed in Chapter 24. In primary hypoparathyroidism, PTH is low or absent and serum phosphorus is often increased because of the loss of the phosphaturic effect of PTH (8,73,74). In the secondary hyperparathyroidism seen in the nonparathyroid causes of hypocalcemia, the opposite occurs because of compensatory secondary hyperparathyroidism that increases the serum PTH and consequently decreases the serum phosphorus. An exception to this rule is pseudohypoparathyroidism, the genetic disease of end-organ resistance to PTH characterized by the biochemical features of hypoparathyroidism, a characteristic somatotype, and a secondary increase in serum PTH (75). Hypocalcemia related to hypomagnesemia can also present an unusual picture. Magnesium deficiency can cause hypocalcemia by impairing PTH secretion (1,73). So the PTH response to the hypocalcemia can be attenuated in the magnesium-depleted patient, with inappropriately low PTH levels in the presence of anatomically normal but functionally impaired parathyroid glands. As briefly discussed earlier, the calcium and skeletal abnormalities of renal failure directly lead to hypocalcemia, which, in turn, leads to secondary hyperparathyroidism. Consequently, the serum PTH and phosphorus are elevated and the serum calcium is low in these patients. Thus the measurement of serum PTH is a key procedure in differential diagnosis of hypocalcemia. With the exceptions discussed above, a decreased serum PTH identifies a parathyroid cause for the hypocalcemia (primary hypoparathyroidism) and an increased serum PTH identifies a nonparathyroid cause for the hypocalcemia accompanied by secondary hyperparathyroidism. As is the case for other parathyroid disorders, most well-characterized immunoassays for PTH will serve to distinguish a m o n g the parathyroid and nonparathyroid causes of hypocalcemia. Thus, the measurement of serum PTH is a most valuable test in the differential diagnosis of hypocalcemia.

Renal Osteodystrophy and Secondary Hyperparathyroidism Renal osteodystrophy is the name given to the complex of skeletal disorders that occur in renal failure

PTH AND PTHrP IMMUNOASSAYS / (71,72). Two abnormalities associated with declining renal function initiate this complex skeletal disease: increased serum phosphorus and decreased renal production of the active vitamin D metabolite, 1,25-dihydroxyvitamin D. The increased serum phosphorus causes hypocalcemia as does the decreased renal production of 1,25-dihydroxyvitamin D, which can also cause osteomalacia. These events lead to hypocalcemia, which in turn increases PTH secretion and, through this secondary hyperparathyroidism, causes the skeletal disease of PTH excess, osteitis fibrosa cystica (72,74). The parathyroid gland escapes the control of its mineral and hormonal regulators in part because of decreased expression of its calcium and vitamin D receptors (76,77). One or more glands can also undergo monoclonal expansion as the gland becomes hyperplastic. As the lowered calcium simulates PTH secretion by the parathyroid gland in renal disease, the increased serum phosphate concentration further increases hormone biosynthesis (71,76). Treatment is directed at reversing this process and returning the serum calcium, phosphorus, and PTH toward normal. Parathyroidectomy is reserved for those few patients whose medical m a n a g e m e n t has failed or whose disease has advanced to tertiary hyperparathyroidism. Renal transplantation is the ultimate treatment (74). The precise and accurate measurement of parathyroid gland secretory activity in renal failure is an important goal, because suppressing the hyperplastic gland toward normal secretory activity is a major end point of treatment (77-80). However, assessment of PTH secretion is complicated by the accumulation of all PTH fragments in renal failure, but especially carboxyterminal fragments that seem to more closely reflect creatinine clearance than parathyroid gland secretory activity (57-61). Assays that measure non-carboxy-terminal PTH fragments are thus easier to interpret in this context (4,8). The important clinical goal of assessing parathyroid secretory function in renal disease rather than measuring biologically irrelevant retained fragments of the hormone has been elusive (58,60). The advent of the intact PTH assay has been welcomed as a solution to this problem (4,8,21,81). However, comparison studies of the intact assay with other PTH assays shows them all to give spuriously elevated values because of their impaired renal clearance, a complex process that may involve the multifunctional clearance receptor, megalin (22,23,57,63). Furthermore, studies have demonstrated that some reportedly intact assays can be questioned regarding their assessment of gland secretion in that they also seem to measure PTH fragments that can be affected by impaired glomerular filtration (22,23). This may be due to the fact that their antibodies are

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not directed against the far termini of PTH, so that less than full-length fragments of the h o r m o n e are recognized (23). In fact, a newly described PTH fragment, PTH (7-84), has been observed to accumulate in renal failure, and it is variably measured by assays that were presumed to measure intact PTH(1-84) (23,63). This fragment may account for up to half of the circulating PTH immunoreactivity in patients on dialysis (22,23,63). So the recently recognized accumulation of PTH (7-84) in renal failure continues the dilemma of PTH assays and renal disease. This dilemma is further c o m p o u n d e d by the possibility that PTH(7-87) may be a PTH antagonist or weak agonist (23). New assays using antigenic determinants in PTH(1-6) are being developed to address this issue (22,23,63). These assays identify only half of the circulating concentrations of PTH in renal failure, as well as in other conditions (63). However, all progress in this area should be evaluated with the realization that the clinical value of PTH immunoassays is especially complex as an assessment tool in the secondary hyperparathyroidism of renal disease because the immunochemical heterogeneity of the molecule is further complicated by the accumulation of PTH fragments, both biologically agonistic, antagonistic, and inert (23,63). As is the case for primary hyperparathyroidism, however, all PTH assays have some value in the clinical m a n a g e m e n t of the patient with renal failure as long as the clinician is familiar with the proper interpretation of assay results. Tertiary Hyperparathyroidism Tertiary hyperparathyroidism is the name applied to secondarily hyperplastic parathyroid glands of renal failure that escape from secretory control of PTH by calcium, secrete even more PTH, and thereby lead to hypercalcemia (9,77,78). It has also been observed in certain vitamin D disorders (77,78). However, because of successful approaches to medical management, tertiary hyperparathyroidism is rare in renal disease. If hypercalcemia does occur in this setting, it is usually due to overtreatment with calcium a n d / o r vitamin D administration (10). So the diagnosis of tertiary hyperparathyroidism should not be made unless predialysis hypercalcemia can be demonstrated after t h e discontinuation of vitamin D and calcium administration. This distinction is important, because true tertiary hyperparathyroidism, a commonly monoclonal expansion of abnormal parathyroid cells, usually requires parathyroidectomy. In addition to calcium and vitamin D, other causes of hypercalcemia should also be ruled out before the diagnosis of tertiary hyperparathyroidism is made in renal disease.

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Adynamic Renal Osteodystrophy This form of low-turnover skeletal disease can be a significant problem in renal failure (78,79). Initially attributed to deposition of aluminum from drugs (e.g., aluminum hydroxide) or dialysate fluid, it is now appreciated that adynamic bone disease can occur in dialysis patients who are never exposed to excessive amounts of aluminum from either aluminum-containing phosphate binders or dialysate aluminum (79,80). Iron deposits may play a role in some patients (72,79,80). Because serum PTH is lower in patients with adynamic bone disease than in other forms of renal osteodystrophy, risk factors still include those that will suppress PTH, such as the use of dialysis fluid with high calcium concentration, use of calcium-containing phosphate binders (e.g., calcium carbonate), as well as the now rare use of aluminum-containing drugs (79,80). This rare variant of renal osteodystrophy should be suspected in the patient with renal disease whose serum PTH is low relative to the increased concentrations generally seen in renal failure (72,79).

Other Skeletal and Mineral Disorders Serum PTH levels are generally normal in most common skeletal disorders such as osteoporosis, unless they are complicated by hyperparathyroidism. How-ever, there may be subtle abnormalities of PTH secretion in some skeletal diseases (81,82). Furthermore, there is a seemingly increased incidence of primary hyperparathyroidism in Paget's disease of bone, involving up to 10% of the patients in some studies (82). Furthermore, most patients with osteomalacia will have secondary hyperparathyroidism because of the accompanying hypocalcemia (73,74). If the responsive increase in PTH secretion substantially corrects the hypocalcemia, serum calcium levels may approximate the normal range. However, in the absence of renal disease, there will be a deceased serum phosphorus caused by the phosphaturic

TABLE 1

effect of the increased PTH (73). The combination of a low serum calcium and phosphorus and a high PTH is the signature of secondary hyperparathyroidism. Table 1 summarizes the c o m m o n laboratory findings that can be useful in establishing the correct diagnosis in the patient with an elevated PTH. The differential diagnosis of hypercalcemia and hypocalcemia is discussed in detail in Chapters 41 and 47.

PTHrP BIOSYNTHESIS, PROCESSING, AND SECRETION Overview Parathyroid hormone-related protein can be characterized as an oncofetal protein (83-86). Originally discovered as a product of breast and lung cancer cells that produced hypercalcemia, PTHrP is now known to be produced by many normal and malignant tissues, with and without hypercalcemia, and regulated by a variety of factors (83-85). The amino terminus of PTHrP reacts with the P T H / P T H r P receptor and has the potential to produce most of the biologic effects of native PTH, including hypercalcemia (84,86). Although multiple, the functions of PTHrP in malignant and normal tissues seem to be related to cell growth and proliferation (87,88). A variety of factors, many of them also growth regulatory, affect the production of PTHrR T h o u g h ambient calcium mediates its effects on PTHrP through the calcium sensor, as it does for PTH, the effects are more complex than for PTH and can be opposite, in that increased ambient calcium can increase PTHrP production (1,87). More details about the physiology and pathophysiology of PTHrP can be found in Chapter 3. Despite many studies demonstrating the high frequency of PTHrP expression in many malignant tumors, secretion studies of PTHrP in blood have had limitations (89). T h o u g h PTHrP expression is c o m m o n

Routine Serum Tests in the Differential Diagnosis of the Patient with an Increased Serum PTH a Diagnosis

Calcium

Phosphorus

BUN/creatinine

Primary hyperparathyroidism Secondary hyperparathyroidism Renal Nonrenal Pseudohypoparathyroidism Tertiary hyperparathyroidism

H

L/N

N

L L L H

H L H/N H

H N N H

all,

High; L, low; N, normal; BUN, blood urea nitrogen.

PTH AND PTHrP IMMUNOASSAYS

Biosynthesis of PTHrP The expression of PTHrP is controlled by a complex gene that resides on chromosome 12 and seems related in evolution to the PTH gene (82,84). The PTHrP gene sequence spans more than 15 kb and is composed of

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three primary regions: a complex promoter region, a coding region, and a multiple 3' noncoding region (83). The gene's promoter region contains three promoter elements, designated P1, P2, and P3. P1 and P3 are "TATA box" like, and the P2 element is a GC-rich region (85). The PTHrP gene expresses three native forms of the polypeptide through alternate mRNA splicing, PTHrP(1-141), a truncated 139-residue form, and a 173-residue form expressed primarily in humans (Fig. 2). Whereas PTHrP(1-139) is quite similar to PTHrP(1-141), PTHrP(1-173) completely diverges from both at its own carboxy terminus (83,85). It is not surprising that the biosynthesis of PTHrP shares many features with the biosynthesis of PTH, because they both follow the biologic rules of polypeptide biosynthesis (85,86). Thus, PTHrP contains a leader or prepro sequence whose components take it through a cellular journey not unlike that of PTH (86). However, PTHrP differs from PTH in that three isoforms of the polypeptide are encoded by the h u m a n PTHrP gene, and each of these exhibits multiple processing sites that can release unique peptides (83). The biologic effects of PTHrP are mediated, at least in part, through the receptor that it shares with PTH, which is a m e m b e r of the seven membrane-spanning and G protein-coupled cell surface receptors (84). PTHrP also contains nuclear localizing sequences that

in cancer and hypercalcemia, it usually occurs in advanced disease (90,91) And many patients who have PTHrP-expressing tumors fail to demonstrate hypercalcemia and abnormally increased serum PTHrP concentrations (89,90). However, new PTHrP assays of improved sensitivity and specificity are being developed to address these clinical limitations; they are becoming important tools in the evaluation of the patient with hypercalcemia (89-92). As for PTH, a detailed background in relevant physiology for a full understanding of the role of PTHrP in cancer is contained in Chapter 41. The following synopsis of the biosynthesis, secretion, and metabolism of PTHrP focuses on the rationale that has been used for PTHrP immunoassay assay development for clinical application. This background synopsis is followed by a discussion of the application of PTHrP immunoassays to specific disorders of calcium and skeletal metabolism, especially hypercalcemia of malignancy.

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156

/

CHAPTER9

may be responsible for some of its biologic effects (93). The most clinically important pathophysiologic effect of PTHrP is hypercalcemia (see Chapter 13 for more details about PTHrP biosynthesis and effects).

Processing of PTHrP In addition to biosynthesis of the three isoforms, PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173), processing of PTHrP into peptides is an important regulatory mechanism (85,93). The processing of PTHrP is complex, because there are many processing sites within each of the PTHrP isoforms (Fig. 2). Enzymes have been identified from mammalian tissues that seem to serve this function, including furin, PC2, and PC3 (86,88), although the specific enzymes involved in PTHrP processing have not been conclusively established. In addition to classic basic amino acid processing sites, PTHrP can also be processed at monobasic sites, such as Arg-36 (93). T h o u g h many peptides are predicted by the processing sites in PTHrP, the presence of only a few has been experimentally demonstrated or theoretically implied. In addition to P T H r P ( 1 - 3 6 ) , they include 1-38, 1-74, 1-94, 1-95, 37-74, 1-101, 67-86, 107-139, 126.144, and 141-173 (83-92). It has not been d e t e r m i n e d which of these peptides is the result of processing, protease degradation, or both. As is the case for PTH, the demonstration of these peptides has resulted from both in vitro and in vivo studies (91,92). But the situation is more complicated than for PTH, for which only one gland is the source of the hormone; PTHrP a can arise from a wide variety of cancers and is thus susceptible to tissue-specific regulation (87,88). Accordingly, most immunoassays for PTHrP have been developed along empirical lines (90,92). Distinct biologic properties have been attributed to the different PTHrP peptides (84,93). For example, P T H r P ( 1 - 3 4 / 3 6 ) mediates the growth-regulating and hypercalcemic effects of the molecule, PTHrP(35-94) promotes placental calcium transfer, and peptides included in PTHrP(109-141) inhibit osteoclast function (88,93). Preliminary studies of PTHrP(140-173) suggest that it also has growth-regulating effects (94,95). Although these PTHrP peptides appear to have distinct biologic properties, their structures do not necessarily conform to those predicted by processing of the native molecules (93). Furthermore, relatively little is known about the tissue-specific processing of PTHrP, but it is u n d e r intense study (89-95). In addition to its fundamental importance, identifying the peptides processed from the three native forms of PTHrP will help in the design of immunoassays with improved specificity, because PTHrP and its related peptides are

expressed and secreted by several c o m m o n cancers and they have the potential to serve as tumor and serum markers for such malignancies (89,90). The biochemical hallmark of the PTHrP-producing cancer is hypercalcemia, an effect mediated by the amino terminus of PTHrP through the receptor that it shares with PTH (84,88) (more details of PTHrP processing can be found in Chapter 3).

PTHrP and Cancer PTHrP and its related peptides are expressed and secreted by many cancers, and they have the potential to serve as tumor and serum markers for such malignancies (83-92). PTHrP is the most c o m m o n mediator of the hypercalcemia of malignancy (90,91). It has a been estimated that 80% of hypercalcemic patients with cancer have elevated serum levels of PTHrP (91,92). In some types of tumors, the percentage is even higher (89,92). T h o u g h PTHrP expression was initially noted to be c o m m o n in squamous cell cancers, it has been subsequently shown that many other cancer types can overexpress PTHrP (90,91 ). The biologic and clinical importance of PTHrP in breast cancer has become well established (87,96-98). PTHrP production and secretion by breast cancers are very common, occurring in 50-60% of cases, with an even higher incidence when the patient is hypercalcemic (87,91). Breast tumors that produce PTHrP are more likely to metastasize, and breast cancers that metastasize to bone are even more likely to produce PTHrP (89,90,92). Breast cancers and their bone metastases commonly express the PTHrP receptor, and breast cell lines and primary cultures also commonly express PTHrP and its receptor (88,96-99). PTHrP and its peptides are secreted into blood by such breast cancers, and they can often serve as tumor and serum markers for this cancer (96--101). Prostate cancers robustly express PTHrP (94,95, 102-104). In the prostate, as in other tissues, PTHrP is processed into distinct peptides that have unique biologic effects (95,103,104). Prostatic expression of PTHrP is associated with regulatory effects and interactions that are important in the development and progression of prostate cancer (94,104). Furthermore, studies provide evidence for a role of PTHrP expression in the development of bone metastases in prostate cancer (102). However, serum measurements of PTHrP are not yet useful for clinical application in prostate cancer, because levels are seldom elevated (95,105). Furthermore, patients with prostate cancer seldom have osteolytic bone metastases and hypercalcemia; in fact, they are more likely to be hypocalcemic with osteoblastic metastases (95,100,102).

PTH AND PTHrP IMMUNOASSAYS /

157

PTHrP is commonly expressed in lung cancer, but, as in breast cancer, increased serum concentrations can be detected only in late stages of the disease (89,106,107). Nevertheless, this oncoprotein has important implications for the pathogenesis, diagnosis, and treatment of lung cancer. Abnormal PTHrP production by lung cancer can be demonstrated with specific immunochemical and nucleic acid probes (91,97,93). PTHrP is often produced in those lung cancers that metastasize to bone, and it is a common causative humoral agent in the patients that have hypercalcemia of malignancy (87,88,106,107). Lung cancer is intermediate to prostate and breast cancer in the incidence of"humoral hypercalcemia" (87,95,106). PTHrP and PTHrP peptides can have profound effects on growth and function of lung cells (107,112). However, as with prostate cancer, the majority of patients with lung cancer, including those with hypercalcemia, do not commonly have increases in serum PTHrP (90,106-108).

terminus through the receptor that it shares with PTH (5,84). Most patients with hypercalcemia and malignancy have PTHrP-expressing cancers (81,92). Despite the common pattern of PTHrP expression in many cancers, clinical secretion studies have revealed that many patients with PTHrP-expressing tumors and eucalcemia do not have elevated serum levels of PTHrP (90,113, 114). In general, most serum PTHrP assays do not yet have the sensitivity a n d / o r specificity to make them a clinically useful biomarker for the early course of the cancer, a problem compounded by the poorly defined normal range of PTHrP, discussed subsequently (89,91,92). In many ways, the current status of the development of PTHrP immunoassays is reminiscent of the comparably early days of PTH assay development, although PTHrP assay development is occurring at a faster pace (89-92). Accordingly, there is substantial promise that clinically useful assays for PTHrP will be eventually developed (see Chapters 3 and 6) for more details about PTHrP secretion).

Secretion of PTHrP

PTHrP Immunoassays

Despite studies demonstrating the common pattern of PTHrP expression in many cancers, especially breast, lung, and prostate, secretion studies have limited impact in that elevated levels of PTHrP are discovered relatively late in the course of the patient with hypercalcemia and malignancy (90,109,111). And the comm o n occurrence of PTHrP expression in prostate cancer is seldom accompanied by either hypercalcemia or elevated serum PTHrP levels (95,105). Thus, many patents who have PTHrP-expressing tumors fail to demonstrate abnormally increased serum PTHrP concentrations that are clinically useful. Most serum PTHrP measurements, especially solution immunoassays, do not have the sensitivity a n d / o r specificity for cancer-produced PTHrP forms to make them valuable biomarkers for the early diagnosis of many malignancies (89-92). However, as for PTHrP, midregion assays seem to be the most sensitive (89,90,113,114). In contrast to PTH, for which serum assays can often detect early parathyroid disease, serum levels of PTHrP are likely to be increased only when the cancer is advanced and the patient is hypercalcemic (91,92,110,111). In summary, PTHrP is produced by many normal and malignant tissues, and in these tissues, PTHrP expression commonly produces abnormal growth and proliferation (84,87,92). Through complex gene expression, mRNA splicing, and peptide processing of PTHrP, the h u m a n PTHrP gene expresses three native forms of the polypeptide and multiple processed peptides with distinct biologic properties (85,93). The hypercalcemic effect of PTHrP is mediated by its amino

It took over 30 years of development for the clinical application of serum PTH assays to be substantially realized, thus it is not surprising that current serum PTHrP assays still have clinical limitations. Nevertheless, there has been promising activity in this area of assay development. To correspond to actual and potential PTHrP processing, immunoassays for PTHrP have been designed to recognize peptides across the linear sequence of the native molecules. Investigators have essentially traversed the PTHrP molecule, developing solution (one-site) immunoassays for PTHrP(1-34), 1-40, 33-67, 37-74, 50-69, 53-84, 67-86, 109-138, 109-141, 127-141, and 141-173, and solid-phase (two-site) immunoassays for PTHrP(1-67), 1-72, 1-74, 1-83, 1-84, and 1-86, all of which have been applied to a variety of clinical studies (115-157). These many assays systems reflect the fact that, because of the complexity of PTHrP biosynthesis and processing, most PTHrP immunoassays have been developed empirically (91,92). It should be noted that PTH, as is the obverse for PTHrP, does not cross-react in these assays systems, because the homology between the two molecules is primarily limited to the first 13 amino acids (83). Despite their format differences, there is substantial uniformity among the different PTHrP assays in their clinical applications and characteristics. Most of the PTHrP assays can identify the majority of patients with hypercalcemia and PTHrP-producing tumors, but only the minority of patients with eucalcemia and cancer, including such patients with tumors demonstrated to

158

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CrtAeTER9

express PTHrP (89,90,98). PTHrP assays also demonstrate that, as is the case for PTH, the circulating forms of PTHrP are immunochemically heterogeneous (89-91). And like PTH immunoassays, PTHrP immunoassays that detect the midregion of the molecule seem to be the most sensitive (91,92,113). Although current assays can identify many patients with PTHrP-producing cancer, they do not yet seem to be able to identify the early course of the malignancy, where they would be most clinically useful (89,92).

PTHrP Immunoassay Development Solution Immunoassays The development of PTHrP immunoassays has, in many ways, mirrored the course of the development of PTH immunoassays, with region-specific solution immunoassays developed first, followed by the development of solid-phase, two-site immunoassays (122,129,156,157). Among the first solution immunoassays were aminoterminal PTHrP assays based on PTHrP (1-34) (91,156). These were followed by solution immunoassays based on the carboxy terminus and the midregion of PTHrP (129,151,157). These regional peptide designations were based on PTHrP(1-141), not PTHrP(1-173), so the carboxy terminus meant a peptide in the vicinity of PTHrP(109-141), and the midregion meant a peptide in the vicinity of PTHrP(36-67) (93,151). In general, these solution immunoassays could identify over half of the hypercalcemic cancer population, although a higher percentage was reported for some of them, with midregion assays identifying the most (89,90). Like PTH, midregion PTHrP assays resulted in higher PTHrP values than did amino-terminal assays (90). These assays also varied in their ability to detect serum PTHrP in normal subjects, ranging from some measuring it in none to others measuring it in all (91,92).

Two-Site Immunoassays Two-site immunoassays first focused on the amino PTHrP region, including PTHrP(1-34), 1-67, 1-74, and 1-84 (89-92,132,140,147,155). They had all of the advantages of the two-site PTH immunoassays discussed earlier, including improved specificity, freedom from serum protein-related artifacts, and technical ease (89). Some of them had improved clinical application, distinguishing better than solution immunoassays between normal subjects and patients with hypercalcemia and malignancy (90-92). With some two-site assays, more than 80% of patients with malignancy-associated hypercalcemia have elevated circulating PTHrP values (90,91). However, assays with greater sensitivity had more false positives, and the assays with lesser sensitivity had fewer false positives (92). There was less overlap between the two groups with intact assays, but the normal PTHrP

ranges tended to be somewhat arbitrary (89,90). The apparently improved clinical performance of two-site assays and their technical advantages have resulted in their wide clinical application (89-93). False positives aside, increased assay sensitivity is desirable for monitoring the course of the patient with a PTHrP-producing tumor, because that patient's pretreatment concentration will serve as the control for evaluation (90,93).

Specific Clinical Application of PTHrP Immunoassays Hypercalcemia and Malignancy PTHrP assay is most commonly used to establish pathogenesis in the patient with hypercalcemia, usually with cancer (91,92). In general, most patients with hypercalcemia and solid tumors have elevated serum PTHrP levels regardless of tumor type (85-92). This picture has evolved from early studies that suggested that squamous cell tumors were more likely to be associated with increased serum levels of PTHrP (89,90). In contrast, most patients with malignancy and normal calcium levels do not have elevated serum levels of PTHrP (87,90). Although there are exceptions to this generality, it holds true for most PTHrP assay systems (91,92). PTHrP seems to play only a minor role in hematologic malignancy, with the exception of adult T cell l e u k e m i a / l y m p h o m a (91,140,141). Although there is variability, in approximately half of these patients the hypercalcemia is associated with increased PTHrP expression by the cancer cells (89,90,140,141). This contrasts to myeloma and hypercalcemia, for which increased serum PTHrP is rare, even when the patient is hypercalcemic (89,141). Incidentally, the same situation seems to pertain to sarcoidosis (158). In contrast to PTHrP production in cancer, PTH expression by malignancy is now known to be so rare that it is reportable (89,91,159-166). In fact, an elevated serum PTH in the patient with malignancy (and the absence of renal failure and secondary hyperparathyroidism) most likely reflects coexisting PHPT. Several studies of PTHrP and hypercalcemia also assessed the presence or absence of skeletal metastases (89,97,126,156). In general, there can be a dissociation among these clinical events. Thus, many tumors producing PTHrP contribute to the development of hypercalcemia regardless of the presence or absence of skeletal metastases (89,100). PTHrP can thus act as a "humor" in the hypercalcemia of malignancy and stimulate osteoclastic bone resorption through the increased circulating levels derived from tumor expression (89,92). Of course, i n d e p e n d e n t of PTHrP production, a metastatic tumor can have the same effect, referred to as local osteolytic hypercalcemia (LOH) to distinguish it from the humoral hypercalcemia of malignancy (160).

PTH AND PTHrP IMMUNOASSAYS / Parathyroid adenomas or hyperplastic glands secondary to renal disease commonly express PTHrP mRNA (167-169). However, most reports demonstrate increased circulating levels of PTHrP only in secondary hyperparathyroidism, where accumulation of fragments can contribute to the measurement (88,109,143). As is the case in the secondary hyperparathyroidism of renal failure, carboxy-terminal PTHrP fragments accumulate more than the other fragments (109,134,143,157). The most important clinical relationship between PTH and PTHrP is the dissociation between the two found in hypercalcemia and malignancy, where serum PTH is suppressed and serum PTHrP is commonly increased (88).

Malignancy and Eucalcemia A more complicated relationship between PTHrP and malignancy seems to be present in cancer patients who are not hypercalcemic, as exemplified by prostate cancer (95,105,114). Prostate cells commonly express PTHrP (103,104). Furthermore, there seems to be a direct relationship between PTHrP expression and abnormal growth in that there is a directed gradient of PTHrP expression from normal cancer cells, through hyperplastic prostate cells, to malignant prostate cells (102,103). Despite this relationship, prostate cancer is rarely associated w i t h hypercalcemia, even though the tumor often metastasizes to bone, where it commonly produces osteoblastosis rather than osteolysis (95,100,102). It is interesting to speculate that this clinical riddle could be due to the prostate-specific processing of PTHrP into a peptide that inhibits osteoclast function, like peptides contained in PTHrP (107-141) (95). Nevertheless, serum PTHrP levels are not elevated in patients with prostate cancer, regardless of the absence or presence of bone metastases (89,95). Despite the disappointing lack of clinical value of serum PTHrP in the early stages of malignancy, where assays would be most valuable, some studies suggest that with appropriate specificity, PTHrP assays could be useful in early diagnosis (90). In one such study, circulating PTHrP levels were examined with three different immunoassays in 48 eucalcemic breast cancer patients (98). These immunoassays were directed against different parts of the PTHrP molecule. The methods used were a radioimmunoassay with antibodies directed against PTHrP(63-78), an immunofluorometric assay with antibodies against PTHrP (1-34) and PTHrP(38-67), and an immunoradiometric assay with antibodies against PTHrP(1-40) and PTHrP (38-72). PTHrP was detected by immunohistochemistry in tumors from nearly all patients. T h o u g h most patients had PTHrP levels indistinguishable from normal when measured by all three methods, 10% had increased serum levels in the IFMA. The IFMA thus

159

identified increased serum PTHrP forms in some patients with PTHrP-expressing breast cancer who were not hypercalcemic and presumably in the early course of their disease. By contrast, two other assays failed to distinguish between normal and breast cancer subjects, one being a commercial assay measuring PTHrP(1-74) and the other a research assay based on PTHrP (63-78). A hypothesis-setting explanation for this finding is that the forms/species of PTHrP secreted by the early breast cancers corresponded more closely to the PTHrP(1-67) form. This could indicate that there is a circulating form of PTHrP extending from the amino terminus to residues 58 or 66, where cleavage sites of basic amino acid residues are situated. This view is supported by studies that found no measurable serum levels of PTHrP(1-86) in unselected patients with breast cancer prior to surgery, although most of them had tumors with positive staining of PTHrP (89,92,157). Because there are cleavage sites at amino acid residues 66 (arginine) and 58 (arginine), one of the assays could be detecting a fragment extending from the amino terminus to one of these amino acid residues (85). However, these results do not explain the absence of hypercalcemia in the patients, because this effect is presumably due to PTHrP(1-34). Thus, it could be valuable to measure a shorter amino-terminal fragment of PTHrP, especially considering that there are several cleavage sites in this peptide and that PTHrP(1-36) is contained in a secretory form of the molecule.

Normal Range of Serum PTHrP and Other Problems The clinical limitations of current PTHrP immunoassays are exemplified by the contradictory studies regarding the circulation of PTHrP in normal individuals (91,92). In some studies, detectable levels of PTHrP are present in only a few normal individuals, while in other studies detectable levels of PTHrP are detected in most individuals (89,90). To add to the confusion, intermediate results have also been reported (91,109). This variability is not a function of assay sensitivity, because conflicting results have been reported by PTHrP immunoassays with both relatively greater and lesser respective sensitivities (89-95). A well-defined normal range for PTHrP, as for any clinical measurement, is obviously critical for meaningful interpretation of assay results. Some of the problems with contemporary PTHrP immunoassays may have a technical basis. Immunologic PTHrP activity may be labile and sensitive to serum proteases, and impaired renal function, c o m m o n in cancer, made confound PTHrP measurements because of the accumulation of PTHrP fragments, especially, as is the case for PTH, carboxy-terminal fragments (91,134). Studies of the normal values of PTHrP may be further

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CHAPTER9 TABLE 2

Routine Serum Tests in the Differential Diagnosis of the Patient with a Decreased Serum PTHa Diagnosis

Primary hypoparathyroidism Humoral hypercalcemia of malignancy (HHM) Nonparathyroid, non-HHM hypercalcemia Adynamic renal osteodystrophy Hypomagnesemia (severe)

Calcium

Phosphorus

BUN/creatinine

L

H/N

H

H/L/N

N N/H

H

H/N

N

L (relative to the degree of renal failure)

H

H

L

H/N

N

all, High; L, low; N, normal; BUN, blood urea nitrogen.

complicated by its lability, especially because these measurements are close to assay detection limits (91,92,170). Accordingly, proteases are used for sample collection in some assays, and, in all cases, the sample should be processed as quickly as practicable, following the guidelines outlined adapted from PTH assays (90,171). With some dramatic exceptions, circulating PTH concentrations in lactating women are essentially within the normal range, even though there are substantial concentrations of PTHrP in breast milk (118,172). In summary, the m e a s u r e m e n t of serum PTHrP is beginning to provide clinically useful information for the m a n a g e m e n t of the patient with cancer, especially when complicated by hypercalcemia (88-93). However, many hurdles have to be overcome before serum PTHrP assays are regarded by the clinician with the same confidence now conferred to contemporary PTH assays. The robust research activity in the development of PTHrP immunoassays promises to address the limitations of current assay systems, just as research during the past several decades has provided PTH immunoassays of great clinical value. C o n t e m p o r a r y assays exhibit increased serum PTHrP in the majority of patients in whom the malignancy causes hypercalcemia. This is usually a late clinical event of some diagnostic value but has relatively little impact on patient m a n a g e m e n t . W h e n developments lead to PTHrP immunoassays that have the specificity and sensitivity to measure putative tumor-specific forms of the native molecules and their derived peptides early in the course of the cancer, the promise of the clinical value of PTHrP assays in clinical cancer m a n a g e m e n t as well as diagnosis may be fulfilled. Table 2 summarizes the c o m m o n laboratory findings that can be useful in establishing the correct diagnosis in the patient with a decreased PTH, a substantial number of whom have increased serum PTHrP due to a PTHrP-producing tumor. The differential diagnosis of hypercalcemia and hypocalcemia are discussed in detail in Chapters 41 and 47).

As assay development proceeds, the combination of accurate and precise PTHrP and PTH immunoassays can help to establish the correct diagnosis in the patient with calcium and skeletal disorders. FDA-approved tests can be found on the Internet at www.accessdata.fda.gov.

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121. Yang KH, deOaoo AE, Soifer NE, Dreyer BE, Wy TL, Porter SE, Bellantoni M, Burtis WJ, Insogna KL, Burtis WJ, Insogna KLM, Broadus AE, et al. Parathyroid hormone-related protein: Evidence for isoform and tissue-specific posttranslational processing. Biochemistry 1994;33:7460-7469. 122. Ratcliffe WA, Norbury S, Scott RA, Heath DA, Ratcliffe JG. Immunoreactivity of plasma parathyrin-related peptide: Three region-specific radioimmunoassays and a two-site immunoradiometric assay compared. Clin Chem 1991;37:1781-1787. 123. Ratcliffe WA, Norbury S, Heath DA, Ratcliffe JG. Development and validation of an immunoradiometric assay of parathyrinrelated protein in unextracted plasma. Clin Chem 1991;37:678-685. 124. Imamura H, Sato K, Shizume K, Satoh T, Kasono K, Ozawa M, Ohmura E, Tsushima T, Demura H. Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice. J Bone Miner Res 1991 ;6:77-84. 125. Kitazawa S, Fukase M, Kitazawa R, Takenaka A, Gotoh A, Fujita T, Maeda S. Immunohistologic evaluation of parathyroid hormonerelated protein in human lung cancer and normal tissue with newly developed monoclonal antibody. Cancer 1991 ;67:984-989. 126. Grill V, Ho P, BodyJJ, Johanson N, Lee SC, Kukreja SC, Moseley JM, Martin TJ. Parathyroid hormone-related protein: Elevated levels in both humoral hypercalcemia of malignancy and hypercalcemia complicating metastatic breast cancer. J Clin Endocrinol Metab 1991;73:1309-1315. 127. Pandian MR, Morgan CH, Carlton E, Segre GV. Modified immunoradiometric assay of parathyroid hormone related protein: Clinical application in the differential diagnosis of hypercalcemia. Clin Chem 1992;38:282-288. 128. Grill V, Murray RM, Ho PW, Santamaria JD, Pitt P, Potts C, Jerums G, Martin TJ. Circulating PTH and PTHRP levels before and after treatment of tumor induced hypercalcemia with pamidronate disodium (APD). J Clin Endocrinol Metab 1992;74:1468-1470. 129. Ratcliffe WA, Bowden SJ, Emly J, Hughes S, Ratcliffe JG. Production and characterization of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein. J Immunol Methods 1992;146:33-42. 130. Blind E, Raue F, Goltzman J, Schmidt-Gayk H, Kohl B, Ziegler R. Circulating levels of midregional parathyroid hormonerelated protein in hypercalcemia of malignancy. Clin Endocrinol. 1992;37:290-297. 131. Ratcliffe WA, Hutchesson AC, Bundred NJ, Ratcliffe JG. Role of assays for parathyroid-hormone-related protein in investigation of hypercalcemia. Lancet 1992;339:164-167. 132. Burtis WJ, Fodero JE Gaich G, Debeyssey M, Stewart AF. Preliminary characterization of circulating amino-and carboxyterminal fragments of parathyroid hormone-related peptide in humoral hypercalcemia of malignancy. J Clin Endocrinol Metab 1992;75:1110-1114. 133. Bucht E, Eklund A, Toss G, Lewensohn R, Granberg B, Sjostedt U, Eddeland R, Torring O. Parathyroid hormone-related peptide, measured by a midmolecule radioimmunoassay, in various hypercalcemic and normocalcemic conditions. Acta Endocrinol 1992; 127:294-300. 134. Savage MW, Fraser WD, Bodmer CW, Ginty AF, Gallagher JA, Robinson J, Williams G. Hypercalcemia due to parathyroid hormone-related protein: Long-term circulating levels may not reflect tumour activity. Clin Endocrino11993;39:695-698. 135. Fraser WD, Robinson J, Lawton R, Durham B, Gallacher sJ, Boyle IT, Beastall GH, Logue FC. Clinical and laboratory studies of a new immunoradiometric assay of parathyroid hormonerelated protein. Clin Chem 1993;39:414-419.

136. Blind E, Raue F, Meinel T, Pecherstorfer M, Rath U, Schmidt-GGayk H, Kohl B, Ziegler R. Diagnostic significance of parathyroid hormone-related protein in tumor patients with hypercalcemia. Dtsch Med Wochenschr 1993;118:330-335. 137. Blind E, Raue F, Meinel T, Bucher M, Manegold C, Ebert W, Vog-Moykopf I, Ziegleer R. Levels of parathyroid hormonerelated protein in hypercalcemia of malignancy: Comparison of midregional radioimmunoassay and two-site immunmoradiometric assay. Clin Invest 1993;71:31-36. 138. Mune T, Katakami H, Morita M, Noguchi S, Ushiroda Y, Matsukura S, Yasuda K, Miura K. Increased serum immunoreactive parathyroid hormone-related protein levels in chronic hypocalcemia. J Clin Endocrinol Metab 1994;78:575-580. 139. Levin GE, Nisbet JA. Stability of parathyroid hormone-related protein and parathyroid hormone at room temperature. Ann Clin Biochem 1994;31:497-500. 140. Ikeda K, Ohno H, Hane M, Yokoi H, Okada M, Honma T, Yamada A, Tatsumi Y, Tanaka T, Saitoh T, et al. Development of a sensitive two-site immunoradiometric assay for parathyroid hormone-related peptide: Evidence for elevated levels in plasma from patients with adult T-cell leukemia/lymphoma and B-cell lymphoma. J Clin Endocrinol Metab 1994;79:1322-1327. 141. Yamaguchi K, Kiyokawa T, Watanabe T, Ideta T, Asayama K, Mochizuki M, Blank A, Takatuski K. Increased serum levels of C-terminal parathyroid hormone-related protein in different diseases associated with HTLV-1 infection. Leukemia 1994;8:1708-1711. 142. Hutchesson AC, Hughes SV, Bowden SJ, Ratcliffe WA. In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma. Ann Clin Biochem 1994;31:35-39. 143. Burtis WJ, Dann P, Gaich GA, Soifer NE. A high abundance midregion species of parathyroid hormone-related protein: Immunological and chromatographic characterization in plasma. J Clin Endocrinol Metab 1994;78:317-322. 144. Sagarra E, Villabona C, Bonnin R, Moliner R, Merino FJ, Sahun M, Soleer J. The value of the parathyrin-related protein (PTHRP) in the diagnosis of cancer-associated hypercalcemia. Med Clin 1995;105:450--454. 145. Ramirez MM, Fraher LJ, Goltzman D, Hendy GN, Matthews SG, Sangha R, Challis JR. Immunoreactive parathyroid hormonerelated protein: Its association with preterm labor. EurJ Obstet Gynecol Reprod Biol 1995;63:21-26. 146. Bucht E, Rong H, Bremme K, Granberg B, Rian E, Torring O. Midmolecular parathyroid hormone-related peptide in serum during pregnancy, lactation and in umbilical cord blood. E u r J Endocrinol 1995;132:438-443. 147. Minebois-Villegas A, Audran M, Lortholary A, Legrand E, Boux De Casson-Raimbeau F, Jallet E Performances of two kits for parathyroid hormone-related peptide (PTHRP) assay in the additional study of malignant hypercalcemias. Pathol Biol 1995;43: 799-805. 148. Caplan RH, Wickus GG, Sloane K, Silva PD. Serum parathyroid hormone-related protein levels during lactation. J Reprod Med 1995;40:216-218. 149. Seguara Dominguez A, Andrade Olivie MA, Rodriguez Sousa T, Terron Alvarez ML, Rodriguez Perez D, Alvarez Novoa R, Garcia-Mayor RV. Plasma parathyroid hormone related-protein levels in patients with cancer, normocalcemic and hypercalcemic. Clin Chim Acta 1996;244:163-172. 150. Sowers ME Hollis BW, Shapiro B, Randolph J, Janney CA, Zhang D, Schork A, Crutchfield M, Stanczyk F, Russell-Auleet M. Elevated parathyroid hormone-related peptide associated with lactation and bone density loss. JAMA 1996;276:549-554. 151. Nagasaki K, Otsubo K, Kajimura N, Tanaka R, Watanabe H, Tachimori Y, Kato H, Yamaguchi H, Saito D, Watanabe T, Adachi I, Yamaguchi K. Circulating parathyroid hormone-

P T H AND P T H r P IMMUNOASSAYS

152.

153.

154.

155.

156.

157.

158.

159.

160.

related protein (109-141) in malignancy-associated hypercalcemia. Jpn J Clin Onco11996;26:6-11. Lippuner K, Zehnder HJ, Casez JP, Takkinen R, Jaeger P. PTH-related protein is released into the mother's bloodstream during lactation: Evidence for beneficial effects on maternal calcium-phosphate metabolism. J Bone Miner Res 1996;11:1394-1399. Wu TJ, Taylor RL, Kao PC. Parathyroid-hormone-related peptide immunochemiluminometric assay. Developed with polyclonal antisera produced from a single animal. Ann Clin Lab Sci 1997;27:384-390. Hirota Y, Anai T, Miyakawa I. Parathyroid hormone-related protein levels in maternal and cord blood. Am J Obstet Gynecol 1997;177: 702-706. de Miguel E Motellon JL, Hurtado J, Jimenez FJ, Esbrit P. Comparison of two immunoradiometric assays for parathyroid hormone-related protein in the evaluation of cancer patients with and without hypercalcemia. Clin Chim Acta 1998; 277:171-180. Budayr AA, Nissenson RA, Klein RF, et al. Increased serum levels of pa parathyroid hormone-like protein in malignancy associated hypercalcemia. Ann Intern Med 1989;111:807-812. Edwards RC, Ratcliffe WA, Walls J, Morrison JM, Ratcliffe JG, Holder R, Bundred NM. Parathyroid hormone-related protein (PTHRP) in breast cancer and benign breast tissue. EurJ Cancer 1995;31:334-339. Zeimer HJ, Greenaway TM, Slavin J, Hards DK, Zhou H, Doery JC, Hunter AN, Duffield A, Martin TJ, Grill V. Parathyroid-hormone-related protein in sarcoidosis. AmJPathol 1998;152:17-21. Deftos LJ, McMillin PJ, Satiano GP, Abuid J, Robinson AG. Simultaneous ectopic production of parathyroid hormone and calcitonin. Metabolism 1976;25:543-550. Iguchi H, Miyagi C, Tomita K, Kawauchi S, Nozuka Y, Tsuneyoshi M, Wakasugi H. Hypercalcemia caused by ectopic production of parathyroid hormone in a patient with papillary adenocarcinoma of the thyroid gland. J Clin Endocrinol Metab

1998;83:2653-2657.

161. Nielsen PK, Rasmussen AK, Feldt-Rasmussen U, Brandt M, Christensen L, Olgaard K. Ectopic production of intact parathy-

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

/

165

roid hormone by a squamous cell lung carcinoma in vivo and in vitro. J Clin Endocrinol Metab 1996;81:3793-3796. Yoshimoto K, Yamasaki R, Sakai H, Tezuka U, Takahashi M, Iizuka M, Sekiya T, Saito S. Ectopic production of parathyroid hormone by small cell lung cancer in a patient with hypercalcemia. J Clin Endocrinol Metab 1989;68:976-981. Strewler GJ, Budayr AA, Clark OH, Nissenson RA. Production of parathyroid hormone by a malignant nonparathyroid tumor in a hypercalcemic patient. J Clin Endocrinol Metab 1993; 76:1373-1375. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990;323:1324-1328. Sherwood LM, O'Riordan JLH, Aurbach GD. Production of parathyroid hormone by nonparathyroid tumors. J Clin Endocrinol Metab 1967;27:140-145. Stewart AF, Horst R, Deftos LJ, Cadman EC, Lang R, Broadus AE. Biochemical evaluation of patients with cancer-associated hypercalcemia: Evidence for humoral and non-humoral groups. N EnglJ Med 1980;303:1377-1383. Danks JA, Ebeling PR, Hayman JA, et al. Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia. J Pathol 1990;16:27-33. Hayman JA, Danks JA, Ebeling PR, Moseley JM, Kemp BA, Martin TJ. Expression of parathyroid hormone-related protein in normal skin and in tumor of skin and skin appendages. J Pathol 1998; 158:293-296. Henderson JE, Shustik C, Kremer R, Rabbani SA, Hendy GN, Goltzman D. Circulating concentrations of parathyroid hormone like peptide in malignancy and in hyperparathyroidism. J Bone Mineral Res 1990;5:105-113. Hutchesson AC, Hughtes SV, Bowden SJ, Ratcliffe WA. In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma. Ann Clin Biochem 1994;31:35-39. Budayr AA, Halloran BP, King JC, Diep D, Nissenson RA, Strewler GJ. High levels of parathyroid hormone-like protein in milk. Proc Natl Acad Sci USA 1989;86:7183-7185. Lepre F, Grill V, Danks JA, et al. Hypercalcemia in pregnancy and lactation due to parathyroid hormone-related protein production. Bone Miner 1990;323:666-667.

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CHAPTER10 Physiology of Calcium H o m e o s t a s i s

EDWARD M. BROWN Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115

INTRODUCTION

briefly discussed is an e m e r g i n g body of data indicating that C a 2+ o homeostasis does n o t operate in isolation but is integrated with o t h e r homeostatic systems, including those regulating the metabolism of water, sodium chloride, and even protein metabolism.

Calcium i o n s ( C a 2+) play n u m e r o u s critical physiologic and biochemical roles, in b o t h the intra- and extracellular spaces. Ultimately, all intracellular C a 2+ originates from C a 2+ within the extracellular fluid. It is not surprising, therefore, that free-living terrestrial organisms, including h u m a n s and o t h e r mammals, have developed a c o m p l e x homeostatic system that maintains near constancy of the level of free extracellular calcium (Ca2o+). F u r t h e r m o r e , all calcium within the extracellular fluids (ECFs) and elsewhere in the body is ultimately derived from dietary calcium. Once ingested and absorbed, excess calcium can be stored within b o n e or lost in the urine w h e n m o r e C a 2+ is available than is n e e d e d for intra- or extracellular processes. Conversely, if absorbed dietary calcium is insufficient for the body's needs, it can be withdrawn from skeletal reserves a n d the loss of urinary calcium can be mitigated by appropriate physiological responses. Thus maintaining C a 2+ o homeostasis involves the carefully orchestrated control of calcium's m o v e m e n t s into and out of the body via the gastrointestinal (GI) tract and kidney, respectively, as well as into and out of bone, so as to ensure near constancy of C a 2+ o while at the same time providing the Ca 2+ n e e d e d for this ion's diverse intracellular and extracellular functions. The purpose of this chapter is to review the mechanisms underlying Ca2o+ homeostasis, particularly those t h r o u g h which changes in the level of C a 2+ o within the bodily fluids are sensed and transduced into changes in the functions of kidney, bone, and GI tract so as to normalize Ca 2+ Also o

The Parathyroids, Second Edition

BIOLOGIC ROLES OF CALCIUM Calcium is an essential e l e m e n t t h r o u g h o u t the phylogenetic tree by virtue of its myriad biologic roles (Table 1). C a 2+ functions as a critical intracellular seco n d messenger that regulates n u m e r o u s cellular functions, including processes as diverse as h o r m o n a l secretion, muscle contraction, n e u r o n a l excitability, glycogen metabolism, and cell division (1-3). Many of these functions are the result of the interaction of C a 2+ with its intracellular binding proteins, e.g., calmodulin, and the c o n s e q u e n t activation of enzymes and o t h e r intracellular effectors systems (1-3). The free cytosolic calcium concentration (CaZi+) in cells u n d e r resting conditions is on the o r d e r o f 100 nM. The level of 2+. Ca i as controlled by diverse channels, pumps, and o t h e r transport systems that regulate the movements of C a 2+ into and out of the cytosol and between various intracellular c o m p a r t m e n t s (1-3). Consistent with its role as an i m p o r t a n t intracellular second messenger, C a 2+ i can increase by as m u c h as lO0-fold (i.e., to a level of 1-10 ~zM) during cellular activation. Such rises in Ca i are the result of uptake of extracellular Ca 2+ t h r o u g h Ca2+-per meable plasma m e m b r a n e channels, release of Ca 2+ from its intracellular stores, such as the endoplasmic

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167

Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER10 TABLE 1 Biologic Calcium Form Intracellular Insoluble

Location

Mass (% of total)

Function

Plasma membrane, endoplasmic reticulum, mitochondria, other organelles

9 g (0.9%)

Structural integrity, storage

Soluble

Cytosol, nucleus

0.2 mg

Action potentials, contraction and motility, metabolic regulation, cytoskeletal function, cell division, secretion

Extracellular Insoluble

Bones and teeth

1-2 kg (99%)

Protection, locomotion, ingestion of minerals and other nutrients, mineral storage

Soluble

Extracellular fluid

1 g (0.1%)

Blood clotting, kinin generation, regulation of plasma membrane potential, exocytosis, a contraction a

aThe activation of exocytosis and muscle contraction, in part, depend on cellular uptake of extracellular calcium.

reticulum, or both. In spite the importance of intracellular Ca 2+ in its various forms in controlling cellular metabolism, this compartment only represents about 1% of total bodily Ca 2+ (4). In contrast to C a 2+ i, Ca 2+ o is on the order o f l mM. It is carefully regulated by a complex homeostatic mechanism with two key elements: (1) cells that secrete the Ca 2+ o -regulating hormones, parathyroid h o r m o n e (PTH), calcitonin (CT), and 1,25-dihydroxyvitamin D [ 1,25 (OH) 2° ] mnamely, the chief cells of the parathyroid glands, thyroidal parafollicular cells, and proximal tubular cells of the kidney, respectively, as well as specialized Ca2+-transporting/translocating cells in the intestine, skeleton, and kidney (4-7). This system controls the flow of calcium ions into and out of the body as well as between various bodily compartments, especially between the skeleton and extracellular fluid. The rigid control of the level of Ca2o+ ensures a constant supply of C a 2+ in bodily fluids for the vital intracellular functions of this crucial divalent cation. Ca2o+ has several other key roles, which include maintaining intercellular adhesion, contributing to the integrity of the plasma membrane, and promoting the clotting of blood, further emphasizing the importance of maintaining near constancy of C a 2+ o . The total quantity of soluble C a 2+ i in the ECF, however, like intracellular C a 2+, o n l y repre-

sents a tiny fraction of total bodily calcium (about 0.1%; see Table 1). Most calcium in the body (>99%) is deposited as calcium phosphate salts within the skeleton, where it serves two key functions. First, the skeleton serves as a protective covering for vital, potentially vulnerable internal organs (i.e., within the cranium or thoracic cavity) and affords a rigid but articulated framework that facilitates locomotion and other bodily movements (6,7). Second, it provides an almost inexhaustible reservoir of calcium and phosphate ions for those times of need when intestinal absorption and renal reabsorption are not sufficient to ensure adequate levels of these ions within the ECF to support the body's numerous bodily functions that are d e p e n d e n t on them (6). Thus, though C a 2+ within all bodily compartments plays critical roles, the c o m p o n e n t that is most closely regulated by the mineral ion homeostatic system and, therefore, affects all other forms of calcium within the body is Ca2o+. A clear understanding of the mechanism involved in extracellular Ca 2+ homeostasis requires a discussion of the various forms of C a 2+ present in the blood and other bodily fluids, a consideration of how Ca 2+ moves between the organism and the envir o n m e n t and a m o n g various bodily compartments (i.e., overall C a 2+ balance), and, finally, the mecha-

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

Calcium is needed for the growth of both soft and hard tissues (9). Therefore, during childhood, more calcium enters the body through the GI tract than is lost it through the kidneys, GI tract, and perspiration (loss of C a 2+ in the sweat is generally not significant). That is, overall calcium balance is positive. Calcium balance is only truly zero (e.g., the C a 2+ homeostatic system precisely balances intake and output o f Ca 2+) for about two decades after skeletal growth ceases, at about age 20 years. Beginning as early as age 30-40, total bodily calcium begins to decrease, primarily because of loss of skeletal C a 2+ in the absence of any significant change in serum ionized C a 2+ ( 5 , 6 ) . Figure 1 illustrates calcium balance in a hypothetical young adult in zero Ca 2+ balance. Of the 1000 mg of elemental calcium that this individual ingests on a daily basis, about 30% (300 mg) actually undergoes intestinal absorption. About 100 mg of C a 2+ is lost by intestinal secretion, so that net absorption is 200 mg. Approximately 500 mg of C a 2+ e n t e r s and leaves the skeleton daily as a result of bone formation and resorption, respectively. To maintain mineral balance, therefore, 200 mg of C a 2+ m u s t exit the body via the kidneys. The C a 2+ lost in the urine comprises only 2% of that filtered daily (i.e., 10 g), which speaks to the kidney's remarkable efficiency in reclaiming, via tubular reabsorption, C a 2+ filtered at the glomerulus (10).

F O R M S O F C A L C I U M IN B L O O D Although the level of Ca2o+ in the interstitial fluids bathing the various tissues of the body is perhaps most relevant to Ca2o+ homeostasis, it cannot be readily measured. Instead, the total or ionized s e r u m C a 2+ activity is the parameter that is determined. Of the serum total calcium concentration, about 47% is in an ionized or free form. An equivalent a m o u n t (---46%) is b o u n d to various plasma proteins, especially albumin, which contains about 75% of b o u n d serum calcium (with the rest being b o u n d to various globulins), and the r e m a i n d e r (5-10%) is complexed to small anions, comprising phosphate, citrate, bicarbonate, and others (8). The fraction of s e r u m C a 2+ that is ultrafilterable comprises both free and complexed Ca 2+ (e.g., that b o u n d to small anions), but only the first of these is metabolically active (i.e., available for uptake by cells).

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O V E R A L L Ca 2+ B A L A N C E D U R I N G T H E L I F E CYCLE

nisms that control these movements. A critical element within the last of these is the system by which the body recognizes and responds to (i.e., "senses") changes in Ca2o+ (7). This Ca 2+ o -sensing mechanism enables direct a n d / o r indirect [i.e., via alterations in PTH, 1,25 (OH)2 D, and CT] regulation of the effector systems in intestine, kidney, and bone that modulate C a 2+ transport into and out of the ECF so as to restore Ca2o+ homeostasis (7).

BONES (and TEETH)

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o 200 mg~

FIG. 1 Overall Ca 2÷ balance in a hypothetical normal individual. Of the 1 g of elemental Ca 2÷ ingested daily, there is net absorption of 200 mg (300 mg gained by absorption, 100 mg lost by intestinal secretion). Balance is achieved, therefore, by renal excretion of 200 mg of Ca 2÷, because equal amounts of Ca 2÷ are laid down and removed from the skeleton on a daily basis in this person. ECF, Extracellular fluid. (Adapted from Brown EM, LeBoff MS. Pathophysiology of hyperparathyroidism. In: Rothmund M, Wells SA, Jr, eds. Progress in surgery. Parathyroid surgery, Vol 18. Basel:Karger, 1986:13-22.)

170

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CICAeTER10

Furthermore, only modest alterations in the percentage of the filtered calcium that is reabsorbed (i.e., a decrease to 1%, or an increase to 3%, for instance) can have a substantial impact on overall Ca 2+ balance (11). The increasing recognition that osteoporosis is a major public health p r o b l e m in later life has e n g e n d e r e d a great deal of interest in defining optimal levels of calcium intake as a function of age, h o r m o n a l status, and o t h e r factors. Currently designated adequate intakes (AIs) of calcium in the United States for various individuals are as follows: 500 mg, 1-3 years old; 800 mg, 4-8 years old; 1300 mg, 9-18 years old; 1000 mg, 19-50 years old; and 1200 mg, > 5 0 years old. For pregnant w o m e n u n d e r 19 years of age the AI is 1300 mg, and is 1000 mg thereafter (12). Intake of these quantities should not be considered as dietary supplements but rather a nutritional r e q u i r e m e n t for skeletal health.

HORMONAL CONTROL C a 2+ o HOMEOSTASIS

that maintains near constancy of Ca2o+ t h r o u g h cal2+. ciotropic h o r m o n e - i n d u c e d and direct, Ca o -induced changes in the GI, renal, a n d / o r skeletal handling of Ca 2+, as illustrated in Fig. 2 (6,7). Overall C a 2+ o homeostasis can be u n d e r s t o o d in terms of three general principles: (1) The first priority of the homeostatic m e c h a n i s m is that it maintains a n o r m a l level of Ca o2+. (2) W h e n there are m o d e r a t e stresses on the system, changes in the intestinal a n d / o r renal h a n d l i n g of Ca 2+ are usually sufficient to sustain Ca2o+ homeostasis without alterations in skeletal Ca 2+ balance. (3) In the presence of severe hypocalcemic stresses, skeletal Ca 2+ is " mobilized in order to m a i n t a i n Ca2o+ homeostasis. This loss of skeletal calcium, if it persists for a sufficiently long time (e.g., m o n t h s to years), eventually compromises the structural integrity of the skeleton. The Ca2o+ homeostatic system, as n o t e d previously, has two essential elements. The first is a group of several different cell types that are capable of sensing changes in Ca2o+ and r e s p o n d i n g with homeostatically 2+ relevant alterations in their o u t p u t of Ca o -regulating h o r m o n e s [PTH, calcitonin, and 1,25(OH)2D] (7). The second key c o m p o n e n t is the effector cells that control the renal, intestinal, and skeletal handling of

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Tetrapods, e.g., reptiles, amphibians, birds, a n d m a m m a l s , posses a c o m p l e x homeostatic m e c h a n i s m

INTESTINE

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FIG. 2 The hormonal regulation of calcium homeostasis by PTH and vitamin D (solid lines and arrows). Ca2o÷ regulates PTH secretion in an inverse manner. PTH, in turn, modulates renal handling of Ca2o÷and phosphate as well as renal production of 1,25(OH)2D. PTH and 1,25(OH)2D act synergistically to mobilize Ca2o÷ and phosphate from bone, whereas 1,25(OH)2D increases intestinal absorption of both ions. Also shown are direct actions of calcium and phosphate ions on tissues that participate in maintaining mineral ion homeostasis (dashed lines and arrows), showing the roles of these ions as extracellular first messengers. For additional details, see text. (Reproduced with permission from Brown EM, Pollak M, Hebert SC. Cloning and characterization of extracellular Ca2+-sensing receptors from parathyroid and kidney. Molecular physiology and pathophysiology of Ca 2+ sensing. Endocrinologist 1994;4:419-426.)

PHYSIOLOGYor Ca 2+ HOMEOSTASIS / C a 2+. As described in more detail below, the transloca-

tion of calcium and phosphate ions into or out of the extracellular fluid by these tissues is regulated by PTH, calcitonin, and 1,25 (OH)2D as well as by mineral ions. The homeostatic system operates as follows: In the example shown (Fig. 2), a slight decrement in Ca2o+ evokes a p r o m p t increase in the secretory rate for PTH by the parathyroid chief cells. This h o r m o n e has several key actions on the kidney, including promoting phosphaturia (6), increasing distal tubular C a 2+ reabsorption, and enhancing the biosynthesis of 1,25 (OH) 2° from 25-hydroxyvitamin D [25(OH)D] (13-15). The ensuing increase in the circulating 1,25(OH)zD level directly stimulates intestinal absorption of C a 2+ and phosphate by i n d e p e n d e n t transport systems (15). PTH and 1,25 (OH)2 ° also synergistically increase the net release of skeletal C a 2+ and phosphate (6). The increased movement of C a 2+ into the ECF from bone and intestine, coupled with PTH-induced retention of this ion by the kidney, restore the circulating level of Ca2o+ to normal, thereby reducing PTH release and closing the negative-feedback loop. 1,25(OH)zD, synthesized in response to PTH, also directly reduces PTH synthesis and secretion (16), and contributes in this m a n n e r to the negative-feedback regulation of parathyroid function by the homeostatic system. Excess phosphate mobilized from bone and intestine undergoes urinary excretion in response to the phosphaturic action of PTH. As indicated in Fig. 2, both C a 2+ and phosphate ions have direct effects on several of the cells and tissues that participate in mineral ion metabolism. For instance, Ca2o+ not only inhibits PTH secretion but also directly reduces the proximal tubular production of 1,25 (OH) 2o (17), enhances the function of osteoblasts (18), and inhibits osteoclastogenesis (19) and osteoclastic bone resorption (20). Phosphate ions diminish the 1-hydroxylation of 25(OH) vitamin D, promote bone formation, inhibit bone resorption (7), and also stimulate several aspects of parathyroid function (21,22), as described in more detail later. These actions may well play important roles in mineral ion homeostasis by enabling not only the hormone-secreting but also the effector elements of the system to sense changes in the local levels of these ions in the ECF and, therefore, to respond in a physiologically relevant way. Indeed, because of their direct effects on cells participating in mineral ion homeostasis, Ca 2+ and phosphate can be viewed as acting in hormonelike roles as extracellular first messengers (7). 2+ Though the cloning of the Cao-sensing receptor (CaSR), which mediates the direct actions of C a o2+ on the functions of parathyroid glands, kidney, and several other tissues, has clarified substantially the m a n n e r in which these cells sense C a 2+ o (23), the mechanisms underlying phosphate sensing remain obscure.

171

R E G U L A T I O N OF PARATHYROID H O R M O N E S E C R E T I O N BY Ca2o+ A N D O T H E R FACTORS The Overall Secretory R e s p o n s e o f the Parathyroid Cell to Alterations in Ca2o+ The parathyroid cell manifests a hierarchy of responses to decreases in Ca2o+ that permit it to m o u n t a progressively larger secretory response that is appropriate for the rapidity, magnitude, and duration of the hypocalcemic stress that elicited this response (24). The most rapid response is the release of preformed PTH stored within secretory granules. This response occurs within seconds and can persist for as long as 60-90 minutes before these stores are completely depleted. This immediate secretory response exhibits a steep inverse sigmoidal relationship between PTH secretion and the level of Ca o2+ (Fig. 3) (24). Such a curve can be described by four parameters--maximal secretory rate at low Ca2o+ (parameter A), slope at the midpoint (parameter B), midpoint or "set point" (parameter C, the level of Ca2o+ half-maximally suppressing PTH), and minimal secretory rate at high Ca2o+ (parameter D) (25). The steepness of the curve contributes importantly to the nearly constant level of C a 2+ o that is maintained u n d e r normal circumstances (e.g., the percent coefficient of variation of Cao2+ is on the order of 1.5% in normal persons). The set point is also a key parameter that is the major determinant of the level at which Ca2o+ is "set," thereby serving as one of the body's key "thermostats" for Ca2o+, or "calciostats" (24). There are several additional features of the acute secretory response of the parathyroid cell to changes in Ca2o+ that may have an impact on the target tissues of PTH and the homeostatic system's overall response to alterations in C a o2+ (24). That is, PTH levels in vivodepend not only on Ca2o+per se, but also on the rate of change of C a o2+, particularly when it is decreasing (26). This "rate-dependence" is apparent when Ca2o+ is falling rapidly, which elicits a more vigorous secretory response than when Ca2+o decreases slowly. This dependence of PTH output on rate of change of C a o2+ may permit the parathyroid cell to m o u n t a more vigorous response, which would be homeostatically appropriate, when C a o2+ is diminishing rapidly. There also appears to be a "direction-dependence" or hysteresis to the secretory response of PTH to changes in C a o2+ (24). This hysteresis is manifested by the higher levels of PTH that are observed when C a o2+ is falling than when it is rising. Moreover, similar to the rate dependence of PTH secretion, it may be homeostatically relevant because it engenders higher levels of PTH when those are needed (i.e., when C a o2+is falling) than when they are not (e.g. , when C a o2+ is increasing). The parathyroid cell exhibits several other adaptive changes that, by virtue of their occurrence in a graded

172

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greater expression of the PTH gene, owing both to increased transcription and enhanced stability of the messenger RNA (mRNA) encoding preproPTH, which takes place within a time frame of several hours to about a day (16,24). The resultant increase in the mRNA level for preproPTH is probably also accompanied by a more general increase in the parathyroid cell's biosynthetic capacity, particularly when hypocalcemia persists for days or longer, which produces the anticipated morphologic alterations (e.g., greater prominence of organelles that participate in hormonal biosynthesis, such as the rough endoplasmic reticulum and Golgi apparatus) (24). Finally, over several days to weeks or longer, the initiation of parathyroid cellular proliferation increases the total number of parathyroid chief cells and, as a consequence, the total secretory capacity for PTH by manyfold. Increases rather than decreases in Ca2o+ produce exactly the opposite changes in these various aspects of parathyroid cell function, although the time course may not be identical. That is, though the inhibition of PTH secretion by a rise in C a 2+ o takes place essentially immediately, the parathyroid gland shows a rather sluggish and, in all likelihood, incomplete capacity to rid itself of excess parathyroid chief cells once a stimulus to chief cell hyperplasia has abated ( 27,28 ).

The Molecular Mechanism of Ca2o+ Sensing by Parathyroid, Kidney, and Other Cells The technique of expression cloning in Xenopus laevis oocytes permitted the isolation of a phosphoMINIMUM

FIG. 3 (A) Relationship between PTH secretion and Ca2o+ in normal human parathyroid cells. Dispersed parathyroid cells were prepared and incubated with the levels of Ca2o+indicated. PTH released into the medium was then determined by radioimmunoassay. (B) The four parameters that describe the relationship between PTH secretion and Ca2o+: maximal (parameter A) and minimal secretory rates (parameter D) at low and high Ca 2+ concentrations, respectively, slope of the curve at its midpoint (parameter B), and set-point (parameter C, the level of Ca2o+ producing half of the maximal inhibition of PTH release). [(A) From Brown EM. In: Brenner BM, Stein H, eds. Contemporary issues in nephrology, Volume 2. Divalent ion homeostasis. New York:Churchill-Livingstone, 1983.]

and sequential manner, increase its overall capacity to produce biologically active PTH (24). The first is decreased intracellular degradation of PTH, so that a larger fraction of the secreted h o r m o n e is intact, bioactive PTH(1-84), which occurs within 20-30 minutes after exposure to hypocalcemia. The next response is

inositide-coupled, Ca 2+ o -sensing receptor (CaSR) from bovine parathyroid that is thought to represent the molecular mechanism by which parathyroid, kidney, and other cells sense Ca2o+ (23). The CaSR has a large amino-terminal extracellular domain, which is the major site where the binding of Ca2o+ takes place. Seven membrane-spanning helices follow that are characteristic of the superfamily of G protein-coupled receptors, and, finally, a cytoplasmic carboxy (C)-terminal domain (Fig. 4). The intracellular portions of the CaSR transduce the Ca2o+ signal into alterations in various intracellular second-messenger systems (29) that regulate key cellular processes involved in Ca2o+ homeostasis, such as hormonal secretion or renal tubular calcium reabsorption. The calcium-sensing receptors play a key role in C a 2+ o sensing by parathyroid and kidney, as proved by the demonstration that the CaSR harbors inactivating or activating mutations in several human genetic diseases manifested by abnormal Ca2o+ sensing (30). In familial hypocalciuric hypercalcemia (FHH), persons with heterozygous inactivating mutations of the CaSR (31) exhibit mild to moderate hypercalcemia accompanied by inappropriately normal (e.g., nonsuppressed) circu-

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

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lating PTH levels and normal or even frankly low levels of urinary C a 2+ excretion (32). In contrast, persons with homozygous FHH present clinically as neonatal severe hyperparathyroidism (NSHPT) and have severe hypercalcemia accompanied by marked hyperparathyroidism (32). Finally, families have been identified with an autosomal dominant form of hypocalcemia that is accompanied by relative hypercalciuria (e.g., inappropriately high for the prevailing level of Ca2o+), in which affected persons harbor activating mutations in the CaSR, providing further proof of its importance in C a 2+ o sensing in parathyroid and kidney (33). In addition to its presence in the parathyroid chief cell, the CaSR is also expressed along nearly the entire renal tubule (34,35). It is present at the highest levels in the cortical thick ascending limb (CTAL; a segment of the nephron that is probably responsible for the abnormal renal C a 2+ handling in FHH) (34,35). The calciumsensing receptor's presence in the kidney likely provides a molecular basis for several of the longrecognized but poorly understood direct actions of

C a 2+ o on renal function, including the impaired urinary concentrating capacity present in some hypercalcemic patients) (36). In the thyroid, the CaSR resides almost solely in the calcitonin-secreting (C) cells and likely mediates the stimulatory effect of high levels of C a 2+ o on CT secretion (37,38).

Other Factors Modulating Parathyroid Function In addition to Ca2o+, several other factors, including vitamin D metabolites (especially 1,25 (OH) 2D), catecholamines and other biogenic amines, prostaglandins and peptide hormones, and phosphate and monovalent cations (e.g., potassium and lithium), also modulate PTH secretion (16,39). Of these, the most physiologically relevant are probably 1,25 (OH) 2o and phosphate. 1,25(OH)zD is thought to play an important role in the longer term (over days or longer) control of parathyroid function, tonically reducing PTH secretion (40,41), diminishing expression of the PTH gene (16,42), and probably inhibiting parathyroid

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cellular proliferation (43). Thus actions of PTH on its target tissues produce negative-feedback regulation of parathyroid cellular function not only by raising Ca2o+ but also by enhancing the synthesis of 1,25 (OH)2 D , which then directly exerts negative feedback actions on parathyroid function. Recent studies in vitro have shown that elevating and decreasing the ambient phosphate concentration will increase or reduce, respectively, PTH secretion (possibly indirectly through alterations in PTH gene expression), PTH gene expression, and parathyroid cellular proliferation (16,22).

EFFECTS OF PTH O N Ca2o+-REGULATING TISSUES Control of Gastrointestinal Ca 2+ Absorption The net amount of Ca 2+ absorbed from the GI tract is the difference between the amounts moving from lumen to plasma (absorption) and plasma to lumen (secretion) (44). The latter, which is called endogenous fecal calcium, is about 100 m g / d a y and varies little as a function of Ca balance. Ca absorption is the result of both passive diffusion across the intestinal mucosa via the paracellular route and active, transcellular transport. The passive, paracellular diffusion of C a 2+ is concentration dependent and nonsaturable; it accounts for absorption of approximately 10-15% of dietary C a 2+ (i.e., 100-150 m g / d a y of ingested C a 2+ when dietary C a 2+ is 1000 mg/day). The active transcellular component of C a 2+ absorption is a saturable, carrier-mediated mechanism regulated by 1,25(OH)2D. It involves apical uptake of calcium by a CaZ+-permeable channel(s) (45), transcellular movement by an incompletely understood mechanism that likely involves the intracellular Ca2+-binding protein, calbindin DgK, and then eventual extrusion of calcium at the basolateral cell surface by the CaZ+-ATPase and, perhaps, the Na+-Ca 2+ exchanger (46). The highest density of sites of active C a 2+ absorption is in the proximal small intestine, i.e., d u o d e n u m (44,47). There is vitamin D-responsive C a 2+ absorption in more distal segments of the intestine as well, including both the small intestine (ileum > jejunum) and the proximal large bowel (44). Because these segments of the GI tract are much longer than the duodenum, they may well contribute significantly to overall C a 2+ absorption. After administering 1,25 (OH)2D to vitamin D-depleted animals, GI absorption of C a 2+ rises over the next several hours. This increase is paralleled, in general, by increases in the levels of several intestinal vitamin D-dependent proteins, including calbindin DgI~, alkaline phosphatase, and Ca -Mg -ATPase (44,47). 1,25(OH)zD appears to stimulate both the influx and the egress of C a 2+ from the intestinal epithelial cells (48,49). Therefore, it will be of •

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interest to examine the effects of vitamin D nutrition on the expression of the Ca -permeable influx channel thought to mediate uptake of Ca 2+ from the GI lumen into the absorptive cells (45). The bulk of phosphate absorption by the intestine takes place in the small bowel through a vitamin D-responsive mechanism, which is distinct from that for Ca 2+ (6). Even in vitamin D deficiency, nearly half of total dietary phosphorus is still absorbed. The less stringent control of phosphate absorption in the GI tract is consonant with the ubiquity of dietary phosphate and the looser regulation of the serum phosphate concentration. A key feature of the C a 2+ o homeostatic system is its capacity to adapt appropriately the efficiency of Ca 2+ absorption to dietary intake. Persons placed on a low-Ca2+ diet elevate their serum concentrations of 1,25 (OH) 2D by 50% within 24-48 hours, whereas exposure to a high-Ca 2+ diet causes a 50% reduction in the circulating level of this metabolite over the same period (50). In experimental animals, the low dietary CaZ+-evoked increase in the 1,25(OH)2D level is largely prevented by prior parathyroidectomy (51), indicating that dietary CaZ+-induced alterations in 1,25(OH)2D concentration are the result of changes in serum C a 2+ concentration, which, in turn, regulate vitamin D metabolism indirectly through alterations in PTH secretion. Nonetheless, decreasing or elevating the level of C a 2+ o also has been shown directly to stimulate or inhibit, respectively, the 1-hydroxylation of 25-hydroxyvitamin D (17). These direct actions of Ca2o+ on renal vitamin D metabolism could potentially be mediated by the CaSR in the proximal tubule (34,35), although this has not yet been proved. The CaSR is also expressed along the entire GI tract, but it remains to be determined whether it directly regulates mineral ion absorption (52,53) Because of the C a 2+ and PTH-elicited, 1,25(OH)zD-mediated modulation of the efficiency of intestinal C a 2+ absorption, the absorption of this ion varies less than does its content in the diet. Absorption of supplemental dietary Ca 2+ may occur principally through the vitamin D-independent, paracellular route. Phosphate intake also regulates the production of 1,25 (OH) 2D , in a physiologically relevant manner, with hypophosphatemia increasing and hyperphosphatemia reducing its renal synthesis (6). 2+

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PTH-induced and direct Ca o -induced changes in renal Ca 2÷ handling play key roles in the overall finetuning of Ca 2÷ balance (54-56). In contrast, vitamin D and its metabolites exert only minor direct effects on renal Ca 2÷ handling. Of the approximately 10 g of Ca 2÷ that are filtered daily by the kidney, about 65% is reabsorbed in the proximal tubule (56). C a 2+ reabsorption in this site is closely coupled to the bulk transport of solutes,

PHYSIOLOGYOF Ca 2+ HOMEOSTASIS / such as sodium and water, and PTH has little effect on Ca 2+ transport in this segment of the nephron. In fact, PTH modestly inhibits, in some studies, proximal tubular Ca 2÷ absorption, perhaps because the hormone reduces sodium reabsorption in this part of the nephron (56). In the more distal portions of the tubule, the descending and ascending thin limbs of Henle's loop transport only small quantifies of Ca 2+ (56). In contrast, the thick ascending limb of Henle's loop (57) and the distal convoluted tubule (DCT) reabsorb about 20 and 10% of filtered C a 2+, respectively. PTH rapidly increases the reabsorption of C a 2+ in both the TAL and the DCT in experimental animals (56). It exerts this effect, similar to its other biologic actions in the kidney, by interacting with its own G protein-coupled receptor that is linked to activation of both adenylate cyclase and phospholipase C (58). cAMP appears to play the dominant role in mediating PTH-induced alterations in renal C a 2+ handling. PTH-sensitive adenylate cyclase

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resides within the proximal tubule, cortical thick ascending limb, and portions of the DCT (59). The location of this enzyme in the proximal tubule mediates the well-known, PTH-induced phosphaturia. PTH-activated adenylate cyclase activity in more distal nephron segments is present in sites where the hormone enhances Ca 2+ reabsorption. Furthermore, exposing renal tubules to cAMP analogs mimics the actions of PTH o n C a 2+ transport, further supporting the mediatory role of cAME In the CTAL, PTH increase the overall activity of the Na/K/2C1 cotransporter that drives transcellular NaC1 reabsorption in this nephron segment (Fig. 5) (36,60). This increased transcellular salt transport elevates the lumen-positive, transepithelial potential difference that drives about 50% of the reabsorption of NaC1 and most of the reabsorption of Ca 2+ and Mg 2+ in the CTAL. In contrast, raising C a 2+ o , by activating the CaSR that resides in the same epithelial cells of the CTAL, decreases overall

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cotransporter activity, probably both by inhibiting the cotransporter as well as by reducing the activity of an apical potassium channel that recycles K + back into the tubular lumen (Fig. 5) (36,61). The resultant decrease in the transepithelial potential gradient diminishes the paracellular reabsorption of both Ca 2+ and Mg 2+. In effect, Ca2o+, by acting in a m a n n e r analogous to the "loop" diurectics (e.g., furosemide), directly controls 2+ its own reabsorption (and that of Mg ) by a CaSRmediated action on the CTAL that antagonizes the effect of PTH on the same cells (36). Although the detailed cellular mechanisms by which PTH regulates Ca 2+ transport in the distal convoluted tubule remain incompletely understood, they likely involve a PTH-stimulated increase in the apical uptake of Ca 2+ through a recently cloned, Ca 2+-permeable channel (62,63). As in the intestine, the ensuing transcellular Ca 2+ transport is likely facilitated by a vitamin D-dependent, Ca2+-binding protein, which in this case is calbindin D2sK. The latter is expressed in the DCT and is distinct from the related Ca2+-binding protein, calbindin DgK, that is present in the intestinal epithelial cells that absorb Ca 2+ (64). An additional small quantity of Ca 2+ (about 5% of the filtered load) is reabsorbed in the collecting duct, but Ca 2+ transport at this site is not PTH regulated. In addition to being present in the CTAL, the CaSR also resides in the DCT (34,35), but its role, if any, in controlling tubular reabsorption of Ca 2+ in this n e p h r o n segment is unknown. The net effect of PTH on renal Ca 2+ handling is to decrease the quantity of Ca 2+ excreted at any given concentration of serum Ca 2+ (55). This relationship has been shown in vivo by measuring renal Ca 2+ excretion as a function of serum Ca 2+ in persons with underactive, normal, or overactive parathyroid function (Fig. 6) (55). In patients with primary hyperparathyroidism, even though the total quantity of urinary Ca 2+ that is excreted per 24 hours may be greater than normal, substantially less urinary Ca 2+ is excreted than in a normal person whose serum Ca 2+ concentration has been elevated to the same extent. In contrast, patients with primary hypoparathyroidism exhibit a renal Ca 2+ "leak," excreting greater than normal quantities of urinary calcium at any given level of serum Ca 2+. Thus when treating patients with hypoparathyroidism with vitamin D and dietary Ca 2+ supplementation, their total serum calcium concentration should be maintained in the range of 8 to 9 m g / d l to avoid hypercalciuria. Figure 6 illustrates the steep positive relationship between the serum and urinary levels of Ca 2+ in these various states, which is probably mediated by the CaSR. This relationship is "reset" d e p e n d i n g on the prevailing state of parathyroid function, thereby shifting to the right or left with chronic increases and decreases, respectively, in circulating PTH levels (55).

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Along with its actions on renal C a 2+ handling in the CTAL and DCT, PTH also reduces phosphate reabsorption in both proximal and distal sites and increases the proximal tubular synthesis of 1,25(OH)2D(5,6 ). The first of these effects, similar to the actions of the horm o n e on the reabsorption o f Ca 2+, is thought to be cAMP mediated. PTH activates phospholipase C (58), an action that may participate in the PTH-mediated stimulation of 1,25(OH)2D synthesis (65). R o l e o f the S k e l e t o n in Ca 2+ H o m e o s t a s i s

During the ongoing remodeling of the skeleton, there is close coupling of bone resorption and formation (6). Osteoblasts play a key role in the generation of osteoclasts from their precursors (66). Osteoclastic resorption of bone, in turn, is tightly coupled to the subsequent replacement of the resorbed bone by osteoblasts. The constant turnover and renewal of bone is thought to play an important role in maintaining the structural integrity of the skeleton. The precision of the coupling between resorption and formation is illustrated dramatically in patients with Paget disease of bone, in whom increases in the rate of skeletal turnover of up to 10-fold are often unassociated with any change whatsoever in s e r u m C a 2+ concentration or overall C a 2+ balance. Studies have identified key mechanisms that participate in regulating the differentiation and function of osteoclasts and osteoblasts; these are briefly described below.

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

PTH and other agents activating bone resorption [e.g., interleukin-11, prostaglandin E2, and 1,25(OH)2D ] stimulate osteoclast maturation and function indirectly by increasing the expression of an osteoclast differentiating factor, most commonly called RANKL (67). RANKL is expressed on the cell surface of osteoblasts and stromal cells. It activates osteoclast development and increases the activity of mature osteoclasts by interacting with its receptor (called RANK) on preosteoclasts, which then differentiate to mature osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) (67). Osteoclastic bone resorption, in turn, is thought to be coupled to the subsequent osteoblastic replacement of the missing bone, at least in part, through the release of skeletal growth factors such as transforming growth factor-J3 (TGF-[3) and insulin-like growth factor-I (IGF-I), which stimulate the recruitment and maturation of preosteoblasts, and, in some cases, the activity of mature, bone-forming osteoblasts (68). As noted earlier, the skeleton provides a virtually inexhaustible reservoir for calcium and phosphate ions (5,6). Because the content of Ca 2+ in the skeleton is 1000-fold higher than that in the ECF, this function of bone as a reservoir can be accomplished by the net movement of relatively small amounts of Ca 2+ into or out of the skeleton. After administering PTH to animals, alterations in the structure of osteoclasts, osteoblasts, and osteocytes (which are osteoblasts trapped within the calcified bone matrix) take place within minutes (5). Those morphologic alterations are accompanied by e n h a n c e d activity of osteoclasts and inhibition of the function of osteoblasts, producing an increase in net skeletal release of Ca 2+ within 2 to 3 hours (5,69). The PTH-elicited increase in the size of the lacunae within which osteocytes reside has also been considered indirect evidence for a role of these cells in promoting release of skeletal calcium. Continued exposure to PTH produces increases in the activity and n u m b e r of osteoclasts, which are ultimately accompanied by a coupled increase in osteoblastic activity, as noted above. The mechanisms by which PTH regulates bone cell function remain to be fully elucidated. Evidence indicates that a PTH-induced increase in the expression of RANKL by osteoblasts probably is an important mechanism through which this h o r m o n e promotes osteoclastogenesis and stimulates the activity of p r e f o r m e d osteoclasts (70). PTH activates adenylate cyclase in osteoblasts through the same G protein-coupled receptor via which it exerts its actions on the renal handling of calcium and phosphate ions (58,71), but it may also act through other second-messenger systems, including those downstream of phospholipase C. In addition to modulating skeletal turnover indirectly, e.g., by altering PTH secretion a n d / o r 1,25 ( O H ) 2 o production, changes in Ca2o+ also directly

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177

control bone cell function in vitro in ways that likely contribute to the control of bone turnover in vivo. The CaSR is expressed in osteoblasts, and high Ca2o+ is known to stimulate the chemotaxis and proliferation of osteoblasts (18,72) as well as the production of osteocalcin, a marker of differentiated osteoblasts. If these actions of Ca2o+ are mediated by the CaSR expressed in osteoblasts, then it is possible that Ca 2+ released during the resorption of bone by osteoclasts serves as one of the signals that are designed to ensure the availability of osteoblasts to replace the missing bone (73). Conversely, elevated levels of Ca2o+ directly inhibit osteoclastic function (20), which could provide a mechanism by which osteoclasts autoregulate their activity as a function of the a m o u n t they resorb of Ca 2+ that has been released into the local ECE Pharmacologic evidence has implicated a mechanism for C a 2+ o sensing by osteoclasts that differs from the CaSR (20). Further studies, however, have demonstrated that the CaSR can be expressed by both osteoclasts and their precursors (19,74). Thus further studies are n e e d e d to clarify the molecular mechanisms by which bone cells sense C a 2+ o. Changes in the level of extracellular phosphate also modulate bone turnover, as noted before. Elevations in phosphate enhance bone formation and inhibit bone resorption, whereas reductions in ambient phosphate concentrations produce the converse effects (75). As with the direct effects of phosphate on parathyroid gland function, the mechanisms underlying the sensing of extracellular phosphate ions by bone cells remain unknown.

P O S S I B L E R O L E S OF T H E CaSR IN I N T E G R A T I N G C a 2+ o METABOLISM W I T H O T H E R H O M E O S T A T I C SYSTEMS CaSR-Mediated Interactions b e t w e e n C a ~ and Water Metabolism In addition to regulating renal handling of Ca 2+ and Mg 2+, the CaSR likely mediates the known action of hypercalcemia to reduce urinary concentrating ability (76). It is thought to do so by two actions. First, by inhibiting NaC1 reabsorpdon in the TAL, it reduces the medullary hypertonicity n e e d e d for passive, vasopressinstimulated reabsorption of water in the collecting ducts. Second, in the inner medullary collecting duct, raising Ca o2+directly reduces vasopressin-stimulated water flow, probably by an action mediated by the CaSR in the apical m e m b r a n e of these cells (77,78). Finally, a b u n d a n t calcium-sensing receptors in the subfornical organ (SFO) (79), an important hypothalamic thirst center, may promote a CaSR-mediated increase in thirst that could minimize dehydration from accompanying renal water loss owing to reduced urinary concentrating

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ability. Thus the CaSR may provide a mechanism for integrating the renal handling of Ca 2+ and water, permitring appropriate "trade-offs" in how these parameters of renal function are regulated u n d e r specific physiologic conditions (76). For instance, when disposing of a systemic C a 2+ load, a CaSR-mediated increase in luminal C a 2+ o in the inner medullary collecting duct (IMCD), particularly in a dehydrated individual, could predispose to CaZ+-containing renal stones were it not for the concomitant CaSR-mediated inhibition of maximal urinary concentrating capacity. Thus there may be multiple layers of CaSR-mediated integration and coordination of water and calcium metabolism that optimize the ability of terrestrial organisms to adapt to intermittent dietary access t o C a 2+ and water (76).

P o s s i b l e C a S R - M e d i a t e d Interactions b e t w e e n Ca2o+ a n d S o d i u m / V o l u m e / Blood Pressure Control Figure 5 shows that activating the CaSR reduces transcellular NaC1 transport in the CTAL by inhibiting the Na/K/2C1 cotransporter and pari passu reducing paracellular NaC1 reabsorption by diminishing V~.Therefore, high C a 2+ o exerts a "loop diuretic-like" action that likely contributes to the volume depletion of severely hypercalcemic persons (e.g., via urinary loss of NaC1) (76). The action of C a 2+ o could also potentially participate in the salutary action of dietary calcium supplementation on certain forms of genetic hypertension in experimental animals (e.g., the spontaneously hypertensive rat) (80,81), and, perhaps, in treating pregnancy-induced hypertension (82) or preventing preeclampsia (83). Because of its exquisite sensitivity to changes in Ca2o+ resulting from alterations in dietary calcium intake, the CaSR in the kidney could potentially modulate NaC1 reabsorption and sensitize the kidney to other agents promoting diuresis. There are additional actions through which the CaSR could modulate blood pressure. It is expressed in perivascular sensory nerve endings in rat mesenteric artery (84) and other vascular beds (e.g., mesenteric branch artery > basilar artery = renal interlobar artery > main renal trunk artery > left anterior descending coronary artery) (85). Moreover, stimulating the CaSR in these nerve endings releases a vasodilatory substancemlikely an endogenous cannabinoid [e.g., N-arachidoylethanolamine (anandamide) ]--which then acts on a cannabinoid receptor in the vascular wall (86). It is possible that the inhibition of renin release from the juxtaglomerular apparatus (]GA) by high Ca2o+ involves the CaSR (87). Further studies, therefore, may reveal additional mechanisms through which the CaSR, by regulating renal fluid and electrolyte metabolism, vascular tone, and, perhaps, central vasomotor control, contributes to overall blood pressure regulation.

P o s s i b l e C a S R - M e d i a t e d Interactions b e t w e e n Ca2o+ a n d P r o t e i n M e t a b o l i s m Protein and Ca 2+ metabolism are linked at a fundamental level. For instance, reducing protein intake below a certain level in normal young women produces secondary hyperparathyroidism despite normocalcemia (88-90), and high dietary protein intake can promote substantial hypercalciuria (91). Studies have shown that the CaSR can "sense" not only polyvalent 2+ but also amino acids at cations , such as Ca2o+ and M go, levels that are not dissimilar from those present in vivo (92). Although a large n u m b e r of amino acids are effective allosteric activators of the CaSR (that is, they activate the receptor in the presence but not in the absence of Ca2o+), the CaSR shows a preference for L-aromatic amino acids (i.e., L-Phe, L-Trp, L-Tyr, and L-His) (92). This pharmacologic profile is similar to that for the stimulation of gastrin and gastric acid secretion from the stomach by amino acids (93), which are physiologic processes that are also stimulated by high Ca2o+, likely by a CaSR-mediated mechanism (94). The finding that the CaSR senses L-amino acids may clarify its widespread distribution in the upper GI tract, including the stomach (53,94), where it is exposed to dietary amino acids (and calcium) and could potentially contribute to physiologic responses, such as the release of gastric acid and pancreatic enzymes. Thus the CaSR may act as a "nutrient sensor" in the lumen of the proximal GI tract, responding to Ca2o+ and amino acids as coagonists that might function together to coordinate digestive responses to ingested nutrients. The pharmacologic profile for the actions of amino acids on the CaSR differs, however, from that of other metabolic actions of amino acids, such as the stimulation of insulin release (92). Therefore, there are probably additional amino acid sensors, one of which might be one or more purinergic receptors (92). It is likely that the calcium-sensing receptor's capacity to sense amino acids is relevant to additional physiologic interactions between Ca2o+ and protein metabolism. L-Amino acid mixtures that emulate those present in fasting h u m a n plasma activate the CaSR, and changes in the levels of this mixture equivalent to those occurring during the transition from the fed to the fasted state can modify the receptor's sensitivity to Ca2o+ significantly (92). As noted above, low-protein intake causes secondary hyperparathyroidism (88,89), whereas high-protein intake promotes hypercalciuria ( 9 1 ) ~ a c t i o n s that could potentially be mediated by the CaSR in the parathyroid and CTAL, respectively. It is of interest in this regard that substantial amounts of both Ca 2+ and protein are laid down during the growth of not only the skeleton, but also soft tissues. For instance, smooth muscle has a calcium content when expressed per wet weight that is about 8 m M / k g , nearly one-half

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

of that in bone (95). These observations raise the possibility that the CaSR, known to be expressed in growth plate chrondrocytes (96) and osteoblasts (73,97), for instance, could integrate information about the availability of key nutrients n e e d e d for growth of cartilage and bone, respectively, in ways that would be relevant to the physiologic control of these processes.

SUMMARY A N D C O N C L U S I O N S Ca2o+ homeostasis requires the coordinated functions 2+ 2+ of both the Ca o -sensing cells that secrete Ca o -elevating o r - l o w e r i n g h o r m o n e s as well as the effector tissues that translocate calcium ions into or out of the ECF in kidney, bone, and intestine. Furthermore, the capacity of not only the cells secreting Ca 2+ o -regulating hormones but also the capacity of these effector tissues to sense C a 2+ o (as well as phosphate ions) add additional layers of regulatory control to the mineral ion homeostatic system. The G protein-coupled CaSR has a central role in systemic C a 2+ o homeostasis by enabling maintenance of near constancy of Ca2o+ via its coordinated actions on the various tissues involved in mineral ion homeostasis. As we learn more about the calciumsensing receptor's roles in the tissues directly participating in C a 2+ o homeostasis, it may turn out that it contributes to the regulation of other processes relevant to mineral ion homeostasis, such as controlling 1-hydroxylation of vitamin D or phosphate reabsorption in the proximal tubule. In any event, the calciumsensing receptor's exquisite sensitivity to even m i n u t e changes in Ca2o+ permits adjustments in the C a 2+ o homeostatic system's responses, for example, to increases or decreases in dietary Ca 2+ intake that produce barely detectable changes in Ca2o+. Finally, the CaSR is present not only in tissues directly involved in C a 2+ o homeostasis but also in those that are not. Thus this receptor may participate in coordinating interactions a m o n g several different homeostatic systems, such as those for regu2+ lating water, M go, Na + , extracellular volume, blood pressure, a n d / o r protein metabolism, which are usually t h o u g h t of as functioning largely i n d e p e n d e n t l y of mineral ion metabolism.

ACKNOWLEDGMENTS The author gratefully acknowledges the support of the following grants for work described in this chapter, as well as for salary support: NIH grants DK41415, DK48330, and DK52005; The National Dairy Council; The Cystic Fibrosis Foundation; The National Space Bioscience Research Institute (NSBRI); and the St. Giles Foundation and NPS Pharmaceuticals, Inc.

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CHAPTER11 Parathyroid H o r m o n e A n a b o l i c and Catabolic Effects o n B o n e and Interactions with Growth Factors JANET M. H O C K Department of Periodontics, Indiana University School of Dentistry, Indianapolis, Indiana 46202 LAWRENCE G. RAISZ Department of Medicine, The University of Connecticut School of Medicine, Farmington, Connecticut 06030 ERNESTO CANALIS Department of Medicine, The University of Connecticut School of medicine, Farmington, Connecticut 06030; and Departments of Research and Medicine, Saint Francis Hospital and Medical Cent~ Hartford, Connecticut 06105

INTRODUCTION

W h e n the synthetic fragment h P T H ( 1 - 3 4 ) became available in the early 1970s, there was renewed interest in evaluating the anabolic effect of PTH as a potential therapeutic to restore bone loss caused by diseases such as osteoporosis (6-8). N u m e r o u s studies since then have showed that synthetic h P T H ( 1 - 3 4 ) , given intermittently, increased bone mass in a variety of animal models, of which the best studied is the rat (9-64). The r e c o m b i n a n t full-length h o r m o n e , h P T H (1-84), as well as a variety of PTH and PTH-related protein (PTHrP) amino-terminal analogs, all induce anabolic effects similar to those induced by h P T H ( 1 - 3 4 ) (6-8, 65). The identification of growth factors and cytokines in the mechanisms by which differing exposures to PTH may induce either anabolic or catabolic effects is predominantly based on in vitro cell and bone organ model systems from embryonic and neonatal rodents. The extent to which these models mirror the mechanisms of in vivo events in osteonal cortical and trabecular bone of diseased h u m a n skeletons remains to be determined. Despite a wealth of literature affirming the anabolic effect of PTH on rat skeletons, many of these studies were done on ovariectomized rats, and the confounding variables associated with ovariectomy were not separated from the mechanisms activated by PTH. Recent studies of ovariectomized monkeys treated for up to 18 m o n t h s with PTH showed that increased bone turnover after ovariectomy was associated with decreased bone mass and strength, whereas increased bone turnover after PTH was associated with increased bone mass and strength (66-68). PTH reversed the changes in serum calcium, phosphate, and calcitriol induced by

The concept that parathyroid h o r m o n e (PTH) has both catabolic and anabolic effects was first proposed in the early n i n e t e e n t h century. The major mechanism for the catabolic action is a selective stimulation of bone resorption. The mechanisms underlying the anabolic response are still not clearly understood, but are likely to involve a cascade of growth factors and cytokines that regulate or support bone formation. Low doses of crude preparations of parathyroid extract (PTE) increased trabecular bone density in rodents, guinea pigs, and rabbits, after an initial dose-dependent episode of resorption with some tissue necrosis, and transient hypercalcemia (1-5). The PTE preparations contained a mix of proteins, and it was not clear which events could be attributed directly to PTH and which represented an inflammatory response to the protein mix. However, the p h e n o m e n a gave rise to the perception that the stimulatory effects on formation had to be preceded by a resorption phase to generate the appropriate growth factors. More recent data have modified this hypothesis, as dose and duration of exposure have become recognized as critical factors in determining the outcome on bone, and differences in responses of the different bone envelopes have become better characterized through studies of large animals with osteonal bone skeletons. The molecular mechanisms and contributing factors that underlie the differences in skeletal responses between the pharmacologic actions of exogenous PTH and the pathologic actions of endogenous PTH in hyperparathyroidism have yet to be identified. The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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ovariectomy to match the values in sham monkeys, in addition to stimulating accumulation of bone on cortical and trabecular endosteal surfaces (67-69). Understanding how the multiple growth factors and cytokines activated by ovariectomy may be modified by exogenous PTH to improve biomechanical properties of bone, and to counteract the negative bone balance associated with ovariectomy, will require innovative approaches and new in vitro models.

SKELETAL G R O W T H FACTORS In vitro, the anabolic effect has been defined on the basis of surrogate markers for bone gain, such as an increase in the rate of type I collagen synthesis, increased bone cell proliferation, or increased formation of bone marrow fibroblastic cell colonies. Cells either briefly or intermittently exposed to PTH exhibit stimulatory effects on osteoblast differentiation (70) and function (71). The extent to which these outcomes may model the in vivo actions of PTH is not well understood yet, because other agents that are not anabolic in vivo induce similar in vitro p h e n o m e n a . Although PTH undoubtedly has direct effects on bone cells, in vitro studies have shown that some of its actions may be mediated by locally produced growth factors. Bone cells synthesize insulin-like growth factors (IGF-I and IGF-II), transforming growth factor-[~ (TGF[31,-[32, a n d - [ 3 3 ) , platelet-derived growth factors (PDGFs), fibroblast growth factors (FGF-1 and FGF-2), bone morphogenetic proteins (BMPs) or osteoinductive factors, and hepatocyte growth factor or scatter factor (72). Of these various factors, TGF-[3 is of particular interest because it both stimulates bone formation and inhibits bone resorption. This suggests it may be involved in the molecular mechanisms of the reversal phase of coupling, that is, inhibition of resorption followed by stimulation of formation. Skeletal cells also synthesize and respond to cytokines known to have primary effects on i m m u n e and hematologic cells, such as interleukin-1 a n d - 6 (IL-1, IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor ot (TNFot), and colony-stimulating factors. Of these, PTH regulates IL-6 and LIF as immediate-early genes in vivo (73-76), but the downstream consequences of this transcriptional regulation are not known. Most recently, PTH was found to regulate transcription of RANKL/OPG, which has been linked to regulation of osteoclast differentiation as an immediate-early gene, after a single injection was given to young male rats (77). Immunohistochemistry showed localization of RANKL protein to cells in close proximity to osteoblasts and osteoblasts, within the primary spongiosa, including the zone enriched for proliferating cells subjacent to the

growth plate (77). A detailed description of the growth factors synthesized by skeletal cells is beyond the scope of this chapter, and our discussion is limited to a selected group of factors.

Insulin-like Growth Factors-I a n d - I I IGF-I a n d - I I are polypeptides with a molecular mass (Mr) of 7600. IGF-I a n d - I I have 66% amino acid sequence homology and have similar biologic activities. IGFs are present in the systemic circulation and are synthesized by skeletal and a variety of nonskeletal cells. The role of IGFs as systemic regulators of bone metabolism has not been demonstrated fully. Mice in which the IGF-I gene has been genetically deleted show abnormal skeletal development, growth retardation, and do not attain puberty (78). Significant abnormalities in serum levels of IGF-I or-II have not been shown in patients with various metabolic bone disorders. In mice in which liver production of IGF-I was genetically abrogated, skeletal growth of long bones did not differ from those of intact controls, suggesting that hepatic endocrine production of IGF-I is not required for normal skeletal development (78,79). It is believed that IGF-I and -II act as local regulators of musculoskeletal cell function (78) through activation of the IGF-I receptor (80). IGFs stimulate bone formation in vitro (81,82). They increase the replication of bone cells, primarily of preosteoblasts, and independently stimulate osteoblastic collagen synthesis and matrix apposition rates (Fig. 1). IGF-I and-II have similar effects, although IGFI is somewhat more potent than IGF-II. In addition to their effects enhancing collagen synthesis, IGFs decrease collagen degradation, probably because they decrease collagenase expression (83). Because of their important effects on bone cell replication and differentiation, it is believed that these polypeptides are major regulators of bone formation and are important in the maintenance of bone mass. Skeletal cells secrete the six known IGF binding proteins (IGFBPs), as well as two of the four known IGFBP-related proteins (IGFBP-rPs) (84-86). The exact role of IGFBPs in bone cell metabolism is not entirely known. It is postulated that IGFBPs are important for the storage of IGF and to prolong its half-life. They may regulate IGF activity by competing with cell surface receptors for IGF binding. Some IGFBPs, such as IGFBP-4, have mostly an inhibitory activity on osteoblastic function, whereas others, such as IGFBP-5, can enhance the anabolic effects of IGF-I (86). In addition to IGFBPs, osteoblasts express IGFBP-rP, the product of the mac25 gene, and IGFBP-rP-2 or connective tissue growth factor, although it is not known if they express IGFBP-rP-3 and-4. PTH induces IGFBP-rP-1 in osteoblasts by transcriptional mechanisms (86).

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Transforming Growth Factor-[[ and Related Polypeptides TGF-[3 is a dimeric polypeptide of M r 25,000. TGF-[3 is secreted as a homodimer. Five TGF-[3 isoforms have been describedmTGF-[3 1 , - 2 , - 3 , - 4 , a n d - 5 (87). Heterodimers of TGF-[3 also have been isolated from various tissues. Skeletal cells synthesize TGF-[31,-2, a n d - 3 and bovine bone has been shown to contain TGF-[31.2 and TGF-[32.3 heterodimers (88-90). The reason for the presence of various TGF-[3 isoforms in bone tissue is not clear, particularly because the various forms tested have virtually the same effects on bone cell function, with modest differences in potency (91). For the most part, studies using cultures of normal osteoblasts and intact calvariae have demonstrated that TGF-[3 stimulates cell replication as well as collagen and noncollagen protein synthesis (88). TGF-[3 enhances biochemical parameters of bone formation as well as matrix apposition rates (92). TGF-[3 is secreted as a complex of large molecular weight consisting of the polypeptide, a precursor, and a binding protein. TGF-[3 is present in the tissue matrix in an inactive form and it is activated by various mechanisms, including lowering of the pH, which could occur in the bone environment during the process of bone resorption (93). Thus, TGF-[3 may be important in the coupling of bone resorption to formation. In addition to the five TGF-[3 isoforms described, there are a n u m b e r of related polypeptides that share up to 30% amino acid sequence homology with TGF-[3 and may have similar biologic activities. These polypeptides include inhibins, activin, Mullerian inhibiting substance, the gene products of Drosophila decapenta-

plegic,

and a variety of BMPs or osteoinductive substances (94). Initially, the primary function of BMPs was considered to be the induction of e n d o c h o n d r a l bone formation following the implantation of demineralized bone matrix in soft tissues. Further studies have indicated that BMPs play a central role in inducing the differentiation of mesenchymal cells into cells of the osteoblastic lineage and e n h a n c i n g the differentiated function of the osteoblast (95,96).

Platelet-Derived Growth Factor Platelet-derived growth factor is a dimer of M r 50,000. PDGF is the product of two genes, PDGF A and B, which give rise to two distinct PDGF chains with 60% homology (97). PDGF exists as a h o m o d i m e r or h e t e r o d i m e r of these two chains, which can combine to form PDGF AA, BB, and AB. PDGF is stored in platelet granules and, as such, it can act as a systemic regulator of cell function. In humans, the circulating forms of PDGF are, for the most part, PDGF BB and AB (98). Osteoblasts and osteosarcoma cells express the PDGF A and B genes (99-101). The three PDGF isoforms stimulate cell replication in intact calvariae and in osteoblast cultures (102-104). PDGF BB is more potent than PDGF AA, and PDGF AB has an intermediate effect. As a consequence of its effects on cell replication, PDGF causes a small increase in bone protein synthesis. However, PDGF does not stimulate the differentiated function of the osteoblast and it is, to some extent, inhibitory (104). At present, the production of specific PDGF binding proteins by bone cells is uncertain, and it is believed that skeletal PDGF is secreted in a biologically active form.

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Fibroblast Growth Factors FGFs are a group of polypeptides with mitogenic activity. FGF-1 (acidic fibroblast growth factor) and FGF-2 (basic fibroblast growth factor) are secreted by osteoblastic cells (105,106). FGFs have a n M r of approximately 17,000, and FGF-1 and-2 have about 55 % amino acid sequence homology. FGFs are not secreted proteins and are likely to be b o u n d to the bone matrix, where they probably act as local regulators of skeletal cell function. FGFs are bone cell mitogens and do not appear to have potent direct stimulatory effects on osteoblastic differentiated function (107,108). Currently, it is unknown if bone cells also secrete specific binding proteins for these factors.

I N T E R A C T I O N S O F P T H A N D SKELETAL G R O W T H FACTORS IN VITRO PTH stimulates IGF-I synthesis by osteoblastic cells (107). This effect is observed at PTH concentrations of 0.1-10 n M and involves the transcriptional regulation of IGF-I synthesis. PTH increases both IGF-I mRNA and polypeptide levels about threefold (Fig. 2). The effect of PTH appears to be mediated, at least in part, by an increase in cyclic AME because other agents known to enhance cyclic AMP production by osteoblastic cells, such as prostaglandin E 2 (PGE2) and forskolin, also increase skeletal IGF-I synthesis (109,110). In contrast, the calcium ionophore (ionomycin) and phorbol esters do not alter IGF-I synthesis by osteoblastic cells. The effect of PTH on IGF-I production is mimicked by parathyroid hormonerelated peptide in bone cells (111). PTH and PGE 2 each increase IGF-I production and PTH can increase PGE 2 production in bone, suggesting possible mechanisms for the anabolic effect (107,112,113). However, in cell culture, indomethacin, which inhibits PGE 2 synthesis, has little effect on the ability of PTH to increase IGF-I production. Nevertheless, it is possible that u n d e r some circumstancesmfor example, in estrogen deficiency, when PGE 2 production is e n h a n c e d m t h a t an increase in PGE2-induced IGF-I could amplify the anabolic effect of PTH (114). FGF may also mediate some of the anabolic effects on bone formation because it has been implicated as a possible mediator for PTH and PGE 2 (115-117). FGF appears to be important in limb develo p m e n t and patterning, due to its effects on growth plate cartilage and linear growth of bones (118). Knockout FGF m u t a n t mice exhibit decreased bone formation (118). In vitro, PTH increases FGF-2 mRNA levels in osteoblastic cells, as well as FGF receptor 1 and 2 transcripts (118). FGF receptors 3 and 4 were neither detected nor regulated by PTH in these studies

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(118). The regulation of FGF by PTH supports the possibility of a complex amplification system. Not only is FGF production stimulated by prostaglandins, particularly PGE2~, but FGF increases PGE 2 production in bone cells (115,117). Although PTH stimulates IGF-I synthesis and IGF-I increases bone collagen synthesis, continuous exposure of bone cells to PTH results in a decrease in bone collagen production (70,111) (Fig. 3). This inhibitory effect seems to be the result of PTH overriding the stimulatory actions of IGF I. When calvarial explants are concomitantly exposed to PTH and IGF-I, only the inhibitory effect of PTH on collagen synthesis is observed. In contrast, transient exposure to PTH, which results in an induction of skeletal IGF-I, causes a stimulation of collagen synthesis (Fig. 4) (71). The stimulatory effect of PTH on collagen synthesis is blocked by IGF-I neutralizing antibodies, suggesting that IGF-I is at least in part responsible for the increase in bone collagen (119).

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In addition to changes in IGF-I synthesis, PTH also modifies the synthesis of IGFBPs and IGFBP-rPs. However, it does not alter IGF-II synthesis or the binding of IGFs to specific cell surface receptors (119). The synthesis of IGFBP-3,-4, a n d - 5 in bone cells is cyclic AMP d e p e n d e n t , and transcripts for IGFBP-4 have been shown to be elevated by PTH (85). It is a p p a r e n t that the induction of cyclic AMP in bone cells not only results in an increased production of IGF-I, but also of selected IGFBPs (85). Although the role of the binding proteins is not entirely known, it is possible that they are i m p o r t a n t in mechanisms regulating the exposure of bone cells to endogenous IGF-I. PTH and other agents that stimulate bone resorption increase the secretion of TGF-[3 activity in bone cultures during this process (93). It has been suggested that this p h e n o m e n o n is due to the activation of TGF-[3 in the osteoclastic microenvironment, possibly the result of lowering the pH. Because PTH does not increase TGF-[3 mRNA levels in osteoblastic cells, it most likely activates previously synthesized TGF-[3. TGF-[3 may play an i m p o r t a n t role in the local control of bone resorption, because it has b e e n shown to inhibit this process as a result of a decrease in the formarion of osteoclast-like cells (120,121). In addition to its effects on TGF-[3 activation, PTH regulates the binding and activity of TGF-[3 in osteoblast cultures (122). PTH increases the n u m b e r of apparent TGF-[3 receptors, but for reasons not entirely understood it opposes the activity of TGF-[3 on bone DNA and collagen synthesis in vitro. The contribution of TGF to PTH effects in more mature postnatal bones has not b e e n studied.

PTH does not modify the concentrations of PDGF AA or BB in bone cell cultures or the binding of PDGF to its bone cell receptors, suggesting that PDGF is not an important m e d i a t o r of PTH function in bone. In vivo, PDGF increases bone mass to the same extent as PTH in rats, but also accelerates maturation of the growth plate and a n u m b e r of extraskeletal side effects (123), suggesting a different pathway of activation than that induced by PTH. There are few studies on interactions between multiple growth factors and PTH. The possibility that the ability of PTH to alter the geometry and connectivity of trabecular bone may be d e t e r m i n e d by a spectrum of local growth factor responses has not been adequately studied, but could represent an i m p o r t a n t feature of the anabolic effect. New models, based on developmental biology models of patterning, could be applied to investigate this hypothesis.

ANABOLIC EFFECT OF PTH ON THE SKELETON

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The anabolic effect of PTH in vivo may be best defined as a net increase in bone mass or bone mineral either of the skeleton as a whole, or of individual bones, associated with a selective increase in bone formation. The increase in bone mass induced by exogenous PTH has been d e m o n s t r a t e d in a wide variety of animal models and confirmed in h u m a n s (6-8,44,65,124,125). Differences in the basic bone biology of different species appear to control the magnitude of the increm e n t in bone mass. T r e a t m e n t with PTH for at least

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6 months increases bone mass by more than two- to fourfold in the spine and proximal tibia, and by more than 20% in the other long bones of ovariectomized rats (52,126). In animals in which osteonal bone is the dominating structure, bone mass gain is in the range of 8-15% in both ovariectomized monkeys after 18 months (66-68) and in osteoporotic women after 2-3 years (6,127-129). The greatest incremental increase in bone mass occurs early in treatment, usually within the first one to two turnover cycles (8,39,64,130,131) (Fig. 5). The anabolic effect of PTH is i n d e p e n d e n t of g e n d e r and sexual maturity; it occurs in middle-aged and osteoporotic male and female h u m a n s (6,127-129) and in intact and castrated osteopenic male and female rats (20,24, 32-34,42,45,47-50,54,60,126,132). PTH also increases bone mass in rats with osteopenia induced by denervation (53,133,134), immobilization (35-37,134,135), or discontinuation of exercise (44,53,136,137). Anabolic

A n a b o l i c E f f e c t s o f P T H in A n i m a l s with Osteonal Bone

In adult h u m a n s and animals with osteonal cortical bone, the response to PTH is d e p e n d e n t on both modeling and remodeling processes in bone. T h o u g h the net outcome is a gain in bone mass, as in rats, the mechanisms by which this is accomplished are apparently quite different, and the bone gain is always less than that observed in rats. W h e t h e r PTH induces similar molecular signaling pathways in bone of different species has not been explored. The doses in humans are considerably lower than those used in rats, and are limited by the need to avoid chronic hypercalcemia. In h u m a n s and large animals with osteonal bone, PTH stimulates modeling as de novo bone formation on trabecular and cortical endosteal surfaces, and stimulates remodeling by increasing activation frequency in both trabecular and intracortical bone (44,141-146). The stimulation of new trabecular bone formation by modeling is present as early as 2 weeks after starting treatm e n t in h u m a n s (147,148). The remodeling process in cortical bone of large animals and h u m a n s replaces intracortical matrix in situ, so bone mass may be maintained or slightly decreased in the course of treatment. Endocortical formation is stimulated but PTH has little or no effect on periosteal bone formation. The increase in intracortical bone turnover is necessarily associated with remodeling

PTH EFFECTS ON BONE ANt) GROWTH FACTORS / transients manifest as increased porosity, signaling the formation of new osteons (130,131). In some species of large animals with cortical osteonal bone, such as intact dogs and rabbits or ovariectomized ferrets and sheep, the increase in activation frequency predominantly reflects an increase in remodeling sites, and there is often no change in overall bone mass (6,125,130,131,149,150). However, in ovariectomized monkeys and osteoporotic humans, the composite response of an increase in trabecular bone mass due to the increase in u n c o u p l e d modeling formation and an increase in the a m o u n t of replaced matrix due to intracortical remodeling results in increased bone mass and resistance to fracture (6,69,130,131). The tissue events supporting the increase in trabecular bone change with time in animals with remodeling bones. In iliac trabecular bone of ovariectomized monkeys, the increase in trabecular bone volume after 6 m o n t h s is due to increased trabecular thickness attributable to new endosteal bone formation (6,69,124,130,131,151). After 15 m o n t h s of treatment, the increased trabecular volume is associated with increased trabecular number. The thickened trabeculae are modified by increased sites of tunneling, indicative of the increase in n u m b e r of r e m o d e l i n g sites, to reestablish connectivity in the trabecular framework and reduce trabecular thickness back to control values (124). The consequence of bone matrix renewal and restructuring by remodeling, c o m b i n e d with the increase in trabecular bone, is a significant improvem e n t in bone quality and biomechanical properties at clinically relevant sites in the spine and f e m u r neck (66-68,124). The growth factors and patterning genes that must be required to regulate the restructuring and r e m o d e l i n g of bone have not been e x a m i n e d in animal models in which osteonal bone structure and remodeling processes predominate.

A n a b o l i c E f f e c t s o f P T H on the S k e l e t o n o f Rodent Models The complex changes in bone structure induced by PTH in animals with osteonal skeletons are only partially r e p r o d u c e d in the rat. The rat lacks osteonal bone structure, and so PTH given at normocalcemic doses given for equivalent turnover cycles only stimulates accrual of bone t h r o u g h cumulative appositional bone growth on cortical and trabecular bone surfaces (61-63,152,153). In rats, PTH stimulates linear bone growth and increases trabecular bone mass by thickening trabeculae of the primary spongiosa and increasing the n u m b e r of osteoblasts per unit area (35,126,154). As the woven bone of the primary spongiosa acts as a template for secondary spongiosa, its increased mass and volume translates into increased bone mass of the secondary spongiosa, which is composed of mostly

189

lamellar bone. In PTH-treated rats, the trabecular osteoblasts stay active for a longer time, so the overall bone-forming surfaces in the metaphyses increase significantly. At the demarcation between metaphyseal bone and diaphyseal hematopoietic bone marrow, trabeculae are terminated by osteoclastic resorption. Although the rate of resorption at this location is not coupled to the formation events at the growth plate, the two processes are usually in equilibrium (35,126,141-144,154). PTH increases the rate of metaphyseal bone turnover so that the metaphyseal trabeculae extend in to the diaphyseal marrow as the osteoclastic resorption of the trabeculae fails to keep pace. This process, which represents a "bulking up" of e n d o c h o n d r a l osteogenesis, underlies the two- to threefold increases in bone mass seen in rats treated with PTH. The anabolic effects of PTH on trabecular bone are reversible when the h o r m o n e is withdrawn and normal e n d o c h o n d r a l osteogenesis resumes (16,47-50). The process by which this occurs in rats is quite different from the more complex response of humans, where remodeling of bone is the d o m i n a n t m e c h a n i s m controlling bone mass. In cortical bone of rats, radial growth occurs t h r o u g h o u t life as bone diameter is increased by periosteal formation, but cortical width is maintained by a parallel resorption at the endocortical surfaces (58,63). PTH may increase endocortical bone formation rate by as m u c h as 400% and periosteal rate by 60-70% (58,63,64). The continued surface accumulation of matrix on both periosteal and endosteal surfaces significantly thickened the cortex by 75% and increased cortical area by 25% in just 10 weeks in aged ovariectomized rats (62,155). The question remains whether in vitro models can differentiate these multiple responses. The models should be designed to determine the critical roles for growth factors specific for PTH induction, versus their role in mediating accelerated bone turnover and skeletal growth. The m a g n i t u d e of the bone response in rats is dose d e p e n d e n t (8,9,15) (Fig. 6). The threshold dose for an anabolic effect on bone in rats has not been determined, although increased bone has b e e n induced in rats after 3-6 months of h P T H (1-34) at low doses ranging from 1.5 to 8 Ixg/kg/day (10,45,134). In older rat models where growth is less, the increase in p e r c e n t bone-forming surfaces is of a m a g n i t u d e similar to that of younger animals, but there is less surface available for formation because bone has been lost with aging. Much of the anabolic effect in aged rats is therefore limited to the cortical e n d o s t e u m and periosteum. The increased bone mass and improved biomechanical properties are attributed to increased cortical width and cortical area (12,13,33,34,60,61,126,132). In adult rats, there appears to be a limit to the magnitude of the dose response in terms of bone mineral

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CHAPTER11 RAT TRABECULAR

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hPTH 1-34

FIG. 6 Anabolic effects of PTH on calcium content and dry weight of cortical and trabecular bone of distal femurs of young male rats treated with vehicle or hPTH(1-34) once daily for 12 days. Reprinted courtesy of Metab Dis Relat Res 1984.

density (BMD). A plateau effect, especially at higher doses of PTH, may be induced after 3 or more months of t r e a t m e n t (10,11,39). The consequences of continuing t r e a t m e n t with PTH once this plateau has been reached, and the interaction of this induced response with the catabolic changes of aging, have not been studied. Published data have only reported on treatment for up to 6 months, and then only in ovariectomized rats (52,126). Interactions with growth factors and cytokines that may be involved in these processes have not been investigated. A n a b o l i c vs. C a t a b o l i c E f f e c t s o f P T H o n B o n e in Vivo

In rats, the timing of exposure of PTH appears to be more critical than dose or peak concentrations in d e t e r m i n i n g whether formation or resorption predominates. The anabolic effect of PTH requires its intermittent administration (27,156-158). Increasing the n u m b e r of injections of PTH per day to 2 - 3 / d a y did not a u g m e n t the anabolic effect on either total body calcium in female rats (19), or hydroxyproline content of femurs of young female rats (158,159). PTH, given by continuous infusion at doses that were anabolic when given once by daily injection, induced hypercalcemia and death in rats a n d d o g s (27,44,137). W h e n PTH was infused continuously, or when repeated

injections were given over extended periods of time each day, the stimulatory effect of PTH on bone turnover was retained but the balance shifted from formation to resorption (27,160,161). The extent of boneforming surfaces decreased and the extent of bone resorption surfaces increased, resulting in decreased bone mass (27,160,161). The matrix metalloproteinase MMP13 (collagenase 3) appears to be required in mice because the resorbing effects of PTH were blocked in calvaria of m u t a n t mice carrying a targeted mutation in the collagen I gene that was resistant to collagenase cleavage of type I collagen (162). In addition, subtle changes were observed in proteins synthesized by the bone lining cells, which were m o r e consistent with e m e r g e n c e of a fibroblastic phenotype when compared to the osteoblast phenotype associated with intermittent t r e a t m e n t (163). Peritrabecular fibrosis, which was not observed in rats or monkeys treated with once-daily PTH, and focal bone resorption were observed when PTH was infused for 6 days in rats (160,163,164), but not when PTHrP was infused (164). Hypercalcemia and increased calcitonin were reported during the infusion period, suggesting additional systemic factors might influence the response (28,165). There was also a shift in IGF and IGF binding proteins. IGF-I, which was detected in osteoblasts of rats treated with once-daily PTH, became undetectable in the bone lining cells following PTH infusion, whereas the intensity of immunostaining for IGF-BP-3,-4, a n d - 5 increased following infusion for 7 days (153). IGF-I infusion in older ovariectomized rats stimulated resorption and impaired the anabolic effect of PTH on bone mass (28). These cumulative data suggest that additional molecular and cellular pathways may be invoked when there is prolonged exposure to PTH. The signaling pathways that result in a switch from bone accumulation to bone loss in rats are not understood. Rats given PTH as two or three injections/day at doses that sustain normocalcemia continued to exhibit an anabolic effect equivalent to that occurring with once-daily injections (19,158,159). When PTH was given at an equivalent daily dose of 80 txg/kg/day, divided into one or six injections, given either within 1 hour or over 6-8 hours, the anabolic effect of six divided injections in 1 h o u r / d a y was equivalent to that measured with one injection/day (166). However, when injections were spread over 6-8 hours, loss of bone mass occurred (166). Similarly, when PTH was infused for at least 2 hours, peritrabecular fibrosis and focal resorption, characteristic of a catabolic outcome, could be detected (160). This would suggest that stimulation of the resorptive effects of PTH is less a consequence of peak blood concentration, and more likely due to a critical cumulative duration of exposure (166). It has been shown that RGS-2 (a m e m b e r of a family of "regulator of G protein

PTH EFFECTS ON BONE AND GROWTH FACTORS // signaling" proteins) increased within 1 hour of an injection of PTH in young rats, suggesting a novel mechanism that can limit G protein signaling, subsequent to effects on the PTH1 receptor (167). In large animals, PTH, irrespective of regimen, stimulates bone resorption in intracortical bone, thereby stimulating a transient increase in remodeling space, manifest as increased porosity (69,130,131,146,149). Activation of resorption associated with porosity was observed mostly within the endosteal region of cortical bone. This regionalization, combined with new bone apposition on cortical endosteal surfaces to thicken cortical width, m e a n t that the increased porosity did not significantly alter bone biomechanical measures of strength (69,130,131,145,146,149). W h e t h e r PTH is anabolic or catabolic in skeletons with cortical osteonal bone depends on the response of the cortical endosteum. In ovariectomized monkeys and intact rabbits, intermittent PTH stimulated endocortical bone formation to increase cortical width and area, thus preserving bone mass and biomechanical measures of strength (69,130,131,145,146,149). In contrast, in dogs, continuous infusion of PTH did not stimulate formation on the cortical endosteum, so there was a transient loss of bone mass associated with porosity during treatment (168).

Reversal o f the A n a b o l i c E f f e c t o n With drawal o f P T H In young rats, the activation of bone formation by PTH was abrogated within 24-48 hours after the last PTH injection (16). This reversal was attributed to inactivation of forming surfaces, because there are m a r k e d decreases in percent calcein-labeled bone surface (DLS/BS) and percent osteoblast surface (ObS/BS), but little change in n u m b e r of osteoclasts and osteoclast surface (16). In a series of studies of spine and long bones of older ovariectomized rats, Shen et al. showed that loss of the bone gain over several weeks could be blocked by c o n c u r r e n t estrogen (47-50). This suggested that in older rats, in which growth is a less significant factor, the loss of bone following withdrawal of PTH may be due in part to activation of previously suppressed resorption (47-50). Large animal studies have provided additional insights in the more complex skeleton in which osteonal bone structure dominates. Ovariectomized (OVX) monkeys were treated once daily with h P T H ( 1 - 3 4 ) for 12 months; t r e a t m e n t was then discontinued and monkeys were given daily vehicle injections for 6 additional months before being euthanized (67,68). Bone gain after this t r e a t m e n t regimen was significantly higher than OVX controls, and similar to that of animals treated for the entire 18 months (67,68). H i s t o m o r p h o m e t r y showed that bone formation rates

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in trabecular bone following t r e a t m e n t withdrawal r e m a i n e d equivalent to that of OVX controls (69,124). In cortical osteonal bone, bone formation rates decreased dramatically to the level of sham controls, and below those of OVX controls. Such a response implies that a significant decrease in turnover of cortical bone occurred once t r e a t m e n t was withdrawn. In this monkey study, as well as an earlier study of intact rabbits, the percent porosity in cortical bone declined after withdrawal of treatment, as the r e m o d e l i n g cycles completed and newly formed matrix mineralized (69,130,131). These events may explain the gain in bone mass and bone quality after withdrawal of treatm e n t in a small clinical study of p r e m e n o p a u s a l women (169). In hyperparathyroid h u m a n s following surgical removal of the parathyroid gland adenoma, adverse cortical changes were reversed and i m p r o v e m e n t in cortical bone mass was observed, especially the first year following surgery (170-172). However, whether these changes alter susceptibility of bones to fracture has still to be determined. Because these sequelae have not been observed in rats or reported in in vitro protocols, new experimental models are n e e d e d to study the local mediators and molecular mechanisms that regulate the different responses of trabecular and cortical osteonal bone following t r e a t m e n t withdrawal.

I N T E R A C T I O N S O F P T H A N D SKELETAL HORMONES AND GROWTH FACTORS IN VIVO E v i d e n c e o f R e q u i r e m e n t for M e d i a t o r s to I n d u c e the A n a b o l i c E f f e c t o f P T H in Vivo The early histologic studies of the bone effects of intermittent PTE in young rats (1-5,125) reported a resorptive phase in the first few days, later followed by a significant gain in bone density. I n vitro studies of avian bones treated by transient exposure to PTH (173,174) showed increases in resorption and the rate of hydroxyproline incorporation in parallel cultures. However, in vivo, blocking resorption with a short-term course of either calcitonin or a bisphosphonate did not modify the anabolic effect of PTH in either rats or h u m a n s (21,27,175). Even in the presence of a loss of body weight and inhibition of resorption associated with 12 days of calcitonin treatment, PTH continued to increase bone mass (21). W h e n PTH was given by continuous infusion and bone resorption was blocked with a bisphosphonate, the inhibition of bone formation associated with bisphosphonate was reversed by the PTH to control levels (21,36,37,41,42). Studies on the early response in h u m a n s also showed that de novo bone

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formation was stimulated by PTH in the absence of any measurable change in resorption (147,148). Although PTH increases the activation frequency of remodeling, the r e q u i r e m e n t for a regulatory mediator in osteonal bone has not been investigated in vivo. In cortical bone, bone formation increased the percent active osteonal surfaces in ovariectomized monkeys treated with h P T H ( 1 - 3 4 ) . Although the percentage of osteons exhibiting resorption increased, this did not appear to be a d o m i n a n t m e c h a n i s m of action, and bone mass was retained or increased in ovariectomized monkeys.

Interaction with IGF-I In vivo, IGF-I stimulates both bone formation and resorption. W h e n given by continuous infusion, IGF-I stimulated tunneling resorption of the diaphyseal cortex in aged female rats (28). IGF-I may stimulate osteoclastic bone resorption in vitro by regulating osteoclast differentiation (176). PTH has been shown to regulate not only IGF-I but also its binding proteins in vitro (82,177). It may be that, in vivo, intermittent PTH transiently modifies the IGF-binding proteins to activate IGF-I and initiate its anabolic action in bone, as suggested by the work of Watson et al. (153). Circulating IGF-I could also play a role in regulating the anabolic effects of PTH. Preliminary data, obtained from young hypophysectomized (HX) rats and aged female rats, showed that although PTH did not alter serum IGF-I, growth h o r m o n e (GH) increased IGF-I, and PTH given with GH partially inhibited this stimulatory effect of GH on serum IGF-I (14,23). Long-term energy restriction of rats results in a 50% decrease in circulating IGF-I (178). Rats, energy restricted in their dietary intake by 60% since weaning, showed an attenuated response in their f e m u r bone mass and tibia bone mineral when treated with PTH alone or in combination with GH (14).

Interaction with Growth H o r m o n e Early in vitro studies in which calvaria from HX rats were treated with high doses of PTE in vivo and then cultured showed less hydroxyproline incorporation than did calvaria from intact rats treated with PTE (179). This suggested the hypothesis that a pituitary h o r m o n e , most likely, growth h o r m o n e , might be required for the anabolic effect of PTH on bone (23). We found that hypophysectomized rats, s u p p l e m e n t e d with corticosterone and thyroxine and treated with the intermittent PTH showed a 50-70% inhibition of the anabolic effect. GH not only reversed this inhibition, but also e n h a n c e d the anabolic effect of PTH on bone (23). To d e t e r m i n e if this was due to GH stimulation of

systemic growth, young intact rats were given PTH and either pair-fed to the dietary intake of age- and weightm a t c h e d HX rats, or fed a 50% calorie-restricted diet to prevent the increase in body weight associated with growth. The PTH-induced increase in bone mass of distal femurs of the restricted rats was equivalent to that of rats fed ad libitum (23). GH secretion declines with age (180-182). If GH is required for the anabolic action of PTH, aged animals should show a loss of responsiveness to PTH in their bones. In a study of aged intact females rats, neither PTH nor GH alone increased bone mass, but the combination of PTH and GH increased femur trabecular bone mass and vertebral trabecular bone volume (25). In another study of aged hypophysectomized rats, PTH increased bone formation rate despite the absence of GH, but the net effect on bone mass, to confirm an anabolic outcome rather than an outcome in which bone turnover but not bone mass increased, was not assessed (183). Further studies are n e e d e d to better resolve the relative r e q u i r e m e n t for GH and IGF-I in the anabolic effects of PTH on bone.

Interaction with Prostaglandins PTH stimulates prostaglandin synthesis in vitro by initiating the synthesis of the inducible cyclooxygenase (COX-2) (112,113). This effect may be involved in the catabolic responses to PTH in vivo because injection of PTH in transgenic mice lacking the COX-2 enzyme show less hypercalcemia than do wildtype animals. Both PTH and PGE 2 can stimulate bone formation in rats (114). PGE 2 appears to produce greater periosteal stimulation c o m p a r e d to PTH and may also produce more woven bone on cortical endosteal surfaces (184-186). In vivo studies, using indomethacin given by injection or orally to block prostaglandins, did not modify the anabolic response to PTH in young rats (187). These experiments were limited because the highest doses produced gastric lesions and the lower doses may not have fully inhibited local prostaglandin production. It seems likely both from in vitro studies (188,189), in which PTH stimulation of bone resorption and mitogenesis are not completely blocked when prostaglandin production is inhibited, and from the in vivo studies cited above that prostaglandins are not essential for anabolic action of PTH. Nevertheless it is possible that prostaglandins as well as fibroblast growth factor, production of which is stimulated by both PTH and PGE 2, can enhance the response to PTH, and that this could be of importance in the therapeutic response. It will now be possible to examine this question more completely by using selective COX-2 inhibitors in humans and COX-2 and FGF knockout animals.

P T H EFFECTS ON BONE AND GROWTH FACTORS

Other Growth Factors The possibility remains that other growth factors, such as TGF-[3, which is known to alter PTH receptors in vitro (88,122,190,191), may also play a contributing role. A flow cytometry study of growth factor profiles of bone cells isolated from the metaphysis after 3 days of PTH treatment showed small but significant increases in the percentage of cells expressing IGF-I, TGF-[3, PDGF, and FGF receptors, whereas cells isolated from the diaphysis showed increases in the percentage of cells expressing IL-4 and EGF receptors (J.M. Hock and N. Falla, unpublished data). In limb development, upregulation of FGF receptors has been associated with patterning and induction phenomena. Although not studied from a molecular aspect, PTH has significant effects in altering the geometry and connectivity of trabecular bone. It may be that PTH regulation of growth factors is the mechanism controlling patterning and localized placement of bone matrix in de novo bone formation on endosteal surfaces.

SUMMARY The roles for the selected growth factors studied in vitro have mostly been in embryonic or fetal rodent bones, or in immortalized or transformed bone cells in which key cell cycle control genes are suppressed. Rodents as an in vivo model contribute to our understanding of the mechanisms of action of PTH on modeling and de novo bone formation, as well as on the growth and development of the skeleton. However, the insights gained from in vitro models need to be validated in vivo, and their context better understood. The latest preliminary data on RANKL should provide additional insights on potential cytokine mediators of the complex actions of PTH in bone. However, the most exciting developments have been in studies of large animal models and osteoporotic humans in which PTH effects on remodeling dominate. New in vitro and in vivo models are needed to provide further insight into how growth factors and cytokines interact with PTH to mediate its effects in osteonal bone.

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150. Li X, Stevens M, Mackey M, Combs K, Tressler D, McOsker J. The ferret: Skeletal responses to treatment with parathyroid hormone. J Bone Miner Res 1994;8 (Suppl. 1):$258. 151. Jerome C, Vafai cj, Kaplan K, Bailey J, Capwell B, Fraser E Hansen L, Ramsay H, Shadoan M, Lees C, Thomsen J, Mosekilde L. Effect of treatment for 6 months with human parathyroid hormone (1-34) peptide in ovareictomized cynomolgus monkeys (Macaca fascicularis) . Bone 1999;25:301-309. 152. Qi H, Li M, Wronski T. A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 1995;10:948-955. 153. Watson P, Lazowski D, Han V, Fraher L, Steer B, Hodsman A. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factor I gene expression in ovariectomized rats. Bone 1995;16:357-365. 154. Toromanoff A, Ammann P, Riond J. Early effects of short-term parathyroid hormone administration on bone mass, mineral content, and strength in female rats. Bone 1998;22:217-223. 155. Baumann B, Wronski T. Response of cortical bone to antiresorptive agents and parathyroid hormone in aged ovariectomized rats. Bone 1995;16:247-253. 156. Podbesek R, Eduoard C, Meunier PJ, Parsons JA, Reeve J, Stevenson RW, ZanelliJM. Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds. Endocrinology 1983;112:1000-1006. 157. Podbesek RD, Mawer EB, Zanelli GD, Parsons JA, Reeve J. Intestinal absorption of calcium in greyhounds: The response to intermittent and continuous administration of human synthetic para thyroid hormone fragment 1-34 (hPTH 1-34). Clin Sci 1984;:591-599. 158. Riond J, Fischer I, G-V, Kuffer B, Toromanoff A, Forrer R. Influence of the dosing frequency of parathyroid hormone (1-38) on its anabolic effect in bone and on the balance of calcium, phosphorus and magnesium. Z Ernahrungswiss 1998;37:183-189. 159. Riond JL. Modulation of the anabolic effect of synthetic human parathyroid hormone fragment (1-34) in the bone of growing rats by variations in dosage regimen. Clin Sci 1993;85:223-228. 160. Dobnig H, Turner R. The effects of programmed administration of human parathyroid hormone fragment (1-34) on bone histomorpometry and serum chemistry in rats. Endocrinology 1997;138:4607-4612. 161. Uzawa T, Hori M, Ejiri S, Ozawa H. Comparison of the effects of intermittent and continuous administration of human parathyroid hormone (1-34) on rat bone. Bone 1995;16:477-484. 162. Zhao W, Byrne M, Boyce B, Krane S. Bone resorption induced by parathyroid hormone is strikingly diminished in collagenaseresistant mutant mice. J Clin Invest 1999;103:517-524. 163. Watson P, Fraher L, Kisiel M, DeSousa D, Hendy G, Hodsman A. Enhanced osteoblast development after continuous infusion of hPTH (1-84) in the rat. Bone 1999;24:89-94. 164. Kitazawa R, Imai Y, Fukase M, Fujita T. Effects of continuous infusion of parathyroid hormone and parathyroid hormonerelated peptide on rat bone in vivo: Comparative study by histomorphometry. Bone Miner 1991;12:157-166. 165. Dobnig H, Turner R. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1995;136:3632-3638. 166. Frolik C, Black E, Cain R, Hock J, Osborne J, Satterwhite J. Pharmacokinetic profile of LY333334, biosynthetic human parathyroid hormone (hPTH) (1-34), and serum biochemistry after anabolic or catabolic injection protocols. J Bone Miner Res 1997;12(Suppl. 1):$319. 167. Miles R, Sluka J, Santerre R, Hale L, Bloem L, Boguslawski G, Thirunavukkarasu K, Hock J. Dynamic regulation of RGS2 in

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bone: Potential new insights into PTH signaling mechanisms. Endocrinology 2000;141:28-36. Malluche H, Sherman D, Meyer W, Ritz E, Norman A, and Massry S. Effects of long-term infusion of physiologic doses of 1-34 PTH on bone. AmJPhysio11982;242:F197-F201. Finkelstein J, Klibanski A, Arnold A, Toth T, Hornstein M, Neer R. Prevention of estrogen deficiency-related bone loss with human parathyroid hormone- (1-34). J Am Med Assoc 1998;280:1067-1073. Christiansen P, Steiniche T, Brixen K, Hessov I, Melsen F, Heikerdorff L, Moskilde L. Primary hyperparathyroidism: Short-term changes in bone remdoeling and bone mineral density following parathyroidectomy. Bone 1999;25:237-244. Christiansen P, Steiniche T, Brixen K, Hessov I, Melsen E Heickerndorff L, Mosekilde L. Primary hyperparathyroidism: Effect of parathyroidectomy on regional bone mineral density in Danish patients: A three-year follow-up study. Bone 1999; 1999:589-595. Duan Y, Luca VD, Seeman E. Parathyroid hormone deficiency and excess: Similar effects on trabecular bone but differing effects on cortical bone. J Clin Endocrinol Metab 1999;84: 718-722. Howard GA, Bottemiller BL, Baylink DJ. Evidence for the coupling of bone formation to bone resorption in vitro. Metab Bone Dis Relat Res 1980;2:131-135. Howard G, Bottemiller B, Turner R. Parathyroid hormone stimulates bone formation and resorption in organ cultures: Evidence for a coupling mechanism. Proc Natl Acad Sci USA 1981;78:3204-3208. Cosman E Nieves J, Woelfert L, Shen V, Lindsay R. Alendronate does not block PTH-induced stimulation of bone formation. J Bone Miner Res 1998. Mochizuki H, Hakeda Y, Wakatusuki N, Usui N, Akashi S, Sato T, Tanaka K, Kumegawa M. Insulin-like growth factor I supports formation and activation of osteoclasts. Endocrinology 1992; 131:1075-1080. McCarthy TL, Centrella M, Canalis E. Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 1989;124:1247-1253. Breese C, Ingram R, Sonntag W. Influence of age and longterm dietary restriction on plasma insulin-like growth factor I, IGF-I gene expression and IGF-binding proteins. J Gerontol 1991;46:B180-B187. Johnston C, Deiss W. Some effects of hypophysectomy and parathyroid extract on bone matrix biosynthesis. Endocrinology 1965;76:198-202.

180. Deslauriers N, Gadreau P, Abribat T, Renier G, Petitclerc D, Brazeau E Dynamics of growth hormone responsiveness to growth hormone releasing factor in aging rats: Peripheral and central influences. Neuroendocrinology 1991 ;53:439-436. 181. Goya RG, Quigley KL, Takahashi S, Reichert R, Meites J. Effect of homeostatic thymus hormone on plasma thyrotropin and growth hormone in young and old rats. Mech Ageing Dev 1989;49:119-128. 182. Takahashi S, Gottshall PE, Quigley KL, Goya RG, Meites J. Growth hormone secretory patterns in young, middle-aged and old female rats. Neuroendocrinology 1987;46:137-142. 183. Schmidt I, Dobnig H, Turner R. Intermittent parathyroid hormone treatment increases" osteoblast number, steady state messenger ribonucleic acid levels for osteocalcin, and bone formation in tibial metaphysis of hypophysectomized female rats. Endocrinology 1995;136:5127-5134. 184. Jee W, Ueno K, Kimmel D, Woodbury D, Price P, Woodbury L. The role of bone cells in increasing metaphyseal hard tissue in rapidly growing rats treated with prostaglandin E2. Bone 1987;8:171-178. 185. Jee W, Ma Y, Li X. The immobilized adult cancellous bone site in a growing rat as an animal model of human osteoporosis. J Histotechno11997;20:201-206. 186. Mori S, Jee w, Li X, Chan S, Kimmel D. Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats. Bone 1990; 11:103-113. 187. Gera I, Hock J, Raisz L, Gunness-Hey M, Fonseca J. Indomethacin does not inhibit the anabolic effect of parathyroid hormone in rats. Calcif Tissue Int 1988;40:206-211. 188. Klein-Nulend J, Pilbeam C, Harrison J, Fall P, Raisz L. Mechanism of regulation of prostaglandin production by parathyroid hormone, interleukin-1 and corticsol in cultured mouse parietal bones. Endocrinology 1991 ;128:2503-2510. 189. Vargas S, Raisz L. Simultaneous assessment of bone resorption and formation in cultures of 22-day fetal rat parietal bones: Effects of parathyroid hormone and prostaglandin E2. Bone 1990; 11:61-65. 190. Pfeilschifter J, Oechsner M, Naumann A, Gronwald RGK, Minne HW, Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factors: A comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor B. Endocrinology 1990;127:69-75. 191. Oursler M, Cortese C, Keeting F, Andersons M, Bonde K, Riggs B, Spelsberg T. Modulation of transforming growth factor-beta in normal human osteoblast-like cells by 17-beta estradiol and parathyroid hormone. Endocrinology 1991 ;129:3313-3320.

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Cellular Actions of Parathyroid H o r m o n e on Osteoblast and Osteoclast Differentiation

JANE E. AUBIN Department of Anatomy and Cell Biology and Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

JOHANN. M.

HEERSCHE Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G 1G6

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOCLAST AND OSTEOBLAST DIFFERENTIATION A N D / O R ACTIVITY IN VIVO: STUDIES IN HUMANS, RATS, AND MICE

other therapeutic factors in humans [for discussion, see Hirano et al. (14) ]. In agreement with results summarized above, intermittent injection of PTH(1-34) increased cancellous bone in the secondary spongiosa of parathyroidectomized rats (15,16), an effect that was i n d e p e n d e n t of resorptive activity (15,17). Other in vivo data from humans (18) and rats have confirmed that intermittent PTH stimulated bone formation de novo, without a prior episode of resorption. Studies done with PTH in fracture healing models in both normal and osteoporotic bones of rats showed that daily subcutaneous injections of PTH(1-34) increased both the ultimate load that could be tolerated before breaking and the callus volume of rat tibial fractures (19). In an interesting gene therapy approach not directly mimicking an intermittent dose regime, degradable (gene-activated collagen matrix) (GAM) sponges were loaded with a plasmid containing PTH (1-34) cDNA and implanted into critical-sized defects made surgically in rat femurs or beagle femurs and tibias (20,21). The matrix carrier appeared to act as a scaffold into which fibroblasts migrated and became infected with the plasmid, and acted as in vivo "bioreactors," locally secreting PTH(1-34), which stimulated fracture healing and new bone formation, but without evidence for stimulation of osteoclast formation in the area (20,21). The authors argued that local PTH concentrations were probably below levels required to see catabolic activity. These and a large n u m b e r of other studies in ovariectomized rats and postmenopausal women have consistently

A prolonged increase in circulating levels of parathyroid h o r m o n e (PTH) is associated with increased bone turnover, i.e., increased osteoclastic bone resorption and increased osteoblastic activity (1-3). In severe hyperparathyroidism, this results in loss of both cortical and cancellous bone (4,5). However, mild hyperparathyroidism is associated with normal or increased bone mineral density and increased bone volume in areas that are primarily cancellous, such as vertebrae (6-9), but bone is still lost in cortical areas (6). Daily injections of h u m a n PTH(1-34) for 6-12 months also increase the cancellous bone area in iliac crest biopsies (10) and decrease femoral cortical bone density of osteoporotic patients (11). Thus, u n d e r certain conditions, PTH can affect cancellous bone and cortical bone differently, with a net increase in bone mass occurring in cancellous bone concomitant with a net loss of cortical bone. Of particular interest is the observation that the adverse effects of treatment with PTH on cortical bone density might be ameliorated by simultaneous treatment with estrogen (12) or calcitriol (13). However, unambiguous conclusions on the effects of PTH on cortical bone remain difficult and c o n f o u n d e d by issues such as differences in models (e.g., rodents versus rabbits or dogs) and the presence and kinds of

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d o c u m e n t e d increased bone mass with intermittent PTH t r e a t m e n t (22-24). It is also of interest to note that the anabolic effects of PTH on bone apposition were abolished in vitamin D-deficient rats and were restored by vitamin D supplementation (25). This interaction seemed to be d e p e n d e n t on the presence of growth h o r m o n e (26). The m e c h a n i s m whereby increased levels of PTH are t h o u g h t to affect bone metabolism in h u m a n s is an increase in remodeling resulting from a 50% increase in activation frequency (27). Why this should maintain or increase cancellous bone volume and decrease cortical bone volume remains to be determined. One explanation, suggested by Parfitt (28) is that the depth of osteoclastic resorption lacunae on the endocortical surface is greater than the d e p t h of the lacunae on cancellous bone surfaces. The reasons for this difference, however, are not known.

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOBLAST AND OSTEOCLAST PROLIFERATION AND DIFFERENTIATION IN B O N E O R G A N C U L T U R E SYSTEMS Early studies with mouse long bone rudiments showed conclusively that PTH affected both osteoblast and osteoclast numbers and activity in a dose- and timed e p e n d e n t fashion (29). With culture in the presence of low concentrations of PTH (0.01-0.00 U / m l ) , decreased activity of osteoblasts was seen after 12-14 hours, whereas multinucleated osteoclasts increased in n u m b e r and became active after 24-28 hours. The three types of tissue represented in such bone rudiments, that is, bone, cartilage, and marrow stroma, all responded to PTH, but timing and dose responsiveness differed. Low concentrations of PTH (0.01-0.00 U / m l ) had no effect on cartilage or marrow stroma, whereas higher concentrations of PTH induced increased proliferation of cartilage and of connective tissue and had more p r o n o u n c e d effects on osteoblasts and osteoclasts. These early experiments uncovered three aspects of PTH action on bone-related tissues that have been topics of major interest over many years: the time delay between PTH effects on osteoblasts and osteoclasts (compatible with a cause-and-effect relationship), reduction of osteoprogenitor differentiation into osteoblasts and e n h a n c e d proliferation of a fibroblastlike cell type, and the effects of PTH on chondrocyte proliferation and differentiation. Progress on the underlying cellular and molecular mechanisms leading to these observations has been made in the past several years in all three of these areas.

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOBLAST PROLIFERATION AND D I F F E R E N T I A T I O N I N VIVO A N D I N VITRO There is m u c h emphasis on d e t e r m i n i n g what the "target" cell in bone is that leads to the anabolic versus catabolic effects of the h o r m o n e . Published studies repeatedly d o c u m e n t the ability of PTH to increase very rapidly the n u m b e r of functional osteoblasts, perhaps m o r e consistent with an effect of PTH on the state of osteoblast differentiation and functional status rather than on proliferation followed by differentiation events. However, it has also been suggested that PTH specifically and bone anabolic agents generally may work, at least in part, through stimulation of cell proliferation (30,31). Both stimulatory and inhibitory effects on proliferation have been reported for PTH. These inconsistent results probably reflect the different sources and kinds of cells used in different studies [e.g., osteosarcoma-derived lines, primary bone marrow stromal populations, primary fetal or neonatal calvariaderived osteoblastic cells, primary trabecular bone (femoral spongiosa cells) cells, etc.] and the many different culture conditions used (e.g., without or with a high percentage of serum). In the UMR-106 osteosarcoma cell line, for example, PTH is known to inhibit proliferation via inhibition of cell cycle progression through a c A M P / p r o t e i n kinase A-mediated process (32) that has been linked to p27Kipl induction (33,34). On the other hand, in the TE-85 osteosarcoma line, PTH stimulates proliferation through stimulation of cdc2 expression via increased levels of free E2F (35). Interspecies differences have also been reported in activation of MAP kinases in different osteoblastic models (36). These differences may be related to true species differences a n d / o r to relative stage of differentiation of the different cell lines (e.g., more or less mature), a possibility that may also explain differences reported in osteoblastic populations isolated from fetal or neonatal animals versus postnatal and m a t u r e rodents. However, osteoblast-like osteosarcoma-derived cells may lack or may express m u t a t e d forms of key cell cycle regulatory genes, such as p53 and Rb, contributing to aberrant proliferation controls and discrepancies seen with multiple agents, including PTH. Thus, it seems crucial to ask whether there are data supporting the ability of PTH either to stimulate or to inhibit proliferation in vivo in any animal model. In young rats, proliferating cells in bone are located subjacent to the growth plate, the cortical endosteum of the metaphysis, and the cortical periosteum of the diaphysis, all locations in which PTH exerts stimulatory effects on bone formation, but without apparently stimulating proliferation [ (37,38); reviewed in (24) ].

PTH AND BONE CELL DIFFERENTIATION / Oniya et al. (38) showed that intermittent PTH treatm e n t appeared to target proliferating cells in the primary spongiosa of young rat distal femur metaphysis, resulting in an increased n u m b e r of osteoblasts, but via and down-regulation of cell proliferation and up-regulation of cell differentiation in trabecular bone with transient stimulation of the early response genes and interleukin-6 (IL-6). In mature rats, PTH stimulates lining cells on quiescent surfaces to function as osteoblasts also without inducing proliferation (39,40). On the other hand, Nishida and colleagues (41) reported that intermittent PTH administration was able to increase the total n u m b e r of fibroblast colony-forming units (CFU-F) and alkaline phosphatase-positive CFU-F recoverable from the bone marrow and capable of growth ex vivo. Similarly, Bikle and colleagues (42) found that PTH treatment of normally loaded, but not unloaded, rats caused a 2.5-fold increase in the n u m b e r of bone marrow stromal cells, with similar increases in alkaline phosphatase activity and mineralization, compared with cultures from vehicle-treated rats. Direct in vitro PTH challenge of stromal cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on stromal cells may be mediated indirectly by a PTH-induced factor. The authors speculated that the factor is insulinlike growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of stromal cells isolated from normally loaded bone, but not from unloaded bone. Taken together, these data suggest that more remains to be done to show conclusively whether and how much proliferation may contribute to the anabolic effects of PTH and to clarify the effects of PTH in vivo on proliferative responses in different subpopulations of osteoblastic cells u n d e r different conditions. The early data with respect to PTH effects on differentiation are as complex, but did provide some evidence for osteoblast differentiation stage-specific effects and differences between chronic versus intermittent exposure to PTH. For example, early data showed that continuous exposure of osteoblasts to PTH in organ culture models decreased osteoblast activity and differentiation of functioning osteoblasts [e.g., (43) ], but that a brief incubation with PTH followed by a return to control medium stimulated collagen synthesis in a m a n n e r d e p e n d e n t on IGF-I (44). In addition, PTH was found to suppress alkaline phosphatase activity in the relatively mature rat osteoblastic cell line ROS 17/2.8 (45), but to stimulate the enzyme in the preosteoblastic mouse osteoblastic cell line MC-3T3-E1 (46). However, as raised previously (31), the ability of osteosarcoma-derived and established/immortalized cell lines to reproduce faithfully a normal differenti-

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ated cell phenotype is not always certain and, more recently, considerable insight has resulted from use of primary cell culture models of normal osteoblasts. Much has been learned from approaches in vitro in which the nature of osteoprogenitors and their more differentiated progeny in primary cultures have been investigated by functional (the nature of the colonies they form, e.g., mineralized bone nodules), immunologic (e.g., immunocytochemistry, Western analysis), and molecular (Northern blots, polymerase chain reaction of various sorts, in situ hybridization) assays of the bone nodules formed in bone marrow and bonederived primary cell populations. The progenitor cells present in these populations u n d e r g o a proliferation-differentiation sequence leading to expression of tissue-specific macromolecules, including the bone matrix molecules (type I collagen, osteocalcin, osteopontin, bone sialoprotein, a m o n g others) and transcription factors that regulate them and commitment/differentiation events (e.g., Cbfal, AP-1 family members, Msx-2, Dlx-5) (47). Ultimately, the differentiated osteoblasts that form are morphologically essentially identical to their counterparts in vivo and the deposited matrix contains the major bone matrix proteins and mineralizes in a regulated manner. These models have become extensively used to investigate the regulation of osteoblast development and activity by hormones, cytokines, and growth factors (Fig. 1) [reviewed in (48,49)]. Part of the value of this model stems from the fact that the bone nodules represent the end product of the proliferation and differentiation of osteoprogenitors present in the starting cell population, their presence and differentiation status can be quantified, and the effects of agents of interest can be studied after either chronic exposure t h r o u g h o u t the developmental sequence or pulsatile exposure during either proliferation or differentiation stages. There are growing data investigating the effects of PTH in this model. O u r labs were the first to use the RC cell bone nodule model to investigate the mechanisms by which PTH might affect osteoprogenitors in vitro and at what developmental stages (50). Continuous exposure to PTH caused a dose-dependent inhibition of bone nodule formation, with half-maximal inhibition at 0.05 nM, and total inhibition at 1 nM, concentrations much lower than those required to elicit a significant cAMP response in RC cells, and without effect on cell growth or saturation density. T h o u g h continuous exposure to 1 n M PTH eliminated bone nodule formation, a single 48-hour pulse administered at any time during the 17-day culture period had no effect. When 1 n M PTH was added on day 1 and removed at different times during the culture period, a time-related release

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Limited proliferation

Limited self-renewal Extensive proliferation~

+

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~

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PTH receptor expression PTH effects on bone nodule formation in vitro

Chronic exposure Repeated 1 hr exposures Repeated 6 hr exposures

No progression past preosteoblaststage and no bone nodules Differentiation and bone nodule formation ~ Differentiation and bone nodule formation +

PTH effects on osteoblast and osteocyte apoptosis in vivo and in vitro

FIG. 1 A schematic of the osteoblast lineage based on cell culture models that recapitulate a proliferation-differentiation program in vitro (see Refs. 31 and 71). These models have suggested that PTH1R is expressed from early osteoprogenitor stages, but increases in levels and activity as osteoblasts mature. In addition, they have provided evidence for differentiation-stage-specific effects of PTH and for anabolic versus catabolic effects on osteoblast development depending on the time and duration of exposure to PTH (see text for further details).

from inhibition was observed. Cultures exposed to 1 n M PTH until nodules had developed in the corresponding control cultures and then switched to m e d i u m without a d d e d PTH rapidly formed clusters of differentiated osteoblasts and nodules within 3 days. PTH added at different times during the culture period and present continuously thereafter suppressed formation of new nodules, the magnitude of the effect being a function of the duration of exposure. These experiments suggest that PTH is a p o t e n t - - b u t reversible-suppressor of osteoblast differentiation and that its effect u n d e r this t r e a t m e n t regime occurs at a late stage in the differentiation of osteoprogenitor cells, probably preventing differentiation of preosteoblasts into osteoblasts (Fig. 1). It was, of course, also of interest to

know what signaling pathway might be coupled to the inhibitory effect of PTH. As m e n t i o n e d above, in our experiments, complete inhibition of bone nodule formation was seen at concentrations of PTH below those required to elicit a measurable cAMP response in RC cells (50). In other experiments, we also found that forskolin at low concentrations that did not affect cAMP or cell architecture were stimulatory, whereas higher concentrations that did increase cAMP and affect cell architecture significantly were inhibitory, whether they were present continuously or in repeated short (1-hour) pulses at each m e d i u m change (each 48 hours) during the entire culture period (51). Taken together, all these results suggest that intermittent elevations in intracellular cAMP have an inhibitory

PTH AYO BoNE CELL DIFFERENTIATION / effect on bone formation in vitro, but that osteoprogenitor cells may be stimulated to differentiate possibly through a non-cAMP-dependent process. That RC cells respond differently depending on their differentiation status and that continuous versus pulsatile exposure of RC cells to PTH may elicit different biologic responses coupled to different signaling pathways was confirmed by Yamaguchi and colleagues (52). These authors treated RC cells either continuously or cyclically with PTH(1-34) for the first few hours of each 48-hour incubation cycle. When cells were exposed to PTH only for the first h o u r of each 48-hour incubation cycle and then cultured for the remainder of the cycle without PTH, osteoblast differentiation was inhibited, as evidenced by suppression of alkaline phosphatase activity, bone nodule formation (Fig. 1), and mRNA expression of alkaline phosphatase, osteocalcin, and receptors for PTH (hereafter referred to as PTHIR, the P T H / p a r a t h y r o i d hormonerelated peptide receptor) (53,54). Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and CaZ+/protein kinase C (PKC) demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, when cells were exposed to intermittent PTH for the first 6 hours of each 48-hour cycle, osteoblast differentiation was stimulated. Both cAMP/PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. These authors further investigated the possible downstream mediators of the PTH effect and found that although both cAMP/PKA and CaZ+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. They found that a neutralizing antibody against IGF-I blocked the stimulatory effect induced by the 6-hour intermittent exposure, but not the inhibitory effect induced by the 1-hour intermittent exposure, again suggesting that PTH catabolic versus anabolic effects are likely mediated through different signaling pathways (52). In a more recent study with the MC-3T3-E1 cell model, Howard and colleagues (55) reported that when continuous PTH treatment was initiated during approximately the first half of the month-long culture period, mineralization decreased, whereas continuous exposure later had little to no effect. However, a 5-day pulse in roughly the middle of the culture period increased mineralization, an effect that also occurred in primary cultures of murine and h u m a n osteoblastic cells. The differences in PTH effects on mineralization did not correlate with P T H I R expression, which was detected early and increased only marginally in the second half of the culture period, nor with the cAMP response to PTH, which increased markedly after day 10 and remained high to the end of the culture. These data confirm that there are differentiation stage-

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specific effects of PTH and that signaling pathways other than stimulation of adenylate cyclase are involved in PTH effects on osteoblast differentiation. Another mechanism has been suggested to contribute to the anabolic effect of PTH, and that is an apoptotic effect. Jilka and colleagues (56) found that daily PTH injections in mice with either normal bone mass (SAMR1) or osteopenia due to defective osteoblastogenesis (SAMP6) increased bone formation without affecting the generation of new osteoblasts and with no evidence for reactivation of lining cells as seen in at least some mature rat studies (39). Rather, in this murine model, PTH appeared to increase the life span of the osteoblasts by preventing their apoptosis (Fig. 1), thereby prolonging the time spent in performing their matrix-synthesizing activity. To determine whether the antiapoptotic effect of PTH was due to direct action of the h o r m o n e on osteoblasts and osteocytes, as opposed to indirect actions mediated by compensatory changes, the effect of PTH on apoptosis was examined in r o d e n t and h u m a n osteoblast and osteocyte models in vitro. Apoptosis, stimulated by glucocorticoids (57,58) in primary cultures of osteoblasts isolated from neonatal murine calvaria, the MC-3T3-E1 murine osteoblastic line, h u m a n osteoblastic MG-63 osteosarcoma cells, or murine MLO-Y4 osteocytic cells (59), was attenuated by PTH(1-34). In osteoblasts, induction of apoptosis by tumor necrosis factor (TNF) was not affected by PTH, which suggested an interesting interference with some, but not all, death pathways (56). However, in a 293 cell model overexpressing PTH1R, PTH induced apoptosis and the TNF receptor and PTH1R pathways appeared to converge (60), suggesting that there may be osteoblast-specific mediators required for the observed antiapoptotic effects in mouse bone. These results on the SAMP6 model are reminiscent of the antiapoptotic effect of PTHrP on chondrocytes during e n d o c h o n d r a l bone development (61,62). Given their putative role as mechanosensors and their abundance compared to other osteoblast lineage cells in adult bone, the observed antiapoptotic effect on osteocytes is also intriguing, but whether it also may contribute to an anabolic effect of PTH is not yet known. As m e n t i o n e d earlier for differentiation effects of PTH, whether the antiapoptotic effect of PTH on osteoblasts is mediated by cAMP or other post-PTH1R signaling events deserves more attention. In the SAMP6 mouse studies, the antiapoptotic effect of PTH was blocked by the P T H I R antagonist bPTH(3-34) and was mimicked by dibutyryl cAMP, suggesting that it was mediated through PTH1R and subsequent activation of adenylate cyclase (56). This is consistent with data showing that periosteal cell apoptosis is inhibited by prostaglandin E through cAMP-dependent stimulation of sphingosine kinase (63). However, in the

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293 overexpressing PTH1R, Turner et al. (60) found that PTH and P T H I R signaling induce apoptosis through Gq-mediated phospholipase C / C a 2+ signaling, rather than Gs-mediated cAMP signaling. Though these discrepancies require clarification, the elucidation of the antiapoptotic effects of PTH in the mouse model in vivo and the evidence that PTH antagonizes the proapoptotic effects of glucocorticoids in vitro provide a possible mechanistic explanation for the efficacy of daily subcutaneous injection of PTH for treatment of glucocorticoid-induced osteoporosis in humans (64).

E X P R E S S I O N OF T H E P T H R E C E P T O R BY CELLS OF T H E O S T E O B L A S T LINEAGE AND SIGNALING THRESHOLDS It seems obvious that a better understanding of when during osteoprogenitor cell differentiation PTH receptors begin to be expressed may help to clarify which osteoblast lineage cells may be targets of PTH activity. PTH receptors were demonstrated on the osteoblast and its immediate precursors in early binding studies (65) and by immunohistochemistry (66). However, in a later binding study, a distinct cell type, not a mature osteoblast but possibly an immediate osteoblast progenitor or preosteoblast, was reported to be the major PTH target (67). On the other hand, by in situ hybridization, Lee et al. found PTH1R mRNA expressed highest in growth plate chondrocytes, followed by osteoblasts (which were higher than preosteoblasts), mononuclear cells along cortical periosteal and endsoteal surfaces, some stromal cells, and plump mononuclear cells lining trabecular bone surfaces, but not flat osteoblasts or in cells embedded in bone matrix (68). Supporting not only mRNA but also PTH1R protein expression, subcutaneous administration of h u m a n PTH (1-84) induced rapid and transient expression of the protooncogene c-fos mRNA in osteoblasts, chondrocytes, and some stromal cells, consistent with the highest PTH1R mRNA expression; only later was c-fos mRNA expressed in the majority of stromal cells and in osteoclasts, implying the latter did not express PTH1R and were probably responding by an indirect mechanism. Other support for high PTH1R expression on mature osteoblasts comes from binding studies in rat calvaria cell cultures whereby PTH binding correlated better with osteocalcin expression than with alkaline phosphatase (69) and from a combined in situ hybridization-PTH binding study in young adult rats (70). Global amplification polymerase chain reaction (PCR) of replica-plated osteoprogenitor cell colonies undergoing differentiation to bone nodule-forming

cells showed that P T H I R is detectable at low levels in very primitive osteoprogenitor cells, but expression increases markedly as the cells mature to functional osteoblasts expressing markers such as osteocalcin (71). McCauley and colleagues also analyzed P T H I R expression in MC-3T3-E1 cells and primary rat calvarial cells undergoing differentiation to bone nodule-forming cells in vitro (72). Their findings indicated that PTH1R expression at the level of mRNA, protein, and biologic activity increased as cells matured and bone nodules formed. Using the same MC-3T3-E1 model, however, Schiller et al. (55) found that PTH1R mRNA was detected early but went through only a modest increase in expression later in the cultures, remaining relatively constant, but that the cAMP response to PTH varied markedly with no response early and a marked response as cells matured. These results point out just some of the inconsistencies in different studies even when similar reagents and cell lines have been used, probably reflecting the variationsmsometimes quite largemin precise culture conditions, medium additives, etc. that have been used. Nevertheless, taken together, they do suggest that PTH1R expression is already detectable on quite primitive osteoblast precursor cells but that levels of expression and cellular responsiveness increase as osteoblastic cells mature, in keeping with earlier data based on cAMP response to PTH in many osteoblast models in vitro (73). Given that PTH1R appears to be present from relatively early stages of differentiation, albeit at apparently lower n u m b e r and with lower consequent cAMP stimulation than in more mature cells, and that continuous versus pulsatile exposure to PTH elicits different biologic effects, one aspect of the molecular mechanism of PTH that deserves more attention is receptor-ligand signaling thresholds. For example, receptors with intrinsic or associated tyrosine kinase activity are known to elicit both proliferative and differentiation responses in factor-dependent cell lines, based on both the duration and the magnitude of extracellular signal-regulated kinase (ERK) activity (74). The fact that the same cytokine can elicit a different outcome simply by changing the relative expression of the corresponding receptor supports the view that the magnitude of signaling (e.g., ERK activation), and not receptor-specific signaling, may determine a biologic outcome (75). Importantly, threshold-dependent regulation may also extend to osteogenesis because both the magnitude and the duration of PTH supplementation modulate bone responses (22). These effects, which result at least in part from the differential stimulation of adenylyl cyclase and phospholipase C, have been shown to be dependent on the density of PTH receptor expression on the cell surface (76,77).

PTH AND BONE CELL DIFFERENTIATION The role of PTH1R in osteoblasts is now also being investigated in another way. A series of clonal murine calvarial osteoblastic cell lines conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and lacking both functional alleles of the P T H I R gene have been made from PTH1R - / - mice made earlier (78). U n d e r nontransforming conditions, these cells stop proliferating, express a series of characteristic osteoblastic genes, and produce mineralized bone nodules in a m a n n e r that is regulated by 1,25-dihydroxyvitamin D 3 but not by PTH(1-84). An unexpected but interesting observation is that osteocalcin expression is lower than expected in P T H I R - / - cells, an observation that suggests that P T H I R expression is required for normal levels of osteocalcin expression and confirms that lower than usual levels of osteocalcin do not inhibit bone formation and mineralization (79). It remains to be determined whether not only altered PTH response but also the low osteocalcin can explain, in part, the increase in osteoblast n u m b e r and matrix accumulation during intramembranous bone formation in the shafts of long bones, the decrease in trabecular bone formation in the primary spongiosa, and the delayed vascular invasion in the

Osteoblastic cell autocrine/paracrin~ feedb

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205

P T H I R - / - mice (80). These cells may also provide a useful model in which to investigate the role of receptor density via transfection of P T H I R to various levels in cells with an otherwise identical osteoblast background.

ARE P T H E F F E C T S O N O S T E O B L A S T DEVELOPMENT MEDIATED THROUGH OTHER FACTORS? It has been suggested at several points that PTH may induce a variety of factors that contribute to the anabolic and catabolic effects of PTH. T h o u g h it is beyond the scope of this chapter to review all of these in detail, mention of a few seems appropriate. Various osteogenic cell models in vitro and in vivo expressing PTH1R respond to PTH treatment by production of the PTHrP holoprotein or processed fragments (81-83) (Fig. 2). The p r o h o r m o n e of PTHrP or certain cleavage products [ones containing the 87-107 nuclear-targeting sequence (NTS)] are intrakines, i.e., peptides that do or do not enter cell nuclei d e p e n d i n g on the presence and activity of cyclin-dependent protein kinases (84,85) and contribute to cell activity, including stimulation

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of expression of the antiapoptosis protein Bcl-2 (61), among other proliferation and differentiation responses (86,87). Such activities of specific fragments of PTH and PTHrP are the basis for drug design of anabolic PTH analogs (88,89). In addition, in numerous models in vitro and in vivo PTH treatment induces production of IGF-I (44,52,90-93), IL-6 (94,95), TGF[3s (TGF-[31 a n d / o r - [ 3 2 , d e p e n d i n g on the signaling pathways stimulated in responding) (96), and FGF-2 (also known as bFGF) (97-99). As m e n t i o n e d earlier, IGF-I was reported to be involved in the stimulatory, but not the inhibitory, effects of PTH on bone formation resulting from pulses of different duration in two different culture models in vitro (44,52). IGF-I, like PTHrP, induces Bcl-2 (100), perhaps contributing to the antiapoptotic effect of PTH, but also may stimulate proliferation of osteoprogenitors and preosteoblasts (101). FGF-2, like PTHrP fragments, is an intrakine factor (102). It is possible to speculate that the combination of these factors induced by PTH acts through multiple mechanisms, e.g., FGF-2 stimulating osteoblast precursor proliferation while IGF-I is promoting the lifetime and activity of the osteoblasts (Fig. 2). There have been many published studies of investigations of the signaling pathways and roles of these multiple factors in mediating PTH effects in vitro, but definitive proof of their role in vivo is often lacking. The PTHrP knockout mice have been used to suggest that locally produced PTHrP is required for osteoblast develo p m e n t and for the skeletal effects of PTH (103). T h o u g h IL-6 is clearly up-regulated in vivo in bones of rats treated with an anabolic PTH schedule (38), it is not clear that IL-6 plays a role in the anabolic response, because neutralization of IL-6 in a mouse model markedly reduced PTH-induced bone resorption but had no effect on bone formation parameters (104), consistent with the lack of effect of IL-6 in the rat calvaria bone nodule system in vitro (105). On the other hand, transgenic mouse calvariae carrying fusion genes of the rat Collal promoter and the chloramphenicol acetyltransferase reporter have been used to show that the inhibitory effect of PTH on Collal expression is mediated mainly by the cAMP signaling pathway and that prostaglandins and IL-6 are not local mediators of the PTH response, at least in this model (106). Transgenic mice overexpressing IGF-I in osteoblasts via the osteocalcin promoter showed increased trabecular bone volume, but no evidence for increased osteoblastic proliferation, suggesting that IGF-I increased the activity of resident osteoblasts; these mice did, in addition, have an increase in osteocyte lacunae occupancy, suggesting that IGF-I may extend the osteocyte life span, consistent with the antiapoptotic effect reported for PTH (56).FGF-2 - / mice have markedly reduced bone mass and bone formarion rates (99), but their responsiveness to PTH chal-

lenge has not yet been reported. A growing number of transgenic and mouse knockout models for factors and mediators proposed to play roles in PTH responses are becoming available and these should provide useful tools to tease out critical players in the PTH pathways.

EFFECTS OF PARATHYROID HORMONE ON OSTEOCLAST DIFFERENTIATION When long bone rudiments or calvariae from embryonic or newborn rats or mice are explanted and maintained in culture for several days, osteoclast progenitors located in marrow spaces or the periosteum proliferate and differentiate to form osteoclasts (107,108). Their differentiation in these culture systems is stimulated by PTH, PGE 2, 1,25(OH)zD ~, and a variety of other bone resorption stimulating agents (29,109). Osteoclast-like cells also develop in bone marrow cultures containing stromal and hematopoietic cells, but generally 1,25(OH)zD 3 is required to obtain significant numbers of osteoclasts (110). Progenitors present in the spleen can also differentiate into osteoclasts, but only in the presence of bone marrow-derived stroma and not in the presence of spleen-derived stromal cells (111,112), indicating that osteoclast differentiation from hematopoietic progenitors is controlled by specific stromal cells present in the marrow cultures. With regard to the effects of PTH on osteoclast differentiation in cocultures of spleen cells and stromal cell lines, it is of interest to note that PTH-induced osteoclast-like cell formation was observed in cocultures with a cell line that responded to PTH with an increase in cAMP (KS-4 cells) (113), but not in cocultures with cell lines not responsive to PTH. However, stromal cell lines not responsive to PTH but responsive to PGE 2 or 1,25(OH)2D 3 did mediate PGE 2- or 1,25-(OH)zD 3induced osteoclast formation in spleen cell coculture systems (114), indicating that several types of stromal osteoblast-like or non-osteoblast-like cells that differ in h o r m o n e responsiveness could be involved in the regulation of osteoclast formation. From the evidence provided in the experiments described above, it has become clear that the effects of PTH, PGE2, and 1,25(OH)zD~ on osteoclast differentiation are mediated through the osteoblastic or stromal cell c o m p o n e n t of the cultures, and that osteoclast differentiation requires direct cell-cell contact between the activated osteoblastic/stromal cell population and the hematopoietic cells (Fig. 2). The m e m b r a n e - b o u n d mediator involved, n a m e d osteoclast differentiation factor (ODF), was subsequently cloned and identified (115); it proved to be identical to the previously identified protein osteoprotegerin ligand (OPGL) (116), which in turn is identical to the ligand for the receptor

PTH AND BONE CELL DIFFERENTIATION / activator of NF-KB (RANKL). The receptor on the osteoclast recognizing ODF (i.e., OPGL/RANKL) turned out to be the RANK receptor, and RANKL has now become the generally adopted term for this ligand (Fig. 2). That RANKL is an absolute requirement for osteoclast formation was proved conclusively by the generation of RANKL knockout mice, which had severe osteopetrosis due to a complete lack of osteoclasts (117). The osteoblastic/stromal cell origin of RANKL was confirmed by setting up cocultures of spleen cells from o p g l - / mice with normal osteoblasts, which generated functional osteoclasts, and cocultures of spleen cells from normal mice with osteoblasts from o p g l - / - mice, which did not form osteoclasts. Thus, lack of RANKL production in osteoblastic cells of R A N K L - / - mice was the cause of the osteoclast deficiency. In agreement with this view, ablation of NF-KB1 and NF-KB2 had the expected results: the mice were osteopetrotic, did not develop osteoclasts, but had an increased n u m b e r of macrophages (118). Virtually simultaneously with the discovery of OPGL (RANKL), it was found that osteoclast formation could be inhibited by a soluble receptor for RANKL, osteoprotegerin (OPG), which is secreted by a large variety of cells and organs, including fibroblasts, osteoblasts, lung, heart, kidney, and intestine (119). The OPGdeficient mice are severly osteoporotic whereas mice overexpressing OPG have an osteopetrotic phenotype (120). OPG acts by binding OPGL, thereby preventing interaction of OPGL with its receptor on osteoclast lineage cells and thus inhibiting osteoclast differentiation. The consensus view now is that osteoclast formation in the bone microenvironment is regulated by the interaction of RANKL and OPG, whereby the a m o u n t of u n b o u n d RANKL available to interact with the RANK receptor on osteoclasts or osteoclast precursors determines the rate of osteoclast formation. That RANKL is involved in the actions of many of the factors known to cause bone resorption and induce hypercalcemia in vivo is clearly shown by the experiments of Morony et al. (121), who found that recombinant h u m a n OPG inhibited the hypercalcemic effects of IL-113, TNFoL, PTH (Fig. 2), PTHrP, and 1,25(OH)2D 3. However, whether these factors directly affect RANKL production or whether their effects on RANKL production are mediated by other factors is not clear. Okada et al. (122) examined this issue by evaluating the effects of disrupting the prostaglandin G / H synthase genes on 1,25(OH)2D ~- and PTH-induced osteoclast formation. They found that both PTH- and 1,25(OH)zD ~- induced osteoclast-formation was reduced by 60-70% in marrow cultures from PGHS-2 ( - / - ) mice, indicating that PGE 2 is a major mediator of PTH-induced and 1,25(OH)zD~-induced increased osteoclastogenesis. In agreement with this, PGE2- , PTH-, and 1,25 (OH)zD3 -

207

induced osteoclast formation was found to be reduced by 86, 58, and 50%, respectively, in marrow cultures of mice in which the receptor for PGE 2 had been disrupted (EP2 - / - mice) (123). Thus, the effects of PTH, and those of many other ligands stimulating osteoclast formation, appear to be mediated via increased PGE 2 production and PGEz-induced RANKL formation in a variety of cell types, among which are the cells of the osteoblastic lineage. However, when the effects of PTH and PGE 2 on OPG production were investigated, it appeared that PTH did stimulate OPG mRNA expression in osteoblast lineage cells in rat femur metaphyseal bone but that PGE 2 did not (124), suggesting that differences between the effects of PTH and some of the other resorption stimulating agents may be caused by differential effects on OPG production. With regard to other mediators of the action of PTH on osteoclasts, IL-6 seems the most likely to play a significant role: IL-6 is clearly up-regulated in vivo in bone treated with an anabolic PTH schedule (38), treatment of mice in vivo with either PTH or PTHrP increases expression of IL-6 by osteoblasts (125), and PTHinduced stimulation of bone resorption both in vitro and in vivo can be blocked by either anti-IL-6-receptor antibody or neutralization of IL-6 (104,126). It is not known whether some or all of the effects of PTH on IL6-production by stromal and osteoblastic lineage cells are direct or indirect. In all likelihood, however, such effects are indirect and mediated to a significant degree by PTH-induced PGE 2 production. Compatible with this view are the observations that PGE 2 e n h a n c e d IL-6 production in ST-2 and MC-3T3-E1 cells (127) and that PGE 2 stimulates IL-6 production in osteoblast-like MC-3T3 cells (128).

CONCLUDING

REMARKS

In summary, though much progress has been made on the cellular and molecular bases of bone cell responses to PTH that lead to catabolic versus anabolic outcomes, much remains unclear and discrepancies continue to abound. It is evident from examples and issues raised in this chapter that continued t h o u g h t must be given to not only the species but the age of animals being used, the concentrations/doses of PTH utilized, the duration of treatment, and the presence or absence of other potential regulators for in vivo, ex vivo, and in vitro studies. It also seems clear that osteoblast lineage cells express PTH1R through multiple stages of their developmental lifetime, and, concomitantly, this implies that PTH response in bone will only be fully understood when the sum of effects on progenitors through apparently terminally differentiated bone cells have been unambiguously dissected.

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ACKNOWLEDGMENTS We thank our current and former lab m e m b e r s and many colleagues for their contributions over many years. This work was supported by grants from the Canadian Institutes of Health R e s e a r c h / C I H R (MT12390 to J.E.A. and MT-14655 to J.N.M.H.) and the Arthritis Society (].E.A.). We apologize to all those colleagues whose work we could not reference directly due to space limitations, but refer readers to many excellent papers summarized in various other reviews included here.

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70. Fermor B, Skerry TM. PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats. JBone Miner Res 1995;10(12):1935-1943. 71. AubinJE. Molecular fingerprinting of osteoblast differentiation: From primitive osteoprogenitor to mature osteoblast. In: Potts JT, Ogata E, Kronenberg HM, eds. The molecular and cell biology of bone, vol. 5. Tokyo: International Bone and Calcium Institute Incorporation, 1996:54-59. 72. McCauley LK, Koh AJ, Beecher CA, Cui Y, Rosol TJ, Franceschi RT. PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis. J Cell Biochem 1996;61 (4):638-647. 73. Rodan GA, Rodan SB. Expression of the osteoblast phenotype. In: Peck WA, ed. Bone and mineral, vol. 2. Amsterdam:Elsevier, 1988:244-285. 74. Traverse S, Gomez N, Paterson H, Marshall C, Cohen E Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 1992;288 (Pt 2):351-355. 75. Traverse S, Seedorf K, Paterson H, Marshall CJ, Cohen P, Ullrich A. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curt Bio11994;4(8):694-701. 76. Takasu H, Guo J, Bringhurst FR. Dual signaling and ligand selectivity of the human PTH/PTHrP receptor. J Bone Miner Res 1999;14(1) :11-20. 77. Guo J, Iida-Klein A, Huang X, Abou-Samra AB, Segre GV, Bringhurst FR. Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells. Endocrinology 1995; 136 (9) :3884-3891. 78. Divieti P, Lanske B, Kronenberg HM, Bringhurst FR. Conditionally immortalized murine osteoblasts lacking the type 1 PTH/PTHrP receptor. J Bone Miner Res 1998;13 ( 12):1835-1845. 79. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996;382:448-452. 80. Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999; 104 (4) :399-407. 81. Kartsogiannis V, Moseley J, McKelvie B, et al. Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 1997;21 (5):385-392. 82. Walsh CA, Bowler WB, Bilbe G, Fraser WD, GallagherJA. Effects of PTH on PTHrP gene expression in human osteoblasts: Upregulation with the kinetics of an immediate early gene. Biochem Biophys Res Commun 1997;239(1):155-159. 83. Zhang RW, Supowit SC, Xu X, et al. Expression of selected osteogenic markers in the fibroblast-like cells of rat marrow stroma. Calcif Tissue Int 1995;56(4):283-291. 84. Aarts MM, Rix A, Guo J, Bringhurst R, Henderson JE. The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation. J Bone Miner Res 1999; 14 (9): 1493-1503. 85. Lam MH, House CM, Tiganis T, et al. Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormonerelated protein negatively regulates its nuclear localization. J Biol Chem 1999;274(26):18559-18566. 86. Goltzman D. Interactions of PTH and PTHrP with the PTH/PTHrP receptor and with downstream signaling pathways: Exceptions that provide the rules [editorial; comment] [see comments]. J Bone Miner Res 1999;14(2):173-177. 87. Karaplis AC, Vautour L. Parathyroid hormone-related peptide and the parathyroid hormone/parathyroid hormone-related peptide receptor in skeletal development. Curr Opin Nephrol Hypertens 1997;6(4):308-313.

88. Stewart AF. PTHrP(1-36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone 1996;19 (4) :303-306. 89. Morley P, Whitfield JF, Willick GE. Design and applications of parathyroid hormone analogues. Curr Med Chem 1999;6 ( 11 ) :1095-1106. 90. PfeilschifterJ, Laukhuf F, Muller-Beckmann B, Blum WF, Pfister T, Ziegler R. Parathyroid hormone increases the concentration of insulin-like growth factor-I and transforming growth factor beta 1 in rat bone. J Clin Invest 1995;96(2):767-774. 91. Hill PA, Tumber A, Meikle MC. Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 1997; 138 (9) :3849-3858. 92. Watson PH, Fraher LJ, Kisiel M, DeSousa D, Hendy G, Hodsman AB. Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat. Bone 1999;24(2):89-94. 93. Watson P, Lazowski D, Han V, Fraher L, Steer B, Hodsman A. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factor I gene expression in ovariectomized rats. Bone 1995;16(3):357-365. 94. Masiukiewicz US, Mitnick M, Grey AB, Insogna KL. Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro. Endocrinology 2000;141 (7):2526-2531. 95. Sanders JL, Stern PH. Protein kinase C involvement in interleukin-6 production by parathyroid hormone and tumor necrosis factor-alpha in UMR-106 osteoblastic cells. J Bone Miner Res 2000;15(5) :885-893. 96. Wu Y, Kumar R. Parathyroid hormone regulates transforming growth factor betal and beta2 synthesis in osteoblasts via divergent signaling pathways. J Bone Miner Res 2000;15(5):879-884. 97. Hurley MM, Tetradis S, Huang YF, et al. Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells. J Bone Miner Res 1999;14(5):776-783. 98. Liang H, Pun S, Wronski TJ. Bone anabolic effects of basic fibroblast growth factor in ovariectomized rats. Endocrinology 1999;140(12) :5780-5788. 99. Montero A, Okada Y, Tomita M, et al. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Invest 2000;105(8):1085-1093. 100. Pugazhenthi S, Miller E, Sable C, et al. Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein. J Biol Chem 1999;274 (39) :27529-27535. 101. Zhang W, Lee JC, Kumar S, Gowen M. ERK pathway mediates the activation of Cdk2 in IGF-l-induced proliferation of human osteosarcoma MG-63 cells. J Bone Miner Res 1999; 14 (4):528-535. 102. Amalric F, Baldin V, Bosc-Bierne I, et al. Nuclear translocation of basic fibroblast growth factor. Ann N Y Acad Sci 1991;638:127-138. 103. Amizuka N, Karaplis AC, Henderson JE, et al. Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev Bio11996;175(1 ):166-176. 104. Grey A, Mitnick MA, Masiukiewicz U, et al. A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 1999; 140 (10): 4683-4690. 105. Malaval L, Gupta AK, Liu E Delmas PD, Aubin JE. LIE but not IL-6, regulates osteoprogenitor differentiation: Modulation by dexamethasone. J Bone Miner Res 1998;13:175-184. 106. Bogdanovic Z, Huang YF, Dodig M, Clark SH, Lichtler AC, Kream BE. Parathyroid hormone inhibits collagen synthesis and the activity of rat collal transgenes mainly by a cAMP-mediated pathway in mouse calvariae. J Cell Biochem 2000;77(1):149-158. 107. Scheven BA, Kawilarang-De Haas EW, Wassenaar AM, Nijweide PJ. Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro. Anat Rec 1986;214( 4):418-423. 108. Burger EH, Van der Meer JW, van de Gevel JS, Gribnau JC, Thesingh GW, van Furth R. In vitro formation of osteoclasts

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CHAPTF a 13

Physiologic Actions of PTH and PTHrP I. Skeletal A c t i o n s

GORDON J. STREWLER VA Boston Healthcare System, West Roxbury, Massachusetts 02132; and Department of Medicine, Harvard Medical School,

Boston, Massachusetts 02114

INTRODUCTION In this chapter the effects of Parathyroid h o r m o n e (PTH) and a PTH-related protein (PTHrP) on the biochemistry and metabolism of individual bone cell types are reviewed, including the most recent analyses of a n u m b e r of the effects of these two peptides. Of necessity, the content overlaps with that in the previous two chapters on osteoblast and osteoclast differentiation ~' and the anabolic and catabolic effects of parathyroid h o r m o n e on bone. The final sections include a synthesis of the cellular effects of the peptides into a more integrated analysis of the skeletal effects of PTH and PTHrE

PTHrP AND RECEPTORS FOR PTH A N D P T H r P IN B O N E Expression o f P T H r P in B o n e PTHrP is expressed and secreted by osteoblast-like osteosarcoma cells (1,2) and by rat long bone explants in vitro (3). Messenger RNA for PTHrP is detected in periosteal cells of fetal rat bones (4). In situ hybridization and immunohistochemistry have localized PTHrP mRNA and protein to mature osteoblasts on the bone surface of fetal and adult bones from mice and rats (5,6), and to flattened bone lining cells and some superficial osteocytes (5) in postnatal mice. In addition, the PTHrP gene is expressed in preosteoblast cells in culture, and in some studies its expression is reduced as The Parathyroids, Second Edition

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preosteoblasts u n d e r g o differentiation (2,7,8). PTHrP is also expressed in tissues adjacent to bone, including growth plate cartilage (5,6) and synovium (9), sites where the peptide could affect bone during endochondral bone formation or destructive rheumatoid arthritis, respectively.

R e c e p t o r s and S e c o n d Messenger Systems for P T H and P T H r P As discussed in detail in Chapter 5, a shared receptor (PTHIR) for PTH and PTHrP is present on bone cells; this is a G protein-coupled receptor that recognizes PTH and PTHrP equally well. The receptor couples its ligands to two cellular effector systems, the adenylyl cyclase/cAMP/protein kinase A pathway and the phospholipase C / p r o t e i n kinase C pathway (Chapters 5 and 7). As will become clear as the individual effects of PTH on bone are presented, PTH and PTHrP utilize cAMP for virtually every action in bone for which a second messenger has been identified. The P T H / P T H r P receptor is expressed widely in the osteoblast lineage. In addition to mature osteoblasts, which are on the trabecular, endosteal, and periosteal surfaces (5,6,10,11) and osteocytes (11,12), the receptor mRNA and protein are expressed in marrow stromal cells near the bone surface (5), a putatively preosteoblast cell population that had previously been shown to bind radiolabeled PTH (13,14). Considering the anabolic effect of PTH on bone formation, it will be important to understand at what point in the osteoblast lineage the receptors for PTH are first expressed. Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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Transcripts for the P T H / P T H r P receptor are absent or n o n a b u n d a n t in STRO-l-positive, alkaline phosphatase-negative marrow stromal cells (15,16), which are thought to represent relatively early osteoblast precursors, but P T H / P T H r P receptor expression can be induced by differentiation of stromal cells, MC-3T3 cells, or C 3 H 1 0 T 1 / 2 cells with dexamethasone or bone morphogenetic proteins (16-21). Other data suggest that PTH receptors are limited to a relatively mature population of osteoprogenitor cells that express the osteocalcin gene (22). It thus appears that the P T H / P T H r P receptor appears at a point in osteoblast differentiation when the cells are acquiring other markers of the mature osteoblast phenotype. Whether receptors for PTH or PTHrP are expressed on the osteoclast is controversial. Initial studies using receptor radioautography failed to demonstrate them (13,23). Further studies also have not identified P T H / P T H r P receptor mRNA or protein on mature osteoclasts (5,6,10). However, relatively low-affinity binding of radiolabeled PTH peptides to osteoclasts or preosteoclasts has been reported (24). The functional importance of such putative receptors is unclear. As discussed in detail below (see Effects on Osteoclasts) the presence of osteoblasts or stromal cells seems to be required to elicit effects of PTH on osteoclasts in vitro (25), and studies have found a requirement for the RANKL/RANK system of cytokines and receptors for bone resorption by PTH or PTHrP (26-29) consistent with the interpretation that stromal or osteoblastic cells expressing the cytokine RANKL are required for the induction of bone resorption by PTH. Both PTH and PTHrP have additional receptors besides the P T H / P T H r P receptor. The PTH2R is a G protein-coupled receptor closely related to the P T H I R (30,31); PTH2R recognizes the amino-terminal domain of PTH but not of PTHrE This receptor is expressed predominantly in brain and has yet to be demonstrated in bone. Evidence for actions of carboxyl-terminal PTH peptides on bone has been presented (32-34), as discussed elsewhere in this chapter, and evidence for a specific receptor for carboxyl-terminal PTH peptides has been presented (35). The polyhormone PTHrP is cleaved to produce a set of peptides: those that contain the amino terminus activate the shared P T H / P T H r P receptors, and additional peptides representing the midregion and carboxyl terminus of PTHrP appear to have distinct biologic actions mediated by their own receptors (36,37). Receptors that are specific for amino-terminal PTHrP and do not recognize PTH have been identified in brain (38) and other tissues (39,40), and midregion peptides of PTHrP have actions on placental calcium . transport that imply a distinct receptor (41,42), but

there is presently no evidence for either receptor in bone. Carboxyl-terminal PTHrP fragments [e.g., PTHrP (107-139)] are reported to inhibit bone resorption (43,44) and stimulate the growth of osteoblasts (45). It is thus likely that a specific receptor for this peptide is present on osteoblasts, and conceivably also on osteoclasts.

EFFECTS OF PTH AND PTHrP O N B O N E CELLS E f f e c t s o n O s t e o b l a s t P r e c u r s o r Cells

In view of the anabolic effects of PTH and PTHrP, evidence for a proliferative effect on osteoblast precursors has been sought. Administration of PTH in vivo does not increase mRNA for the proliferation marker histone H4 (46). Immediate-early gene expression is increased after in vivo administration of PTH in osteoblasts and osteocytes (17,47), but the immediateearly gene response is delayed in stromal cells, suggesting that they may respond secondarily to factors elaborated by osteoblasts (47). Effects on Osteoblasts

Transcription Factors PTH induces the expression of the immediate-early genes c-fos and c-jun in osteoblastic cell lines and in osteoblasts in vivo (47,48). The effect on c-fos is largest and best studied. PTH induces c-fos mRNA in a fashion that does not require protein synthesis and is mediated by phosphorylation of the transcription factor CREB by protein kinase A (49-51), to induce binding to a CRE in the c-fos p r o m o t e r (49,50). The protein kinase C signaling pathway does not appear to be involved in this response (49,52). Because many bone cell genes are regulated by PTH, as discussed below, interactions of PTH with osteoblastspecific transcriptional regulation are likely. A splice variant of the runt-domain transcription factor Cbfal called OSF2 is required for determination of the osteoblast phenotype and confers osteoblast-specific expression on the osteocalcin gene (53,54). Although it is not known how PTH interacts with Cbfal at the osteocalcin promoter, a Cbfal site in the collagenase-3 (MMP13) promoter is required along with an AP-1 site for stimulation of collagenase-3 gene transcription by PTH (55,56). It has also been reported that PTH and other agents that elevate cAMP levels in MC-3T3 cells reduce the level of Cbfal and the activity of Cbfal-dependent genes by activating the destruction of the transcription factor b y t h e ubiquitin-proteosome pathway (57).

SKELETALACTIONS OF PTH AND PTHrP

Cytokines Insulin-like Growth Factors Bone is a rich source of insulin-like growth factors (IGFs) secreted by osteoblasts, with IGF-I predominating in rodent bone and IGF-II in h u m a n bone (58). The secretion of IGF-I by rat (59) and IGF-I and IGF-II by mouse (60) osteoblasts in vitro and in vivo (61) is stimulated by PTH. PTH appears to utilize cAMP as the p r e d o m i n a n t intracellular second messenger to stimulate IGF gene expression, because its effects are mimicked by cAMP analogs or agents that increase cAMP, but not by calcium ionophores or phorbol esters (62). Two sets of results raise the possibility that effects of PTH on IGF-I secretion may be essential for its overall anabolic effect on bone. Continuous exposure to PTH, which has catabolic effects on bone in vivo, inhibited collagen synthesis by isolated rat calvariae; but exposure to PTH for the first 24 hours of a 72-hour experim e n t markedly increased collagen synthesis (63). The stimulation of collagen synthesis by PTH is blocked by antibodies to IGF-I, but the stimulation of [3H]thymidine incorporation is not (64). Moreover, treatment of intact rats with PTH u n d e r conditions where it has an anabolic effect on bone leads to an increase in mRNA for IGF-I (61) and the bone matrix content of both IGF-I and transforming growth factor-J31 (TGF-[31) (65). Finally, skeletal unloading leads to resistance to the anabolic effect of PTH, and also resistance in vitro to IGF-I, a result that was interpreted as suggesting that resistance to IGF-I may account for the resistance of the unloaded skeleton to PTH (66). PTH and PTHrP also affect the secretion of binding proteins for IGFs. There are six IGF binding proteins (IGFBP), and all are present in bone (58). IGFBP-4 inhibits IGF action, but IGFBP-5 seems to function predominantly to anchor IGFs to the extracellular matrix, and may in some circumstances have stimulatory effects on IGF action. Exposure of bone cells to PTH or PTHrP increases the secretion of IGFBP-4 (67) and IGFBP-5 (68) by cAMP-dependent mechanisms, and also increases the level of a related protein, IGFBP-rP-1 (69). Both IGFBPs are subject to proteolysis and there is limited evidence to suggest that IGFBP protease activity may be regulated by PTH (70,71). It is not clear whether the effects of PTH on IGFBP levels are biologically significant.

Transforming Growth Factor-J3 PTH and PTHrP increase both the secretion of TGF[3 by osteoblast-like bone cells and the release of TGF-[3 from calvarial explants, the latter may represent in part the release of preformed TGF-[3 during bone resorption (72-75). Intermittent PTH treatment of rats

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increases the bone matrix content of TGF-[31 as well as IGF-I (61), raising the possibility that the anabolic effects of PTH observed with intermittent administration could be mediated, at least in part, by increased secretion of this potent osteoblast growth and differentiation factor.

Interleukin-6 Family Cytokines The cytokines interleukin (IL-6 and IL-11), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotropin 1 (CT1) bind to related receptors and share a signal transduction pathway (76,77). The pathway involves the c o m m o n receptor subunit gp130, binding of JAK family protein kinases, and phosphorylation and nuclear translocation of the STAT family of transcription factors. Of this cytokine family, three members are prominently stimulated by PTH and PTHrP in bone cells, IL-6 (78-81), IL-11 (53,82), and LIF (79). Both PTHrP(1-34) and PTHrP(107-139) are reported to induce the expression of IL-6 (83). The production of IL-6 is also increased by PTH in mouse calvaria (84) and in vivo (46,85). PTH activates transcription of the IL-6 gene (84,86) using cAMP as its principal signaling pathway (84,86,87). It has been suggested from neutralization experiments that the induction by PTH of osteoblast secretion of IL-6 (87,88) or IL-11 (89), both of which are boneresorbing cytokines, may be one mechanism by which the osteoblast transmits the bone-resorbing signal of PTH to the osteoclast. However, studies have shown that blockade of the intracellular signaling pathway, using dominant negative STAT factors (90) or an IL-6 receptor antagonist (91), fails to inhibit bone resorption by PTH, even though bone resorption by IL-6 is blocked. Compelling evidence is now available to indicate that the principal mediators of the bone-resorbing effect of PTH are another set of cytokines, RANKL and osteoprotegerin, or OPG. This issue is further discussed below (see RANK Ligand and Osteoprotegerin).

Other Cytokines and Prostaglandins PTH induces osteoblasts to secrete granulocytemacrophage colony-stimulating factor (GM-CSF) (92,93). PTH also stimulates the production of the prostaglandin PGE 2 by mouse calvarial osteoblasts (94-96). The direct target of PTH is the enzyme prostaglandin G / H synthase (PGHS-2) (97), the protein levels of which are increased by PTH. Another isoform, PGHS-1, is expressed constitutively but is not affected by PTH. The effect of PTH is mediated by cAMP as the d o m i n a n t second messenger (96). PGE 2, in turn, has diverse effects on bone (98).

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RANK Ligand and Osteoprotegerin One of the most important new insights into the regulation of bone metabolism in recent years has been the delineation of a new system for osteoblast-osteoclast cross-talk. It has three major elements. The first is a new m e m b e r of the tumor necrosis factor (TNF) family of cytokines that is expressed on the osteoblast and stromal cell surface; this cytokine is variously known as RANK ligand (RANKL), osteoprotegerin ligand (OPGL), osteoclast differentiation factor (ODF), and TNF-related activation-induced cytokine (TRANCE) (99). By binding to a receptor on osteoclast precursors, RANKL provides an essential feeder function for osteoclastogenesis, accounting for earlier observations that coculture of bone marrow cells and stromal cells is required for osteoclastogenesis (100); RANKL also activates bone resorption by mature osteoclasts and inhibits osteoclast apoptosis (26,101-104). RANKL is both necessary and, with M-CSF, sufficient for osteoclastogenesis; disruption of the RANKL gene leads to severe osteopetrosis (105). The second element of this system is the receptor for RANKL on the surface of osteoclast precursors and mature osteoclasts. This receptor is called RANK (receptor activator of NF-KB) or ODAR (osteoclast differentiation and activation receptor). Disruption of the receptor gene also produces severe osteopetrosis (106). The third element is a decoy receptor, osteoprotegerin or osteoclastogenesis inhibitory factor (OCIF) (104,107,108). Targeted deletion of OPG produces severe osteoporosis, (107,109) but overexpression leads to osteopetrosis (107). Both genetic and cell biologic approaches to this system have yielded decisive results. Following an early c o m m i t m e n t step u n d e r the control of M-CSF, binding of RANKL to RANK is both necessary and sufficient for osteoclastogenesis. The system has a second function to regulate the activity of the mature osteoclast, and exposure of osteoclasts to RANKL inhibits their apoptosis. The decoy receptor OPG must also be important to modulate the tone of the system, because elimination of OPG produces a severe form of osteoporosis. It is conceivable that a parallel system exists, because MCSF-dependent osteoclast formation from cultured mouse bone marrow cells is induced by TNF~ and is blocked by antibodies to its receptor, but not by OPG or antibodies to RANK (110). The bone resorbing effects of PTH, long known to require the intermediation of osteoblasts (25), appear to occur principally through activation of the RANKL/RANK system. Exposure to PTH increases the expression of RANKL in murine bone marrow cultures, cultured osteoblasts, and mouse calvariae (27,104,111), and simultaneously decreases the expression of OPG (27). Stimulation of osteoclastogenesis by PTH is

blocked by antibodies to RANKL (112) or by OPG (103,104). Infusion of OPG into animals blocks the hypercalcemic response to PTH or PTHrP (28,29). Bone resorption by mature osteoclasts in response to PTH has long been recognized as requiring coculture with osteoblasts or marrow stromal cells (25), but when purified cultures of isolated osteoclasts that were unresponsive to PTH were exposed to RANKL, the cytokine was sufficient to induce bone resorption (26). It thus appears that both the stimulation of new osteoclast formation and the activation of the mature osteoclast by PTH and PTHrP take place by binding of the ligand to receptors on osteoblasts, followed by simultaneous induction of the presentation of RANKL on the osteoblast surface and inhibition of secretion of OPG. It is conceivable that the effect of PTH on RANKL and OPG is indirect, involving other cytokines as intermediate steps. It is also possible that a parallel pathway exists, in which other cytokines such as IL-6 or IL-11 could mediate part of the effect of PTH on bone resorption, but if so it is likely to be of secondary importance.

Cell Proliferation and Apoptosis Continuous exposure to PTH(1-34) or PTHrP(1-34) exerts an antiproliferative action on osteoblast-like UMR-106 osteosarcoma cells (113-115). This effect is cAMP-mediated and results, at least in part, from increased levels of p27Kipl, a regulator of Gl-phase cyclin-dependent kinases (115). However, in some cell lines (73,116,117) and primary cultures (118) PTH appears to increase osteoblast or preosteoblast proliferation. In the preosteoblast cell line TE-85, the mitogenic response to PTH requires an increase in levels of the cyclin-dependent kinase cdc2, probably brought about by increased levels of E2F (117). Treatment of rats with intermittent injections of PTH is reported in some studies to increase the number of osteoprogenitor cells (66,119), but not the proliferation of osteoprogenitors (46,120). In an important study, continuous labeling of bone with [3H] thymidine during a period of intermittent treatment with PTH(1-34) resulted in no increase in labeled osteoblasts, despite a marked increase in osteoblast n u m b e r (121). This indicates that the anabolic effect of PTH does not require proliferation of osteoblast precursors or of mature osteoblasts on the bone surface. The large increase in osteoblast n u m b e r produced in this study by intermittent treatment with PTH was attributed to activation of preexisting bone lining cells to osteoblasts (121), but is also possible that PTH induces the commitment of late osteoprogenitors to the osteoblast lineage without a requirement for mitosis. Another alternative explanation for the increase in osteoblast n u m b e r with intermittent PTH treatment is

SKELETAL ACTIONS OF P T H AND P T H r P

provided by work that indicates that treatment of mice with intermittent PTH inhibits osteoblast apoptosis (122). Prolongation of the osteoblast life span by PTH could account for the observed increase in osteoblast number, although it is not clear how large a quantitative effect on osteoblast survival would result from the observed inhibition of apoptosis. The integrated effects of PTH on bone formation are further discussed below and in Chapters 11 and 55.

Effects on Ion Channels In several bone cell types, PTH induces multiphasic changes in membrane potential, most often depolarization followed by sustained hyperpolarization (123-126). Depolarization has been attributed to cAMP-dependent inactivation of quinine-sensitive K channels (124). Depolarization of bone cells induces calcium entry through L-type voltage-sensitive Ca channels (126-129). Sustained hyperpolarization may result in return from opening of Ca-sensitive K channels (130), which may be identical to stretch-activated cation channels also activated by PTH (131).

Effects on Cell Shape PTH treatment of cultured osteoblasts induces a marked retraction of the cell (132) and similar changes have been observed with treatment in vivo (133). The change in cell shape is cAMP mediated (134) and is associated with disassembly of actin stress fibers (135). It can be blocked by inhibitors of the protease calpain (136). The significance of changes in cell shape is unknown, but it has been suggested that osteoblast retraction could have a role in bone remodeling by baring portions of the bone surface in response to PTH.

Effects on Gap Junctions PTH increases intercellular communication of bone cells by increasing connexin-43 gene expression (137) and opening gap junctions (138,139). The significance of intercellular communication to the overall effects of PTH on osteoblasts is not clear, although it is reported that reduction of connexin-43 levels by transfection of antisense cDNA markedly inhibited the cAMP response to PTH (140).

Effects on Bone Matrix Proteins and Alkaline Phosphatase The most abundant protein of bone matrix is type I collagen. Given acutely, PTH consistently inhibits' collagen synthesis in cultured rat calvaria and in cultured bone cells (141,142) by decreasing transcription of the

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COL1A1 gene (143). PTH treatment of calvaria inhibits transcription of a 2.3-kb fragment of the COL1A1 promoter, indicating that at least one major cis-acting element required for inhibition of gene transcription resides in this portion of the promoter (144). PTHrP and agents that increase cAMP have effects similar to those of PTH (145,146). Acute infusion of PTH into humans also inhibits collagen synthesis (147). In contrast, treatment of calvaria with PTH intermittently can stimulate collagen gene expression (63). The stimulatory effect of PTH on collagen synthesis in calvaria is attributed to stimulation of IGF-I production because it is blocked by IGF-I antibodies (64). Moreover, when given intermittently in an anabolic regimen, treatment with PTH in vivo increases bone collagen gene expression (148). The reversal of direction of the PTH effect in vivo can probably be attributed, at least in part, to increased bone remodeling and increases in osteoblast n u m b e r induced by the chronic regimen. Treatment of osteosarcoma cells with PTH has a stimulatory effect on several other bone matrix proteins, including osteocalcin (bone Gla protein, BGP) (149-152); administration of PTH or PTHrP acutely inhibits osteocalcin release from isolated rat hindlimb, but chronic administration of PTH is stimulatory (153). Exposure to PTH stimulates bone sialoprotein gene expression in embryonic chick bone cells (154). PTH treatment inhibits expression of the osteopontin gene in rat osteosarcoma cells (155). Amino-terminal peptides derived from PTH can either stimulate or inhibit secretion of alkaline phosphatase from bone cells, depending on the cell line (156-161). It is reported that carboxyl-terminal PTH fragments can stimulate alkaline phosphatase (32,33), and PTHrP(107-139) is also reported to inhibit alkaline phosphatase (162). Treatment of women with anabolic regimens of intermittent PTH(1-34) injections increases alkaline phosphatase (163), presumably at least in part owing to an increase in osteoblast number. Effects on Proteases of Bone

PTH stimulates the secretion of a n u m b e r of proteases from osteoblasts (164,165). These include stromelysin (166), gelatinase B (166), and collagenase3 (MMP-13) (167-171). Stimulation of the collagenase3 promoter by PTH requires interactions of an AP-1 site and a binding site for runt-domain transcription factors such as OSF2; the effect of PTH is on AP-1 (55,56). Bone resorption by PTH is markedly abrogated in mice with a mutation in the COL1A1 gene that renders the helical domain of type I collagen resistant to cleavage by collagenase (172). It has been suggested that collagenase action on a hypomineralized layer of collagen on

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bone surfaces may be necessary for osteoblast attachment, although multiple other explanations for the observation are also possible. PTH treatment also increases secretion of the inhibitor TIMP (166). Finally, activity of the serine protease plasminogen activator is increased by PTH in bone cell cultures (173,174). There is controversy as to whether the plasminogen activator is urokinase or tissue-type plasminogen activator and whether the effect of PTH is to increase the level of the protease or decrease the level of its inhibitor PAI-1 (165,175,176).

Effects on Osteocytes As noted previously, PTHrP and the P T H / P T H r P receptor appear to be expressed on osteocytes (5,11,12). Exposure to PTH induces ultrastructural changes in osteocytes (177). Although it was long thought that osteocytes, together with bone lining cells, participate in the acute release of calcium from bone in response to PTH (178), this remains conjectural and seems unlikely in view of evidence that the RANK/RANKL system in osteoclasts is involved (28). The current view of osteocytes has for them a predominant role in mechanotransduction, and it is not known how PTH interacts with the mechanotransduction system (179).

INTEGRATED EFFECTS OF P T H A N D PTHrP O N B O N E PTH, PTHrP, and Bone Resorption Cellular Basis of t ~ H Action PTH and PTHrP increase bone resorption by stimulating both the appearance of new osteoclasts and the activity of existing osteoclasts. The mechanistic details of osteoclastogenesis (100) and osteoclast activation (180) are beyond the scope of this chapter, but have been summarized elsewhere; in neither case does PTH have a distinctive effectmrather, the distal cellular responses of osteoclast precursors and mature cells to all bone resorbing agents seem to represent a final common pathway. Both the stimulation of osteoclastogenesis and the activation of the mature osteoclast appear to require the participation of stromal cells or osteoblasts (25,100,181). To recapitulate what has been summarized in previous sections of this chapter, osteoclasts have not been shown to possess high-affinity P T H / P T H r P receptors (5,6,10,13,23), although several groups have identified low-affinity receptors (24). It appears that the effects of PTH are predominantly

mediated by increased expression of the cytokine RANKL (OPGL, ODF, TRANCE) on the cell surface of stromal cells (27,103,104,111,112), perhaps together with a decrease in expression of the decoy receptor OPG (104). The precise target cell in the osteoblast lineage that is responsible for mediating the bone resorbing effects of PTH and PTHrP has not been identified, but various marrow stromal cell lines will suffice in vitro (100) and bone resorption is still active when mature osteoblasts have been ablated (182). By binding to its cognate receptor (RANK) on osteoclast precursors and mature osteoclasts, RANKL stimulates both osteoclastogenesis and the activity of mature osteoclasts. Osteoclast activation by RANKL is apparently responsible both for bone resorption at the cellular level and for hypercalcemia, because both are blocked by the decoy receptor OPG (28,29). Although it was previously suggested that the early phase of the increase in the plasma concentration of ionized calcium, e.g., within 1-2 hours, might have an osteoclastindependent mechanism involving release of calcium by bone lining cells (178), even early responses to PTH in animal models are blocked by inhibiting the RANK/RANKL system (28).

Comparative Effects of PTH and PTHrP The bone-resorbing effects of amino-terminal PTH and PTHrP are essentially indistinguishable when studied using isolated osteoclasts (183,184), bone explant systems (185,186), or infusion into the intact animal (187,188). PTHrP may be somewhat less potent than equimolar infusions of PTH to induce hypercalcemia in humans, probably owing to differences in plasma half-life (189). As discussed in Chapter 3, PTHrP is a polyhormone, the precursor of multiple biologically active peptides. Carboxyl-terminal peptides that are predicted to arise from cleavage of PTHrP in the polybasic region PTHrP(102-106) have been synthesized and shown to inhibit bone resorption in several explant systems (43,190,191), although not all (192), and also in vivo (44). On this basis, the minimal peptide that inhibits bone resorption, PTHrP(107-111), has been called osteostatin.

Effects o f P T H and PTHrP on Bone Formation The anabolic effects of PTH and PTHrP have been discussed in Chapter 11, and their involvement in the pathogenesis of bone changes in primary hyperparathyroidism will be presented in Chapters 24 and 26. The following discussion is a synthesis of a view of the effects of PTH and PTHrP on bone formation from the perspective of the individual cellular actions of the

SKELETAL ACTIONS OF P T H AND P T H r P

h o r m o n e s that have been summarized in the preceding sections of this chapter. Continuous exposure to PTH leads to a coupled increase in bone formation and bone resorption, with a net loss of bone mass in most circumstances, whereas intermittent t r e a t m e n t with injections of PTH once daily, or less frequently, produces a net anabolic effect (193) (see Chapters 11 and 55 for a review). In contrast, the initial interpretation of bone histomorphometry in malignancy-associated hypercalcemia was that, unlike primary hyperparathyroidism, bone resorption was u n c o u p l e d from bone formation (194), raising the possibility that the effects of PTHrP on bone formation differed radically from the effects of PTH. However, in animal models of h u m o r a l hypercalcemia, increases in bone resorption were appropriately coupled to increases in bone formation (195). It has been shown that intermittent administration of PTHrP(1-36) in h u m a n s for 2 weeks leads to increases in biochemical markers of bone formation and a decrease in markers of bone resorption (196). Moreover, a carboxylsubstituted analog of PTHrP(1-34) also mimics the anabolic action of PTH in the rat (197,198). Thus, the anabolic effects of PTH and PTHrP, administered intermittently, appear similar. Any attempt to u n d e r s t a n d the cellular basis for the anabolic actions of PTH and PTHrP must take into account their histomorphologic effects. The increase in bone formation is best correlated with m a r k e d increases in bone formation surfaces and activation frequency (199-202). Thus, a major effect of PTH is to increase the n u m b e r of active, bone-forming osteoblasts. Increases in mineral apposition rate are also seen but tend to be smaller (199-202). The duration of the active bone formation phase is not prolonged in dogs treated with PTH (199) but is increased in primary hyperparathyroidism (200). An increase in the n u m b e r of active osteoblasts could occur in several ways, and PTH may not have the same effect in all circumstancesmits p r e d o m i n a n t effect on growing bone in a young r o d e n t may differ from its p r e d o m i n a n t effect in aged bone. First, PTH could increase the birth rate or proliferation of osteoblast precursors in bone marrow. In the rat, an anabolic regimen of PTH does not increase the proliferation of osteoblast precursors, based on the absence of an increase in labeled nuclei on the bone surface after continuous labeling with [~H]thymidine (121). This is compelling evidence against the view that a proliferative effect of PTH is decisive in increasing osteoblast number. However, intermittent exposure to PTH could increase h o m i n g to the bone surface of late, postmitotic osteoblast precursors in the bone marrow, which are recognized as having PTH receptors (5,13).

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Second, PTH t r e a t m e n t could activate bone lining cells to again become active osteoblasts. There is no direct evidence for or against this hypothesis. However, bone lining cells cover a relatively large bone surface per cell because of their flattened, spread shape, and it is not clear that the numbers of bone lining cells are adequate to account for the increase in osteoblast number that is observed with PTH treatment. Third, an anabolic PTH regimen could increase the life span of the active osteoblast. In the mouse, intermittent t r e a t m e n t with PTH reduces the rate of osteoblast apoptosis (122). However, it is not certain whether the reduction in cell death is quantitatively sufficient to account for the anabolic activity of PTH. If it were, increases not only in m e a n wall thickness but also in the duration of the active formation period would be expected. It is reasonably clear that m e a n wall thickness is increased by anabolic PTH regimens or in primary hyperparathyroidism, but whether the duration of the active formation period is also increased has not been fully resolved (199,200). In order to d e t e r m i n e the m e c h a n i s m by which PTH or PTHrP increases osteoblast number, and thereby has its anabolic effect, it will ultimately be necessary to learn the origin and fate of osteoblasts that participate in the anabolic effects by d e t e r m i n i n g their precise cellular kinetics.

PERSPECTIVES As evident from the previous discussion of anabolic effects of PTH and PTHrP, there is m u c h to be learned about how the individual effects of the h o r m o n e s on bone cells are integrated to produce the final effects of the h o r m o n e s on the physiology of the skeleton. Moreover, there is a large lacuna in our u n d e r s t a n d i n g of the skeletal role of PTHrP. Bone cells both secrete and respond to P T H r E PTHrP is a major regulator of cartilage (the precursor of e n d o c h o n d r a l bones). It is tantalizing to speculate that PTH evolved as a systemic h o r m o n e to overdrive the local regulation of bone metabolism by its sister peptide. However, the physiology of PTHrP in the skeleton has been refractory to study. Genetic models, so powerful in unraveling the role of PTHrP in the cartilaginous phase of endochondral bone formation (Chapter 15), have yielded little information about bone per se, because any changes observed in bone when the P T H r P / r e c e p t o r systems are p e r t u r b e d are potentially explained by perturbations in e n d o c h o n d r a l bone formation. To apply genetic methods to the study of PTHrP in bone, what is now necessary is tissue-specific targeting of PTHrP and its receptor in bone, and such studies are underway. By ablating PTHrP or the P T H / P T H r P receptor in bone

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only, and ultimately restoring sequence-specific tions of the polyhormone PTHrP to such animals, eventually be possible to determine what is the role of PTHrP in bone, and how PTH and PTHrP act as regulators of skeletal physiology.

porit will local inter-

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and other matrix metalloproteinases in response to osteotropic hormones and cytokines. J Cell Sci 1992;103 (Part 4):1093-1099. Walker DG, Lapiere CM, Gross J. A collagenolytic factor in rat bone promoted by parathyroid extract. Biochem Biophys Res Commun 1964;15:397-402. Partridge NC, Jeffrey jj, Ehlich LS, Teitelbaum SL, Fliszar C, Welgus HG, et al. Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells. Endocrinology 1987; 120:1956-1962. Scott DK, BrakenhoffKD, ClohisyJC, Quinn CO, Partridge NC. Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis. Mol Endocrino11992;6:2153-2159. Winchester SK, Bloch SR, Fiacco GJ, Partridge NC. Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts. J Cell Physio11999;181:479-488. Quinn CO, Scott DK, Brinckerhoff CE, Matrisian LM, Jeffrey JJ, Partridge NC. Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. J Biol Chem 1990;265:22342-22347. Zhao W, Byrne MH, Boyce BF, Krane SM. Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice. J Clin Invest 1999;103:517-524. Hamilton JA, Lingelbach S, Partridge NC, Martin TJ. Regulation of plasminogen activator production by boneresorbing hormones in normal and malignant osteoblasts. Endocrinology 1985; 116:2186-2191. Leloup G, Peeters-Joris C, Delaisse JM, Opdenakker G, Vaes G. Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone. J Bone Miner Res 1991 ;6:1081-1090. Catherwood BD, Titus L, Evans CO, Rubin J, Boden SD, Nanes MS. Increased expression of tissue plasminogen activator messenger ribonucleic acid is an immediate response to parathyroid hormone in neonatal rat osteoblasts. Endocrinology 1994;134:1429-1436. Fukumoto S, Allan EH, Yee JA, Gelehrter TD, Martin TJ. Plasminogen activator regulation in osteoblasts: Parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA. J Cell Physio11992;152:346-355. Krempien B, Friedrich E, Ritz E. Effect of PTH on osteocyte ultrastructure. Adv Exp Med Biol 1978;103:437-450. Talmage RV, Doppelt SH, Fondren FB. An interpretation of acute changes in plasma 45Ca following parathyroid hormone administration to thyroparathyroidectomized rats. Calcif Tissue Res 1976;22:117-128. Burger EH, Klein-Nulend J. Mechanotransduction in b o n e - role of the lacuno-canalicular network. FASEB J 1999;13 (Suppl.):S101-$112. Duong LT, Rodan GA. The role of integrins in osteoclast function. J Bone Miner Metab 1999;17:1-6. Akatsu T, Takahashi N, Udagawa N, Sato K, Nagata N, Moseley JM, et al. Parathyroid hormone (PTH)-related protein is a potent stimulator of osteoclast-like multinucleated cell formation to the same extent as PTH in mouse marrow cultures. Endocrinology 1989;125:20-27. Corral DA, Amling M, Priemel M, Loyer E, Fuchs S, Ducy P, et al. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. P r o c Natl Acad Sci USA 1998;95:13835-13840. Evely RS, Bonomo A, Schneider HG, Moseley JM, Gallagher J, Martin TJ. Structural requirements for the action of parathyroid hormone-related protein (PTHrP) on bone resorption by isolated osteoclasts. J Bone Miner Res 1991;6:85-93.

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184. Murrills RJ, Stein LS, Fey CP, Dempster DW. The effects of parathyroid hormone (PTH) and PTH-related peptide on osteoclast resorption of bone slices in vitro: An analysis of pit size and the resorption focus. Endocrinology 1990;127:2648--2653. 185. Yates AJ, Gutierrez GE, Smolens P, Travis PS, Katz MS, Aufdemorte TB, et al. Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 1988;81:932-938. 186. Raisz LG, Simmons HA, Vargas SJ, Kemp BE, Martin TJ. Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones. Calcif Tissue Int 1990;46:233-238. 187. Thompson DD, Seedor JG, Fisher JE, Rosenblatt M, Rodan GA. Direct action of the parathyroid hormone-like human hypercalcemic factor on bone. Proc Natl Acad Sci USA 1988;85:5673-5677. 188. Kitazawa R, Imai Y, Fukase M, Fujita T. Effects of continuous infusion of parathyroid hormone and parathyroid hormonerelated peptide on rat bone in vivo: Comparative study by histomorphometry. Bone Miner 1991;12:157-166. 189. Fraher LJ, Hodsman AB, Jonas K, Saunders D, Rose CI, Henderson JE, et al. A comparison of the in vivo biochemical responses to exogenous parathyroid hormone-(1-34) [PTH(1-34)] and PTH-related peptide-(1-34) in man. J Clin Endocrinol Metab 1992;75:417-423. 190. Fenton AJ, Kemp BE, Hammonds RG, Jr, Mitchelhill K, Moseley JM, Martin TJ, et al. A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111]. Endocrinology 1991 ;129:3424-3426. 191. Fenton AJ, Martin TJ, Nicholson GC. Long-term culture of disaggregated rat osteoclasts: Inhibition of bone resorption and reduction of osteoclast-like cell number by calcitonin and PTHrP [ 107-139]. J Cell Physio11993;155;1-7. 192. Sone T, Kohno H, Kikuchi H, Ikeda T, Kasai RKY, Takeuchi R, et al. Human parathyroid hormone-related peptide-(107-111) does not inhibit bone resorption in neonatal mouse calvariae. Endocrinology 1992;131:2742-2746. 193. Tam CS, Heersche JNM, Murray TM, Parsons JA. Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continuous administration. Endocrinology 1982;110:506-512. 194. Stewart AF, Vignery A, Silverglate A, Ravin ND, LiVolsi V, Broadus AE, et al. Quantitative bone histomorphometry in humoral hypercalcemia of malignancy. J Clin Endocrinol Metab 1982;55:219-227. 195. Strewler GJ, Wronski TJ, Halloran BE Miller SC, Leung SC, Williams RD, et al. Pathogenesis of hypercalcemia in nude mice bearing a human renal carcinoma. Endocrinology 1986;119:303-310. 196. Plotkin H, Gundberg C, Mitnick M, Stewart AF. Dissociation of bone formation from resorption during 2-week treatment with human parathyroid hormone-related peptide-(1-36) in humans: Potential as an anabolic therapy for osteoporosis. J Clin Endocrinol Metab 1998;83:2786-2791. 197. Vickery BH, Avnur Z, Cheng Y, Chiou SS, Leafier D, Caulfield JP, et al. RS-66271, a C-terminally substituted analog of human parathyroid hormone-related protein (1-34), increases trabecular and cortical bone in ovariectomized, osteopenic rats. J Bone Miner Res 1996;11:1943-1951. 198. Frolik CA, Cain RL, Sato M, Harvey AK, Chandrasekhar S, Black EC, et al. Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(I-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats [see comments]. J Bone Miner Res 1999;14:163-172.

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199. Boyce RW, Paddock CL, Franks AF, Jankowsky ML, Eriksen EE Effects of intermittent hPTH(1-34) alone and in combination with 1,25(OH)(2)D(3) or risedronate on endosteal bone remodeling in canine cancellous and cortical bone. JBone Miner Res 1996;11:600-613. 200. Dempster DW, Parisien M, Silverberg SJ, Liang XG, Schnitzer M, Shen V, et al. On the mechanism of cancellous bone preservation in postmenopausal women with mild primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:1562-1566.

201. Shen V, Dempster DW, Birchman R, Xu R, Lindsay R. Loss of cancellous bone mass and connectivity in ovariectomized rats can be restored by combined treatment with parathyroid h o r m o n e and estradiol. J Clin Invest 1993;91: 2479-2487. 202. Lane NE, Kimmel DB, Nilsson MH, Cohen FE, Newton S, Nissenson RA, et al. Bone-selective analogs of human PTH(1-34) increase bone formation in an ovariectomized rat model. J Bone Miner Res 1996;11:614-625.

CHAPTER14 Physiologic Actions of P T H and PTHrP II. Renal Actions

E RICHARD BRINGHURST Endocrine Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

PTHR EXPRESSION, SIGNALING, AND REGULATION IN THE KIDNEY

INTRODUCTION The kidney is the focal point for the physiologic regulation of mineral ion homeostasis by circulating parathyroid hormone (PTH). By directly controlling renal tubular reabsorption of calcium and phosphate and the synthesis of 1,25-dihydroxy vitamin D (1,25(OH)zD), PTH exerts control over both the intestinal absorption and the urinary excretion of these key mineral ions. Renal tubular responses to P T H deficiency, PTH or PTH-related protein (PTHrP) excess, or defects in function of the type 1 P T H / P T H r P receptor (PTHR) lead to alterations in blood calcium, phosphate, or 1,25 (OH)zD that are the hallmarks of numerous clinical disorders, described in Section III of this volume. This chapter reviews current understanding of the mechanisms whereby PTH (and PTHrP) control renal tubular epithelial function. The discussion focuses principally on the known actions of PTH, because relatively little is known of the possible physiologic actions of PTHrP in the kidney. Because the PTHR recognizes the active amino termini of both ligands equivalently, however, it is likely that the effects described for PTH would pertain to PTHrP as well. Expression and action of PTHrP in the kidney are discussed in the last section of this chapter. Whereas species of PTH or PTHrP receptors distinct from the PTHR have been discovered (see Chapter 5), the roles of these, if any, in normal renal physiology currently are unknown. Though not unequivocally proved in each case, it is likely that the effects of PTH and PTHrP described here are mediated by the PTHR. The Parathyroids, Second Edition

The PTHR is widely expressed within the kidney among cells with dramatically different physiologic roles. The responses to PTHR activation observed in individual renal cells are a complex function of the number and location of expressed PTHRs on the cell surface; the cell-specific expression of effectors capable of coupling to the PTHR; the cell-specific repertoire of PTHR-regulated genes; enzymes, channels, and transporters; the local concentrations of PTH or PTHrP ligand; exposure to other agents that regulate PTHR function heterologously; and the pattern of recent exposure to PTHR ligands.

PTHR Expression within the Kidney The PTHR is widely but not universally expressed by the various cell types that collectively comprise the mammalian nephron. Early work, based on measurements of regional cAMP responses (1-5) and PTH radioligand binding in vivo (6), indicated that PTHRs are expressed in glomeruli, proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs), the cortical thick ascending loop of Henle (CTAL), and portions of the distal nephron, including the distal convoluted tubules (DCTs), connecting tubules (CNTs), and early portions of the cortical collecting ducts (CCDs). More recently, these functional observations have been confirmed by in situ hybridization of tissue sections or by reverse transcriptase and polymerase 227

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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chain reaction (RT-PCR) of microdissected nephron segments, using probes derived from the cloned PTHR cDNA (7-9). Minor disparities regarding PTHR expression in Henle's loop and CCDs arising from use of these sensitive molecular techniques likely reflect methodologic issues (7-9). Given that circulating PTH peptides may be filtered at the glomerulus and appear in the tubular urine, it is of interest that PTHRs are expressed on the apical (luminal) as well as the basolateral membranes of proximal tubular cells (10-12). On the other hand, these apical membrane receptors appear not to be coupled tightly, if at all, to adenylyl cyclase (10,11). Moroever, a high-capacity apical peptide-uptake mechanism, mediated by the multifunctional endocytic clearance receptor megalin (13), likely would limit access of filtered bioactive PTH peptides to these receptors. PTHRs also are expressed within the vasculature of the kidney, including peritubular (but not glomerular) endothelial cells and vascular smooth muscle cells (12). As discussed further below, such receptors may mediate local or systemic vascular effects of PTHrP and PTH, respectively. As described in more detail in Chapter 5, the PTHR gene incorporates multiple promoters and 5' untranslated exons and therefore can generate multiple transcripts via alternative promoter usage and different patterns of RNA splicing (12,14-17). It is of interest that certain promoters (i.e., P1 in mouse and P3 in human) seem to be used exclusively in kidney cells, whereas a different promoter (P2) is employed to generate those PTHR mRNAs that are widely expressed in other tissues and organs (12,16,17). Whether these differences simply reflect opportunities for tissue-specific gene regulation or lead to expression of structurally different forms of the PTHR (15,16) remains to be established.

PTHR Signal Transduction in Renal Cells The PTHR is known to couple to multiple intracellular signal transducers and effectors, including but perhaps not limited to G s and the Gq/G11 family of heterotrimeric G proteins (18) (see also Chapter 7). Administration of PTH in vivo leads to the rapid generation of nephrogenous cAMP (19,20) and to activation of protein kinase C (PKC) in basolateral renal cortical membranes (21). This signaling plurality via the PTHR has been abundantly confirmed and further characterized in extensive studies in vitro, which have involved isolated renal tubules or slices, primary renal cortical cell cultures, a widely employed established opossum kidney cell line with characteristics of PCTs (OK cells), immortalized immunoselected distal tubular cells, and various established epithelial cell lines of renal origin (i.e., COS-7, HEK293, LLC-PK1), devoid of endogenously expressed PTHRs, which have been transfected with cDNA encoding the cloned PTHR (18,22-45).

Collectively, these studies indicate that PTH can activate adenylyl cyclase, protein kinase A (PKA), phospholipase C (PLC), PKC, and cytosolic free calcium (CaZi+) transients, as well as phospholipase A 2 (PLA2) (46-48) and phospholipase D (PLD) (43,45). The repertoire of PTHR signaling appears to differ depending on the region of the nephron in which it is expressed. For example, cells of proximal tubular origin manifest an acute spiking C a 2+ i response that likely is triggered by inositol trisphosphate released via PLC activation. Cells of distal tubular origin, in contrast, exhibit a very delayed and sustained CaZi+ response (probably due to apical C a 2+ entrymsee below) and show PKC activation in the absence of PLC stimulation (43). The PKC response to PTH in these DCT cells may be mediated by PLD (45). The coupling of specific PTHR-generated signals to the various physiologic responses to PTH or PTHrP that occur in different renal epithelial cells has not yet been fully clarified and will be discussed further below.

Regulation of PTHR Signaling in Renal Cells As in other P T H / P T H r P target cells, the responsiveness of renal epithelial cells to PTH or PTHrP may be regulated, both by previous or chronic exposure to the homologous ligand and by other agonists that do not interact directly with the PTHR. Desensitization of renal cellular responsiveness during continuous exposure to high concentrations of PTH or PTHrP has been well documented and extensively studied. Chronic hyperparathyroidism (either primary or secondary to calcium or vitamin D deficiency) and acute infusion of PTH lead to PTH resistance in humans or animals, manifested by impaired cAMP and phosphaturic responses (19,21,49-51). In humans, the cAMP response may be more readily desensitized than the phosphaturic response at low doses of hormone (52). Similar desensitization is observed in cultured renal epithelial cells (23,53-56). Several factors may contribute to this renal resistance to PTHR activation, including a reduced number of cell surface PTHRs, persistent occupancy of PTHRs by ligand, and defective coupling between available PTHRs and the G proteins that mediate activation of effectors such as adenylyl cyclase or PLC (i.e., a "postreceptor" defect). The relative roles of these factors in causing PTHR desensitization appear to vary according to the specific situation and experimental system (49,57-61). As reviewed in more detail in Chapter 5, it is clear that PTHRs are rapidly internalized following ligand occupancy and activation, a response that lowers cell surface receptor expression and that is due to PTHR phosphorylation by both PTHR-dependent activation of "signal kinases" (PKA, PKC) and by the action (s) of generic G protein-coupled receptor kinases

RENAL ACTIONS OF P T H AND P T H r P

(62-64). The particular PTHR-generated signals that mediate PTHR desensitization in renal epithelial cells may be cell type specific. For example, in OK proximal tubular cells, homologous desensitization of the PTHR cAMP response is PKC dependent (53), whereas in PTHR-transfected LLC-PKa cells, desensitization is pathway specific--i.e., adenylyl cyclase is fully desensitized by cAMP-dependent signaling only whereas desensitization of the PLC response is linked to prior PLC activation (56). Control of receptor expression may be an important mechanism for modulating the relative, as well as the absolute, intensities of signaling along the various transduction pathways coupled to the PTHR. Thus, as shown in a series of PTHR-transfected LLC-PK1 renal epithelial cell subclones that comprised a broad range of receptor expression, the magnitude of the PLC response was much more strongly influenced by changes in cell surface PTHR density than was the adenylyl cyclase response (37). This was interpreted as evidence that the coupling between G s and the PTHR in these cells is more efficient than that between the PTHR and the Gq that presumably mediates PLC activation. In any event, it is clear that changes in PTHR expression may allow differential modulation of PTHR signaling responses in a given renal cell. Expression of PTHRs on the surface of kidney cells also is controlled by the rate of PTHR gene transcription, although current understanding of this process is incomplete. Hypoparathyroidism, induced by either parathyroidectomy or dietary phosphate depletion, strongly up-regulates PTHR mRNA levels in rat renal cortex (65). Curiously, the opposite effect, i.e., suppression of PTHR mRNA by exposure to high concentrations of PTH, has not been observed, either in vivo or in vitro (55,65). Renal PTHR mRNA expression is reduced in rats with renal failure, but this apparently is due to some aspect of uremia or renal disease other than secondary hyperparathyroidism per se, because it is not prevented by parathyroidectomy (66-68). In rats with secondary hyperparathyroidism due to vitamin D deficiency, renal cortical PTHR mRNA levels actually were found to be twice as high as normal, a change that could not be corrected by normalizing serum calcium (61). This experiment has been interpreted as evidence of a suppressive action of vitamin D on PTHR gene transcription in the proximal tubule, although this may not be true of all renal epithelial cells. For example, PTHR expression is upregulated severalfold by 1,25(OH)zD ~ in immortalized DCT cells (69). In cultured OK cells, TGF-[31 was shown to diminish PTHR mRNA expression, but the possible physiologic significance of this effect in vivo has not been clarified (70). PTHR mRNA expression was not affected by the mild secondary hypoparathyroidism induced by ovariectomy in rats nor by subsequent estrogen treatment (71).

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CALCIUM AND MAGNESIUM EXCRETION The action of PTH to maintain blood calcium was among the first to be described, and early observations in animals or patients with hypoparathyroidism or hyperparathyroidism clearly implicated abnormalities in renal calcium handling (see Chapters 27 and 47). Alterations in serum magnesium concentrations frequently are encountered also in patients with parathyroid disorders, which led to the understanding that PTH participates in magnesium homeostasis as well (see Chapter 48). The mechanisms whereby Ca 2+ and Mg 2+ are reabsorbed are similar and interrelated in some regions of the nephron but different in others.

Sites and Mechanisms of Calcium and Magnesium Reabsorption Calcium and Mg 2+ are reabsorbed at many sites along the nephron (72,73). Approximately 60% of illtered Ca 2+, but only 20% of filtered Mg 2+, is reabsorbed by the proximal tubule. Reabsorption here is almost entirely passive, driven by both the ambient lumen-positive voltage and the progressive concentration of these ions within the tubular urine as Na + and water are reabsorbed along the proximal segments (72-74) (Fig. 1). In the proximal tubule, the route of reabsorption for both Ca 2+ and Mg 2+ is almost entirely paracellular, and differences in permeability of the intercellular tight junctions for the two cations presumably account for the preferential reabsorption of Ca 2+ here. Both Ca 2+ and Mg2+ also are passively reabsorbed in the CTAL of Henle's loop, although here the permeability for Mg 2+ may be greater than that for Ca 2+, because 60% of Mg 2+ but only 20% of Ca 2+ is reabsorbed in this segment, The lumen-positive transepithelial voltage gradient that drives Ca 2+ and Mg 2+ transport in the CTAL is maintained by, and proportional to, the rate of Na+/K+/CI2 transport, which is dependent, in turn, on the activities of the NKCC2, C1C-Kb, and ROMK transporters (75). The calcium-sensing receptor (CaSR) also is especially strongly expressed in Henle's loop, and activation of this receptor by high peritubular Ca 2+ or Mg 2+ concentrations inhibits Ca 2+ and Mg 2+ reabsorption in the CTAL, presumably by reducing the transepithelial voltage gradient (76) (see Chapter 8). It also is possible that the CaSR may mediate inhibition by Ca 2+and Mg 2+ of the cAMP response to PTH (7%79). Paracellin-1, a novel member of the claudin family of tightzjunction proteins that is expressed only in Henle's loop and the DCT, was identified as the cause of an autosomal recessive renal magnesium' and Ca2+-wasting disorder (80). Though not yet demonstrated directly, it seems likely that expression of paracellin-1 may control the passive permeability of the CTAL for both Ca 2+ and Mg 2+. There is some evidence also for active, transcellular transport of Ca 2+ by the

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FIG. 1 Calcium and magnesium reabsorption in (left) the proximal convoluted tubule (PCT) and (right) the cortical thick ascending loop of Henle (CTAL). In the PCT, Ca 2+ and Mg 2÷ are passively reabsorbed via paracellular routes at rates driven by the lumen-positive transepithelial voltage and limited by the conductance of the intercellular junctions for these cations. The transpeithelial voltage, depicted as positive at the apical (Ap) relative to the basolateral (BI) side of the epithelium, is generated by paracellular diffusion of CI- ions, which, like Ca 2+ and Mg 2+ ions, are progressively concentrated along the lumen by active transcellular Na + reabsorption. Major mechanisms of Na + reabsorption shown include Na+/H ÷ exchange, Na+-dependent cotransport of anions (phosphate, amino acids, sulfate, etc.), and a small apical Na + conductance, all driven by the low intracellular Na + concentration established by the Na+/K +ATPase, which pumps three Na + ions out for each two K + ions that enter the cell. The stoichiometry of the basolateral electrogenic Na+/HCO~ cotransporter (one Na + per three HCO~ ions) allows for active basolateral extrusion of some Na + because of the negative intracellular potential (not shown) and favorable HCO~ concentration gradient that drive HCO~ exit. PTHRs expressed in PCTs inhibit Na + transport by multiple mechanisms (see text) and thereby moderately impair Ca 2+ and Mg 2+ reabsorption (dashed lines indicate responses about which some uncertainty exists). In the CTAL (right panel), Ca 2+ and Mg 2+ reabsorption will again occur mainly via voltage-dependent paracellular transport, although transcellular Ca 2+ transport, presumably mediated by apical Ca 2+ channels and basolateral Ca2+-ATPases, also has been described (question mark). Apical NKCC2 cotransporters and ROMK K + channels maintain the lumen-positive transepithelial voltage necessary for cation transport, which is inhibited by Ca2+/Mg2+-dependent activation of the CaSR and by the loop diuretic furosemide. Chloride exits across the basolateral membrane via one or more CI- channels, including CIC-Kb (not shown). The channel protein paracellin-1 appears to be critical for paracellular cation transport in the CTAL and could be a target for CaSRs and PTHRs, which, respectively, reduce and augment cation transport in this nephron segment.

CTAL (81). Calcium-sensitive cation channels have been found in CTAL apical membranes (82), as have Ca '2+ATPases that would be necessary for extrusion across the steep basolateral electrochemical gradient (83). Finally, small but critical fractions of filtered Ca2+and 2+ Mg uapproximately 5-10% eachmare reabsorbed in the distal nephron (i.e., the DCT, CNT, and early CCD) (Fig. 2). The mechanism of Mg 2+ reabsorption by the distal nephron is obscure, but it seems to be closely related to that of NaCI, in that both pharmacologic (thiazide diuretics) and genetic (Gitelman's syndrome) inhibition

of the thiazide-sensitive NaC1 cotransporter (TSC) impairs Mg 2+ reabsorption. In contrast, Ca 2+ reabsorption in the distal nephron, which involves transcellular active transport against an unfavorable electrochemical gradient (84-86), is promoted by TSC inhibition, which hyperpolarizes the apical cell membrane. Cells of the distal nephron express several proteins that are required for effective transcellular active Ca 2+ transport (73). Calcium enters the apical membrane via multiple C a 2+ channels (87,88), one of which, ECaC, has been cloned and shown to be expressed in the distal tubule, to be acti-

RENAL ACTIONS OF PTH AND PTHrP

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FIG. 2 PTH regulation of distal tubular calcium reabsorption. In the DCT, Ca 2+ reabsorption involves apical Ca 2+ entry via voltage-sensitive Ca 2+ channels and subsequent basolateral extrusion by Ca2+-ATPases and, uniquely, Na+/Ca 2+ exchangers driven by the Na÷/K+-ATPase. Multiple Ca 2+ channels may be expressed here, including the ECaC channel that is activated by hyperpolarizing voltages (increased I Vml). Inhibition of the thiazide-sensitive NaCI transporter, with continued basolateral CI- exit, hyperpolarizes the cell toward the K + equilibrium potential, which then increases Ca 2+ entry by ECaC and other channels activated by hyperpolarizing potentials. Calbindin-D28K binds and shuttles Ca 2÷ from the apical membrane to the basolateral sites of active Ca 2÷ extrusion, thereby buffering the cytoplasm from high concentrations of transported Ca 2+. Calbindin-D28K is induced by 1,25(OH)2D 3 and may directly activate apical Ca 2+ channels, which otherwise are inhibited by intracellular Ca 2+ ions. PTHR activation leads to insertion of additional apical Ca 2+ channels, hyperpolarization of the cell (question mark) via enhancing basolateral CI- exit, and, thus, activation of Ca 2÷ channels, increased calbindin-D28K expression, and stimulation of the basolateral Ca2+-ATPase. The routes and mechanisms of Mg 2+ reabsorption in the DCTs are unknown.

vated by hyperpolarizing voltages, and to be inactivated by intracellular C a 2+ (89,90). These cells also express the vitamin D-dependent calbindin-D28K calcium binding protein, which can transport Ca 2+ across the cytoplasm while buffeting the submicromolar cytosolic free C a 2+ concentration against the high mass flux of transported C a 2+ (91-93). Calbindin-D28K also may directly activate apical membrane C a 2+ channels (94). Extrusion of transported Ca 2+ across the basolateral membrane can occur via both a direct CaZ+-ATPase and a high-capacity Na+/Ca 2+ exhanger driven by the transmembrane Na + gradient.

PTH Regulation of Renal Calcium and Magnesium Excretion Administration of PTH in vivo increases the net renal reabsorption of both Ca 2+ and Mg 2+ (72,95-99).

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PTH augments Mg 2+ reabsorption in the CTAL (100-102) and possibly in the distal n e p h r o n as well (103), but the mechanisms involved are obscure. PTH may increase the transepithelial voltage that drives paracellular Mg 2+ (and Ca 2+) transport in the CTAL, but this is controversial (100,101) and, in any event, is unlikely to explain the magnitude of the PTH effect (72). Other experiments indicate that the PTH response probably is mediated by an increase in paracellular Mg 2+ permeability (104). In this regard, it will be of interest to learn if PTH up-regulates paracellin-1 expression or permeability. M t h o u g h PTH increases net renal C a 2+ reabsorption overall, it actually moderately inhibits C a 2+ reabsorption in the PCT (105-107). As will be discussed further below, this results from a PTH-induced reduction in Na + reabsorption (via inhibition of both NaP i cotransport and N a + / H + exchange) and of Na+/K+/ATPase activity, processes that otherwise support net solute and water reabsorption and thereby establish the elevated intraluminal concentrations of C a 2+ and C1- required for effective paracellular movement of C a 2+ in the PCT. In contrast, PTH augments C a 2+ reabsorption in the CTAL and in the distal n e p h r o n , especially in the CNT (84,106,108-110), and it is these actions that account for the overall positive effect of PTH on renal C a 2+ reabsorption. The mechanism of the PTH effect in the CTAL has not been intensively studied but likely proceeds via an increase in transepithelial voltage and e n h a n c e d paracellular C a 2+ transport (101), although some evidence suggests a c o m p o n e n t of transcellular transport as well (111). The distal n e p h r o n clearly is the major site at which PTH regulates C a 2+ transport. PTH exerts several specific actions in these cells that independently contribute to increased C a 2+ reabsorption. PTH increases C a 2+ uptake across apical membranes of distal tubular cells, an effect that can be observed in apical membrane vesicles isolated following PTH administration in vivo or to isolated tubules in vitro (112,113). In cultured cells obtained from the mouse CTAL and DCT, PTH induced a delayed (10 minutes) and sustained increase in cytosolic C a 2+ that was of extracellular origin, was blocked by dihydropyridine C a 2+ channel antagonists, and appeared to result from exocytosis of membranes harboring preformed but functionally latent intracellular C a 2+ channels (114). These channels were of low conductance and were activated by hyperpolarizing voltages (87), features also reported for the subsequently cloned ECaC channel (90) (although more information is needed to determine if the ECaC channel serves as the main route of regulated C a 2+ entry in distal tubular cells). Importantly, PTH acutely hyperpolarizes distal tubular cells, at least in part by increasing basolateral C1- conductance (115). This action would activate the ECaC channel (90) and increase both the

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driving force for apical membrane C a 2+ entry and the rate of Na+/Ca 2+ exchange at the basolateral membrane (116). Increased Na+/Ca 2+ exchange has been demonstrated following PTH administration in vivo and in vitro (117,118). Moreover, activation of Na+/Ca 2+ exchange is critical for the action of PTH to increase C a 2+ reabsorption, because this could be blocked completely in rabbit CNTs and DCT cells either by disrupting the Na + gradient that drives the Na+/Ca 2+ exchanger with ouabain or monensin or by removing extracellular C a 2+ f r o m the basolateral compartment (116,119). The fact that this exchanger is expressed only in the distal, and not the proximal, nephron may explain, at least in part, why distal and not proximal tubular cells can conduct transcellular C a 2+ transport (113,116,120,121). PTH also may increase C a 2+ e x t r u s i o n by activating the basolateral CaZ+-ATPase (122), although this is not observed in all systems (120). Finally, expression of the calbindin-D28K protein in renal cortex has been shown to decrease following parathyroidectomy and to increase following PTH infusion into intact rats (123). The powerful inductive effect of 1,25(OH)zD ~ on calbindin-D28K expression in the distal nephron (124,125) may be involved in mediating this action of PTH, given that PTH augments 1,25(OH)zD 3 synthesis (see below) and that 1,25(OH)zD ~ directly accelerates the distal tubular calcium reabsorptive response to PTH in vitro (126). Other evidence indicates that PTH can increase calbindinD28K independently of 1,25(OH)zD ~ or serum calcium, however (123).

PTHR Signal Transduction in Regulation of Calcium and Magnesium Excretion The particular PTHR-generated signals responsible for these various effects of PTH on components of the distal tubular CaZ+-reabsorptive response are not fully clarified. The initial entry of C a 2+ a c r o s s the apical membrane seems to require activation of both PKA and PKC in immortalized murine DCT cells (43,45). In many experimental systems, the PTH effect on distal Ca 2+ transport can be mimicked by cAMP analogs or phosphodiesterase inhibitors (112,119,127), although in isolated rabbit CNT/CCD tubules, in which this cAMP mimicry also is true, the effect of PTH was prevented by chelerythrine, a PKC inhibitor, but not by dideoxyadenosine, an adenylyl cyclase inhibitor that did block PTH-dependent cAMP accumulation (128). Further evidence implicated a Ca2+-independent ("atypical") PKC as a mediator of this PTH effect (128). Similarly, the ability of dibutyryl cAMP to promote C a 2+ transport in rabbit distal tubules was greatly potentiated by phorbol esters, which exerted no effect alone, and PKC inhibitors did block the effect of the combination

of phorbol and cAMP analog as well as that of the cAMP analog alone (129). PTH stimulation of Na+/Ca 2+ exchange, transepithelial hyperpolarization, and, in canine cells, CaZ+-ATPase also is reproduced by cAMP analogs (110,117,118,120,122), although, as just noted, such evidence clearly does not exclude a role for other PTHR messengers in these processes as well. Considering that PTH may have to orchestrate a series of independent "elemental responses" to achieve effective distal tubular C a 2+ reabsorption, including membrane hyperpolarization, increased exocytosis of latent C a 2+ channels, increased calbindin-D28K expression, increased Na+/Ca 2+ exchange [this possibly secondary entirely to the hyperpolarization and increased cytosolic free C a 2+ (73)], and increased CaZ+-ATPase activity, and that these responses may not all occur in the same cells, it is perhaps not surprising that some ambiguity persists regarding the roles of PKA versus PKC (or other PTHR-activated effectors) in controlling overall distal tubular C a 2+ transport. Apparent requirements for multiple effectors may reflect a convergence of several signals on a single mechanism, independent actions of different effectors on one or more of the elemental cellular responses that contribute to the overall Ca2+-reabsorptive response, or both.

PHOSPHATE EXCRETION Phosphaturia was one of the earliest recognized actions of PTH (130-133). With the advent of micropuncture analysis, it became clear that the effect of PTH to inhibit phosphate reabsorption occurs almost entirely in the proximal tubules, especially in the late portion of the PCT (105-107,134-137). Some evidence points to a small component of PTH-inhibitable phosphate reabsorption in the distal nephron as well (107,136,138-140).

Mechanisms of Proximal Tubular Phosphate Reabsorption Extensive experimentation with isolated perfused tubules, renal membranes, and membrane vesicles over the past 25 years, exhaustively reviewed by Murer and colleagues (141-143), has provided a clear picture of the mechanisms of proximal tubular phosphate reabsorption. Phosphate (Pi) is moved across the apical membrane of the cell, against a steep electrochemical gradient (due mainly to the negative intracellular potential), by Na+/Pi cotransporters driven by the transmembrane Na ÷ gradient (Fig. 3). Detailed biochemical analyses had indicated that multiple such Na+/Pi cotransporters, with distinct kinetic, allosteric, and physical properties, are located within the renal

RENAL ACTIONS OF PTH AND P T H r P

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dietary phosphate (150,154-158). Thus, regulation of NaPi-2 activity is the principal mechanism whereby PTH controls phosphate reabsorption in the PCT.

PTH Regulation of Proximal Tubular Phosphate Reabsorption

Pi'=I tnM

I I "~''PPTTHI4rP

Early work had demonstrated that PTH rapidly lowers the maximal rate of NaPi cotransport in brush border membrane vesicles and that recovery from this effect requires new protein synthesis, suggesting that PTH causes degradation of NaP~ cotransporters (141, 159-161). Recent functional and immunohistochemical analyses of NaP~-2 protein expression in rat kidney and in cultured OK cells have confirmed that PTH induces a rapid (15 minutes) movement of NaPe2 protein into the subapical endocytic apparatus, followed by a microtubule-dependent delivery to lysosomes and proteolytic degradation (154,156,158,162,163) (see Fig. 4). These effects of PTH on NaP~-2 protein are not associated with suppression of NaPi-2 mRNA expression, although parathyroidectomy does increase both NaPi-2 protein and mRNA severalfold (65,155,164,165).

FIG. 3 Phosphate reabsorption in the proximal tubule. Phosphate (P~) must be actively transported across the apical membrane of the PCT cell because of the strongly interiornegative potential and the fact that cytosolic P~ concentration (1 mM) is roughly 100-fold above equilibrium. This transport is accomplished by an electrogenic type II NaP~ cotransporter [stoichiometry = 3 Na ÷ ions per P~(mono- or dibasic) ion] that is energized by the steep transmembrane Na ÷ gradient established by the basolateral Na+/K+-ATPase. Activity of this cotransporter is reduced by PTHR activation. Mechanisms of basolateral P~ exit are not well understood, but an anion exchanger could allow P~to leave the cell passively.

PTHR Signal Transduction in Regulation of Phosphate Excretion

cortex (144,145). Some of these may be so-called housekeeping cotransporters, presumed to reside on the basolateral membranes, that are ubiquitously expressed by all cells and involved in maintaining intracellular P~ concentrations, whereas others are epithelial-specific and devoted to the specialized function of transepithelial phosphate transport (142). Three major classes (types I, II, and III) of Na+/Pi ("NaPi") cotransporters, products of different genes, have been cloned and shown to be expressed in PCT cells (146-149). Both the type I and type II cotransporters are localized to the apical brush border membrane of PCT cells (150). Type III NaP~ cotransporters, originally identified as cell surface virus receptors (Glvr-1 and Ram-l), are widely expressed (151) and, like type I cotransporters, are not regulated by PTH or dietary phosphate (152). Type III cotransporters are expressed by DCT cells and thus could play a role in phosphate reabsorption in the distal nephron (153). Type II cotransporters are 80- to 90-kDa glycoproteins that are predicted to span the membrane eight times, with both their amino and carboxyl termini oriented into the cytosol (148). These cotransporters are electrogenic and transport Na + and HzPO 4 in a molar ratio of 3:1 (148). Expression and activity of the type II NaP i cotransporters (NaPe2, in rat) are strongly regulated by both parathyroid status and

Determination of the PTHR signals involved in mediating regulation of NaP i activity has been extensively studied. Early experiments in vivo or with isolated renal membranes indicated a role for cAMP-dependent actions of PTH in regulating phosphate excretion, based mainly on mimicry of the PTH effect by cAMP analogs or cAMP phosphodiesterase inhibitors (106,141,160). Many of these studies were conducted before the cAMP-independent signaling features of the PTHR were recognized (166). Analysis of this question in vitro has been pursued almost exclusively using the OK opossum kidney cell line, which expresses both the type II NaP i cotransporter and the type 1 PTHR (167) and manifests PTHdependent inhibition of NaP i cotransport along with other features typical of PCT cells (34,142,168-172). There is general agreement that direct pharmacologic activation of either PKA or PKC can inhibit NaPi activity in OK cells. The importance of the cAMP response of the PTHR was highlighted by experiments in which expression of a dominant-negative inhibitor of PKA (mutant PKA regulatory subunit gene) in OK cells completely blocked NaPi down-regulation by PTH (170) and by the demonstration that NaP~ regulation by PTH was unaffected when PLC/PKC activation was completely inhibited by the drug U73122 (34). On the other hand, a role for PKC is suggested by findings that NaP i activity can be at least partially regulated by PTH analogs, such

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CHAPTER14

Na+ Pi" I.! Na+llPi"

X-~)

PlasmaMembrane Submembranesomes

X/~k~

~ \Microtubules N

"q~'qk~ PTHrP PTH FIG. 4 Regulation of NaP~ cotransport by PTH. Activation of PTHRs on the basolateral membrane of PCT cells stimulates PKA and PKC. PKC induces a rapid decrease in activity of NaP~-2 transporters expressed on the apical surface, an effect that is mimicked by PTH(3-34). This may involve phosphorylation of one or more intermediary proteins (X), because consensus PKC phosphorylation sites within the NaP~-2 protein can be eliminated without affecting this regulatory effect of PKC. Activation of PKA also impairs NaP~ cotransport, but this effect is more delayed and involves retrieval of surface NaPe2 cotransporters by a microtubule-dependent process of endocytosis, lysosomal fusion, and degradation. The responsible PKA substrates and details of their actions currently are unknown (question marks).

as PTH(3-34), at concentrations that do not activate adenylyl cyclase or PKA but that do stimulate PKC (169,172-174). Moreover, NaP i regulation in OK cells may be observed at concentrations of PTH(1-34) that also activate PKC but are too low to measureably stimulate PKA, and NaP i regulation by PTH can be blocked by pharmacologic inhibition of PKC (29,169,175,176). In fact, the NaPi-2 protein can be phosphorylated, it contains several consensus sites for PKC, and its activity is inhibited by pharmacologic PKC activation when it is expressed in Xenopus oocytes (150,177). Because mutation of these sites does not interrupt PKC-dependent down-regulation of NaPi-2 activity, however, it is possible that PKC-dependent phosphorylation of other proteins, which act to regulate NaPi-2, may be the direct mediator of this effect (150,177). It also seems that PKA and PKC activation may lead to temporally and qualitatively distinct changes in NaPi-2 protein expression and activity (171,172) (Fig. 4). For example, PTH(3-34) initially inhibited NaP i activity comparably to PTH(1-34) but did so with no, or much less, induced clearance of the protein from the cell surface, which suggested that the main effect of PKC was to reduce the activity of the cotransporter directly or indirectly, whereas that of PKA may relate more to the physical removal of the protein from the apical membrane via endocytosis (171,172). Similarly, a study in intact rats showed no internal redistribution of membrane NaPi-2

protein in response to PTH(3-34) under conditions in which the peptide was shown to be bioactive, whereas PTH(1-34) provoked an 18% redistribution within 1 hour (163). On the other hand, direct pharmacologic activation of PKC caused membrane retrieval of NaPi-2 transporters expressed in Xenopus oocytes (178). Thus, a coherent view has yet to emerge in this area, but it seems reasonable to conclude at present that though activation of PKA and PKC via the PTHR each can separately downregulate NaPi activity, these kinases likely exert distinct regulatory effects, and activation of both may be necessary to achieve the full response to the hormone.

SODIUM AND HYDROGEN EXCRETION Studies in vivo and with isolated renal tubules in vitro have established that PTH produces an acute natriuresis and diuresis and rapidly inhibits proximal tubular acid secretion (HCO~ reabsorption) (105,179-182). As illustrated in Fig. 1, Na + reabsorption in the PCT proceeds via both the active, transcellular route and the passive, paracellular pathway. These mechanisms account for roughly 60 and 40%, respectively, of Na + reabsorption (183). Much of the transcellular Na + reabsorption in PCTs involves Na+-dependent cotransport of anions such as phosphate, sulfate, and amino acids or the operation of apical N a + / H + exchangers.

RENAL ACTIONS OV PTH AND PTHrP

PTH Regulation of Proximal Tubular Sodium and Hydrogen Excretion Effective reabsorption of Na ÷ and HCO~ in the proximal tubule requires the concerted activities of apical type 3 N a + / H + exchangers (NHE3s), basolateral Na+/K+-ATPases (to maintain the transmembrane Na + gradient), and electrogenic basolateral Na+-3HCO~ cotransporters, among others (184). PTH exerts at least three or four independent actions that conspire to powerfully inhibit Na + and HCO~ reabsorption. These include inhibition of apical N a + / H + exchange, apical Na+/Pi - cotransport, basolateral Na+/K+-ATPase activity, and, possibly, basolateral Na+-HCO~ cotransport (see Fig. 1). PTH strikingly inhibits the activity of the amiloridesensitive NHE3 in proximal tubular apical brush border membranes and in OK cells (185-188), directly impairing both Na + reabsorption and H + excretion. Conversely, parathyroidectomy increases NHE3 exchanger activity (189). The possibility that PTH may inhibit basolateral base exit via regulation of Na+-3HCO~ cotransporters is unsettled, because this has been observed in proximal tubules of rats (190) but not of rabbits (191). On the other hand, in vivo or in vitro ~administration of PTH greatly reduces the activity of the basolateral Na +/K+-ATPase in rat proximal tubules (46-48,192).

PTHR Signal Transduction in Regulation of Sodium and Hydrogen Excretion The mechanisms whereby PTHRs regulate these various effectors of proximal tubular Na + and H + excretion are both different and complex. Within 30-60 minutes of PTH exposure in vivo, NHE3 is phosphorylated and inactivated, after which it is sequestered (but not destroyed) via a more delayed internalization to a highdensity intracellular membrane fraction (163,186,193). Experiments in OK cells also indicate that PTH reduces the sensitivity of the exchanger to the intracellular H + concentration (194). Recent functional analysis of expressed recombinant NHE3 exchangers (195) supports previous evidence (106,185,191,196) that NHE3 is a direct substrate for PKA. Involvement of the cAMP/PKA signaling cascade in PTH regulation of NHE3 was suggested by the demonstration that PTH(1-34), but not a PTH(3-34) analog devoid of PKA activity, induced NHE3 internalization in rat proximal tubules (163). Also, PTHrP (1-34) inhibited NHE3 activity in an OK cell subclone in which this peptide could increase cAMP but not cytosolic Ca2i+, PLC, or PKC (197). On the other hand, others have obtained clear evidence, using both kinase inhibitors and signal-selective PTH analogs in OK cells, for involvement of both the PKA and PKC PTHR signaling pathways in NHE3

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235

regulation (36,39,185,188). By analogy with mechanisms of PTH-regulated PCT phosphate and DCT calcium excretion, it is likely that these two PTHR signal kinases exert cooperative but distinct effects in controlling NHE3 expression and activity. In the case of the basolateral Na+/K+-ATPase, analysis of PTH regulation has disclosed a novel pathway of PTHR signaling. Administration of PTH(1-34) in vivo causes a rapid inactivation of proximal tubular basolateral Na+/K+-ATPase activity without inducing destruction or sequestration of the pump proteins (163). In this case, PTH(3-34) does mimic the action of PTH(1-34) by activating PKC (not PKA) (47,192). This occurs via PTHR coupling to a Gq/G11 family member and leads to a series of further responses, which include activation of PLA 2, generation of arachidonic acid, and metabolism of arachidonate via the P450 monooxygenase pathway to produce active eicosanoids, notably 20-hydroxy-eicosatetraenoic acid (20-HETE) (46-48,192). In a manner as yet unknown, 20-HETE then leads to inhibition of Na+/K +ATPase activity (48,192). This monooxygenase-dependent pathway accounts for most of the PTH regulation of Na+/K+-ATPase activity, although a portion of the response is attributable to cAMP/PKA activation (46).

PTH Regulation of Sodium and Hydrogen Excretion beyond the Proximal Tubule Though it is true that PTH strongly inhibits proximal tubular HCO~ reabsorption, this is compensated to some extent by its effect to increase HCO~ reabsorption in Henle's loop and H + secretion in the CD (180,198,199). Moreover, the phosphaturia induced by PTH also contributes to net acid secretion (200), and PTH actually can increase net renal acid secretion during metabolic acidosis (201). Similarly, in perfused mouse CTAL, PTH may exert an antinatriuretic effect, manifested as augmented paracellular transport driven by an increased transepithelial voltage (202). Thus, the overall effect of PTH on renal acid and sodium excretion may vary markedly depending on the particular physiologic state of the organism.

VITAMIN D METABOLISM Synthesis of 1,25(OH)2D ~ is increased by PTH and reduced by parathyroidectomy (203). This results from regulated expression, in proximal tubular cells, of the 25(OH)Ds le~-hydroxylase gene, the promoter for which is rapidly induced by PTH in vitro (204-206). This effect of PTH can be overridden in vivo by the direct suppressive action of hypercalcemia on le~-hydroxylase expression (207). It is variably impaired in older animals or humans, even though indices of

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CI-IAPTWR14

PTHR signaling per se remain normal (208,209). PTH induction of l e~-hydroxylase mRNA is transcriptional, additive to that of calcitonin, occurs in the genetic absence of the vitamin D receptor, and is antagonized by coadministration of 1,25(OH)zD 3, which directly inhibits expression when given alone (206). The signaling pathways employed by the PTHR to increase 1,25(OH)zD 3 synthesis have been examined extensively in vivo and in vitro. Involvement of cAMP is suggested by the fact that the PTH effect can be mimicked by cAMP analogs, forskolin or phosphodiesterase inhibitors (210-216). Moreover, in a transformed murine proximal tubular cell line, transcriptional induction of the l oL-hydroxylase occurred with either PTH or forskolin, and the effects of both were blocked by the PKA-selective inhibitor H89 (206). On the other hand, careful studies of the effects of added PTH in isolated perfused rat proximal tubules have correlated rapid (30-60 minutes) increases in 1,25(OH)zD ~ synthesis with PKC activation on the basis of (1) concentration dependence (PKC and 1,25(OH)zD ~ syntheses were increased at PTH concentrations 100- to 1000-fold lower than required for PKA activation), (2) selective inhibition by PKC inhibitors, and (3) activation by truncated PTH analogs [i.e., PTH(3-34), PTH(13-34)] that can trigger PKC but not PKA in this system (32,217). More information clearly is needed, but available data seem most consistent with both a predominant effect of cAMP/PKA on transcriptional regulation of lo~-hydroxylase gene expression and a more rapid, posttransciptional effect of PKC on l e~-hydroxylase enzyme activity. The 25(OH)D 24-hydroxylase also is regulated by PTH. In kidney homogenates, cultured proximal tubular cells, and certain proximal tubular cell lines, PTH inhibits 24-hydroxylase activity by mechanisms that may involve cAMP (214,215,218-220). It also antagonizes the inductive effect of 1,25(OH)zD 3 on both 24-hydroxylase and vitamin D receptor expression (221). Interestingly, PTH leads to opposite effects on 24-hydroxylase and vitamin D receptor expression in proximal and distal tubules. Thus, PTH augments 1,25(OH)zD3-dependent induction of 24-hydroxylase in DCT cells, possibly by increasing expression of the vitamin D receptor (222), whereas it inhibits expression of both the 24-hydroxylase and the receptor in proximal tubules, as noted above.

O T H E R R E N A L EFFECTS OF P T H A variety of other effects of PTH on renal metabolism, secretion, and membrane function have been described, the physiologic roles of which currently are less clear than those described elsewhere in this chapter. Examples include rapid microvillar shortening in cultured proximal tubular cells (223); increased renin

release from perfused rat kidneys (224); increased proximal tubular gluconeogenesis, ammoniagenesis, and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression (225-227); activation of an apical C1- channel in rabbit proximal tubular cells (228); and stimulation of ecto-5'-nucleotidase activity in apical membranes of OK cells, an effect that is mimicked by PTH(3-34) but not by forskolin and which is blocked by PKC inhibitors (38).

RENAL EXPRESSION AND ACTIONS OF P T H r P PTHrP is expressed in the glomeruli, distal tubules, and collecting ducts of fetal kidneys and in PCTs, DCTs, and glomeruli of the adult kidney (229,230). In one study in rats, PTHrP mRNA was found in glomeruli, PCTs, and macula densa but not in CTAL, medullary thick ascending loop (MTAL), DCTs, or CDs (9). It is unlikely that PTHrP is critical for normal renal development, because the kidneys of mice missing functional PTHrP genes appear histologically normal. When tested, the active amino-terminal fragments of PTHrP generally exhibit renal actions identical to those of PTH, including stimulation of cAMP production and regulation of P~ transport, C a 2+ excretion, and 1,25(OH)zD ~ synthesis (99,231,232). On the other hand, longer PTHrP fragments may possess unique properties. For example, in an assay of HCO~ excretion by the perfused rat kidney, hPTHrP(1-34) was equipotent with hPTH(1-34), whereas hPTHrP(1-84), hPTHrP(1-108), and hPTHrP(1-141) each were less active than hPTH (1-34) (233). As discussed in Chapters 3 and 6, the PTHrP gene can generate multiple transcripts and protein products, some of which may undergo unique nuclear localization. It is quite possible, therefore, that locally expressed PTHrP may exert actions in the kidney that are not shared with PTH, although this has not yet been adequately addressed. A possible role for locally produced PTHrP in the renal response to ischemia has been suggested by findings that PTHrP expression is induced by ischemia or following recovery from ATP depletion (68,234,235). PTHrP is expressed in the intimal and medial layers of human renal microvessels and in the macula densa (236). PTHrP (like PTH) increases renin release from the juxtaglomerular apparatus and also stimulates cAMP in renal afferent and efferent arterioles, leading to vasodilation and enhanced renal blood flow (224,237-242). Evidence for involvement of both cAMP and nitric oxide in PTHrP-induced vasorelaxation in vitro has been derived from use of specific inhibitors (236). Thus, enhanced local PTHrP production induced by inadequate renal perfusion or ischemia may be involved in both local and systemic autoregula-

RENAL ACTIONS OF P T H AND P T H r P

tory mechanisms, whereby direct local vasodilatory actions are supplemented by systemic activation of angiotensinogen that increase arterial pressure and further sustain renal blood flow.

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17. Bettoun JD, Minagawa M, Hendy GN, et al. Developmental upregulation of human parathyroid hormone (PTH)/PTHrelated peptide receptor gene expression from conserved and human-specific promoters. J Clin Invest 1998;102:958-967. 18. Abou-Samra AB, Juppner H, Force T, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: A single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 1992;89:2732-2736. 19. Tomlinson S, Hendy GN, Pemberton DM, O'Riordan JL. Reversible resistance to the renal action of parathyroid hormone in man. Clin Sci Mol Med 1976;51:59-69. 20. McElduff A, Lissner D, Wilkinson M, Cornish C, Posen S. A 6hour human parathyroid hormone (1-34) infusion protocol: Studies in normal and hypoparathyroid subjects. CalcifTissue Int 1987;41:267-273. 21. Bellorin-Font E, Lopez C, Diaz K, Pernalete N, Lopez M, Starosta R. Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone. Kidney Int 1995;47:38-44. 22. Meltzer V, Weinreb S, Bellorin-Font E, Hruska KA. Parathyroid hormone stimulation of renal phosphoinositide metabolism is a cyclic nucleotide-independent effect. Biochim Biophys Acta 1982;712:258-267. 23. Henry HL, Cunningham NS, Noland TA, Jr. Homologous desensitization of cultured chick kidney cells to parathyroid hormone. Endocrinology 1983;113:1942-1949. 24. Teitelbaum AP, Strewler GJ. Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line. Endocrinology 1984;114:980-985. 25. Goligorsky MS, Loftus DJ, Hruska KA. Cytoplasmic calcium in individual proximal tubular cells in culture. Am J Physiol 1986;251 :F938-F944. 26. Hruska KA, Goligorsky M, Scoble J, Tsutsumi M, Westbrook S, Moskowitz D. Effects of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures. Am J Physiol 1986;251:F188-F198. 27. Hruska KA, Moskowitz D, Esbrit P, Civitelli R, Westbrook S, Huskey M. Stimulation of inositol trisphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone. J Clin Invest 1987;79:230-239. 28. Tamura T, Sakamoto H, Filburn CR. Parathyroid hormone 1-34, but not 3-34 or 7-34, transiently translocates protein kinase C in cultured renal (OK) cells. Biochem Biophys Res Commun 1989;159:1352-1358. 29. Quamme G, Pfeilschifter J, Murer H. Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: Generation of second messengers in the regulatory cascade. Biochem Biophys Res Commun 1989;158:951-957. 30. Coleman DT, Bilezikian JE Parathyroid hormone stimulates formation of inositol phosphates in a membrane preparation of canine renal cortical tubular cells. J Bone Miner Res 1990;5:299-306. 31. Nemani R, Wongsurawat N, Armbrecht HJ. Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates. Arch Biochem Biophys 1991 ;285:153-157. 32. Janulis M, Tembe V, Favus MJ. Role of protein kinase C in parathyroid hormone stimulation of renal 1,25-dihydroxyvitamin D 3 secretion. J Clin Invest 1992;90:2278-2283. 33. Bringhurst FR, Juppner H, Guo J, et al. Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology 1993;132:2090-2098. 34. Martin KJ, McConkey CL, Jacob AK, Gonzalez EA, Khan M, Baldassare JJ. Effect of U-73,122, an inhibitor of phospholipase

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178. Forster I, Traebert M, Jankowski M, Stange G, Biber J, Murer M. Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes. J Physiol 1999;517:327-340. 179. Schneider EG. Effect of parathyroid hormone secretion on sodium reabsorption by the proximal tubule. Am J Physiol 1975;229:1170-1173. 180. Bank N, Aynediian HS. A micropuncture study of the effect of parathyroid hormone on renal bicarbonate reabsorption. J Clin Invest 1976;58:336-344. 181. PuschettJB, Zurbach P, Sylk D. Acute effects of parathyroid hormone on proximal bicarbonate transport in the dog. Kidney Int 1976;9:501-510. 182. McKinney TD, Myers E Bicarbonate transport by proximal tubules: Effect of parathyroid hormone and dibutyryl cyclic AME Am J Physio11980;238:F166-F174. 183. Rector E Sodium, bicarbonate, and chloride reabsorption by the proximal tubule. AmJPhysio11983;244:F461-F471. 184. Mpern RJ. Cell mechanisms of proximal tubular acidification. Physiol Rev 1990;70:79-114. 185. Kahn AM, Dolson GM, Hise MK, Bennett SC, Weinman EJ. Parathyroid hormone and dibutyryl cAMP inhibit Na+/H + exchange in renal brush border vesicles. Am J Physiol 1985 ;248:F212-F218. 186. Hensley CB, Bradley ME, Mircheff AK. Parathyroid hormoneinduced translocation of Na-H antiporters in rat proximal tubules. Am J Physio11989;257:C637-C645. 187. Pollock AS, Warnock DG, Strewler GJ. Parathyroid hormone inhibition of Na+-H + antiporter activity in a cultured renal cell line. Am J Physiol 1986;250:F217-F225. 188. Helmle-Kolb C, Montrose MH, Murer H. Parathyroid hormone regulation of Na+/H + exchange in opossum kidney cells: Polarity and mechanisms. Pfluegers Arch 1990;416:615-623. 189. Cohn DE, Klahr S, Hammerman MR. Metabolic acidosis and parathyroidectomy increase Na+/H + exchange in brush border vesicles. A m J Physiol 1983;245:F217-F222. 190. Pastoriza-Munoz E, Harrington RM, Graber ML. Parathyroid hormone decreases HCOs reabsorption in the rat proximal tubule by stimulating phosphatidylinositol metabolism and inhibiting base exit. J Clin Invest 1992;89:1485-1495. 191. Sasaki S, Marumo E Mechanisms of inhibition of proximal acidification by PTH. AmJPhysio11991 ;260:F833-F838. 192. Ominato M, Satoh T, Katz M. Regulation of Na-K-ATPase activity in the proximal tubule: Role of the protein kinase C pathway and of eicosanoids. J Membr Bio11996;152:235-243. 193. Fan L, Wiederkehr MR, Collazo R, Wang H, Crowder LA, Moe OW. Dual mechanisms of regulation of N a / H exchanger NHE3 by parathyroid hormone in rat kidney. J Biol Chem 1999;274:11289-11295.

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194. Miller RT, Pollock AS. Modification of the internal pH sensitivity of the Na+/H + antiporter by parathyroid hormone in a cultured renal cell line. JBiol Chem 1987;262:9115-9120. 195. Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, Moe OW. Acute inhibition of N a / H exchanger NHE-3 by cAME Role of protein kinase a and NHE-3 phosphoserines 552 and 605. J Biol Chem 1999;274:3978-3987. 196. Weinman EJ, Dubinsky WP, Shenolikar S. Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H + exchanger. J Membr Biol 1988;101:11-18. 197. Maeda S, Wu S, Green J, et al. The N-terminal portion of parathyroid hormone-related protein mediates the inhibition of apical Na+/H + exchange in opossum kidney cells. J Am Soc Nephrol 1998;9:175-181. 198. Bichara M, Mercier O, Paillard M, Leviel E Effects of parathyroid hormone on urinary acidification. Am J Physiol 1986;251 :F444-F453. 199. Paillard M, Bichara M. Peptide hormone effects on urinary acidification and acid-base balance: PTH, ADH, and glucagon. Am J Physiol 1989;256:F973-F985. 200. Mercier O, Bichara M, Paillard M, Prigent A. Effects of parathyroid hormone and urinary phosphate on collecting duct hydrogen secretion. AmJPhysiol 1986;251 :F802-F809. 201. Bichara M, Mercier O, Borensztein P, Paillard M. Acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat. j Clin Invest 1990;86:430-443. 202. Wittner M, Di Stefano A. Effects of antidiuretic hormone, parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron. Pfluegers Arch 1990;415:707-712. 203. Fraser DR, Kodicek E. Regulation of 25-hydroxycholecalciferol1-hydroxylase activity in kidney by parathyroid hormone. Nature 1973;241:163-166. 204. Garabedian M, Holick ME Deluca HE Boyle IT. Control of 25hydroxycholecalciferol metabolism by parathyroid glands. Proc Natl Acad Sci USA 1972;69:1673-1676. 205. Kong XE Zhu XH, Pei YL, Jackson DM, Holick ME Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin Dflalpha-hydroxylase gene. Proc Natl Acad Sci USA 1999;96:6988-6993. 206. Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D~ lalpha-hydroxylase gene by parathyroid hormone, calcitonin, and lalpha,25(OH)zD 3 in intact animals. Endocrinology 1999;140:2224-2231. 207. WeisingerJR, Favus MJ, Langman CB, Bushinsky DA. Regulation of 1,25-dihydroxyvitamin D3 by calcium in the parathyroidectomized, parathyroid hormone-replete rat. J Bone Miner Res 1989;4:929-935. 208. FriedlanderJ, Janulis M, Tembe V, Ro HK, Wong MS, Favus MJ. Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D 3 production in aging does not involve protein kinase A or C pathways. J Bone Miner Res 1994;9:339-345. 209. Halloran BE Lonergan ET, Portale AA. Aging and renal responsiveness to parathyroid hormone in healthy men. J Clin Endocrinol Metab 1996;81:2192-2197. 210. Larkins RG, MacAuley SJ, Rapoport A, et al. Effects of nucleotides, hormones, ions, and 1,25-dihydroxycholecalciferon on 1,25-dihydroxycholecalciferol production in isolated chick renal tubules. Clin Sci Mol Med 1974;46:569-582. 211. Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E. In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of lalpha,25-dihydroxyvitamin D~ synthesis in rats. Endocrinology 1977;101:969-974. 212. Rost CR, Bikle DD, Kaplan RA. In vitro stimulation of 25hydroxycholecalciferol lalpha-hydroxylation by parathyroid

213.

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223.

224.

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226.

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hormone in chick kidney slices: Evidence for a role for adenosine 3',5'-monophosphate. Endocrinology 1981;108:1002-1006. Armbrecht HJ, Forte LR, Wongsurawat N, Zenser TV, Davis BB. Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro. Endocrinology 1984;114:644-649. Henry HL. Parathyroid hormone modulation of 25-hydroxyvitamin D~ metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology 1985;116:503-510. Shigematsu T, Horiuchi N, Ogura Y, Miyahara T, Suda T. Human parathyroid hormone inhibits renal 24-hydroxylase activity of 25hydroxyvitamin Ds by a mechanism involving adenosine 3',5'monophosphate in rats. Endocrinology 1986;118:1583-1589. Korkor AB, Gray RW, Henry HL, Kleinman JG, Blumenthal SS, Garancis JC. Evidence that stimulation of 1,25(OH)2D 3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis. JBone Miner Res 1987;2:517-524. Janulis M, Wong MS, Favus MJ. Structure-function requirements of parathyroid hormone for stimulation of 1,25dihydroxyvitamin D3 production by rat renal proximal tubules. Endocrinology 1993;133:713-719. Tanaka Y, DeLuca HE Rat renal 25-hydroxyvitamin D~ 1- and 24-hydroxylases: Their in vivo regulation. Am J Physiol 1984;246:E 168-E 173. Matsumoto T, Kawanobe Y, Ogata E. Regulation of 24,25dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12). Biochim Biophys Acta 1985;845:358-365. Shinki T, Jin CH, Nishimura A, et al. Parathyroid hormone inhibits 25-hydroxyvitamin D~-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine. J Biol Chem 1992;267:13757-13762. Reinhardt TA, Horst RL. Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo. Endocrinology 1990;127:942-948. Yang W, Friedman PA, Kumar R, et al. Expression of 25(OH)Ds 24-hydroxylase in distal nephron: Coordinate regulation by 1,25(OH)zD 3 and cAMP or PTH. AmJPhysio11999;276:E793-E805. Goligorsky MS, Menton DN, Hruska KA. Parathyroid hormoneinduced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells. J Membr Biol 1986;92:151-162. Saussine C, Massfelder T, Parnin F, Judes C, Simeoni U, Helwig JJ. Renin stimulating properties of parathyroid hormone-related peptide in the isolated perfused rat kidney. Kidney Int 1993;44:764-773. Wang MS, Kurokawa K. Renal gluconeogenesis: Axial and internephron heterogeneity and the effect of parathyroid hormone. AmJPhysiol 1984;246:F59-F66. Chobanian MC, Hammerman MR. Parathyroid hormone stimulates ammoniagenesis in canine renal proximal tubular segments. A m J Physiol 1988;255:F847-F852. Watford M, Mapes RE. Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney. Arch Biochem Biophys 1990;282:399-403. Suzuki M, Morita T, Hanaoka K, Kawaguchi Y, Sakai O. A C1channel activated by parathyroid hormone in rabbit renal proximal tubule cells. J Clin Invest 1991;88:735-742. Philbrick WM, Wysolmerski JJ, Galbraith S, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996;76:127-173. Aya K, Tanaka H, Ichinose Y, Kobayashi M, Seino Y. Expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney. Kidney Int 1999;55:1696-1703. Yates AJ, Gutierrez GE, Smolens P, et al. Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium

RENAL ACTIONS OF P T H AND P T H r P

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homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 1988;81:932-938. Pizurki L, Rizzoli R, MoseleyJ, Martin TJ, Caverzasio J, Bonjour JE Effect of synthetic tumoral PTH-related peptide on cAMP production and Na-dependent Pi transport. Am J Physiol 1988;255:F957-F961. Ellis AG, Adam WR, Martin TJ. Comparison of the effects of parathyroid hormone (PTH) and recombinant PTH-related protein on bicarbonate excretion by the isolated perfused rat kidney. JEndocrinol 1990;126:403-408. Soifer NE, Van Why SK, Ganz MB, Kashgarian M, Siegel NJ, Stewart AF. Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia. J Clin Invest 1993;92:2850-2857. Garcia-Ocana A, Galbraith SC, Van Why SK, et al. Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion. J A m Soc Nephro11999;10:238-244. Massfelder T, Stewart AF, Endlich K, Soifer N, Judes C, Helwig JJ. Parathyroid hormone-related protein detection and interaction with NO and cyclic AMP in the renovascular system. Kidney Int 1996;50:1591-1603.

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237. Schor N, Ichikawa I, Brenner BM. Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat. Kidney Int 1981;20:442-451. 238. Nickols CA, Metz MA, Cline WH, Jr. Endothelium-independent linkage of parathyroid hormone receptors of rat vascular tissue with increased adenosine 3',5'-monophosphate and relaxation of vascular smooth muscle. Endocrinology 1986;119:349-356. 239. Musso MJ, Barthelmebs M, Imbs JL, Plante M, Bollack C, Helwig JJ. The vasodilator action of parathyroid hormone fragments on isolated perfused rat kidney. Naunyn-Schmiedeberg'sArch Pharmacol 1989;340:246-251. 240. HelwigJJ, Musso MJ,Judes C, Nickols GA. Parathyroid hormone and calcium: Interactions in the control of renin secretion in the isolated, nonfiltering rat kidney. Endocrinology 1991;129:1233-1242. 241. Simeoni U, Massfelder T, Saussine C, Judes C, GeisertJ, Helwig JJ. Involvement of nitric oxide in the vasodilatory response to parathyroid hormone-related peptide in the isolated rabbit kidney. Clin Sci 1994;86:245-249. 242. Endlich K, Massfelder T, Helwig JJ, Steinhausen M. Vascular effects of parathyroid hormone and parathyroid hormonerelated protein in the split hydronephrotic rat kidney. J Physiol 1995;483:481-490.

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CHAPTER 15

Endochondral

Bone Formation

Regulation by Parathyroid Hormone-Related Peptide, Indian Hedgehog, and Parathyroid Hormone GINO V. SEGRE AND KAECHOONG LEE Endocrine Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical School,

Boston, Massachusetts 02114

ENDOCHONDRAL

BONE DEVELOPMENT

Spatially, chondrocyte differentiation in the growth plate is oriented longitudinally (Fig. 1). The proliferating zone at the distal end of the developing bone consists of small, round chondrocytes that have a high

Development of the vertebrate skeleton is a highly regulated process that takes place by at least two distinct mechanisms: intramembranous and endochondral ossification. The axial and appendicular skeletons develop by endochondral ossification; membranous bones are largely restricted to the skull and parts of the mandible and clavicles. Membranous bones form directly from mesenchymal progenitors, whereas endochondral bones form after cartilaginous primordia are first generated. These primordia consist of aggregated undifferentiated mesenchymal cells, whose position, shape and size prefigure the future skeletal elements. Cells in the central cores of these condensates differentiate into chondrocytes, as evidenced by their synthesis and secretion of cartilage matrix proteins, such as type II collagen and specific proteoglycans, and the cartilage model is surrounded by a sheath of apparently poorly differentiated cells, the perichondrium. Proliferation of cells within the condensates and the perichondrium, and deposition of matrix proteins, account for the initial growth of these skeletal elements. Cells in the center mature into hypertrophic cells, a process characterized by cellular enlargement, the exit of these cells from the cell cycle, and the secretion and deposition of a distinct extracellular matrix that progressively calcifies. Generally cartilage differentiation and replacement by bone occur earlier in proximal bones of the appendicular skeleton than in distal bones, and they progress from cephalic to caudal portions of the axial skeleton. The Parathyroids, Second Edition

'roliferation -lypertrophy Vlineralization 31ood Vessel Invasion Primary Spongiosa

Secondary Spongiosa Bone Marrow (Hemopoietic cells)

FIG. 1 A schematic representation of the growth plate. Chondrocytes proliferate, mature, and organize into columns, and undergo hypertrophic differentiation. As the hypertrophic chondrocytes undergo programmed cell death, the cartilage matrix is invaded by blood vessels and is then resorbed by chondroclasts. The remnant longitudinal septae provide the scaffolding for osteoblasts to lay down bone matrix to form the primary spongiosa. Differentiation of growth plate chondrocytes drives the longitudinal growth of bone.

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capacity to divide. These cells mature and assume a columnar architecture, each column representing the expansion of a single clone. Examination of the distribution of bromodeoxyuridine (BrdU) in fetuses, after BrdU was given shortly before delivery to their mothers, first showed this clonal expansion (1). This has been confirmed by elegant lineage studies in which the distribution of [3-gal-expressing cells in the growth plate of chimeric mice was determined after introducing embryonic stem cells transfected with this gene into blastocysts from normal mice (2). The columnar appearance and clonal distribution are maintained in the hypertrophic zone, although the cells no longer divide. The matrix surrounding the maturest hypertrophic cells becomes mineralized, the cells undergo p r o g r a m m e d cell death, or apoptosis, and are replaced by bone. Although evidence suggests that hypertrophic cells may "transdifferentiate" into osteoblast-like cells, this must involve no more than a small minority of hypertrophic cells, if it occurs (3). The net result is lengthening of the bone, whereas the thickness of the growth plate remains relatively constant. With continued bone lengthening, proliferating chondrocytes and the growth plate become progressively restricted to the two ends of the skeletal element. The processes by which the cortices of axial and appendicular bones develop are called appositional ossification. Cortical bone formation in the shafts of endochondral bone initiates in perichondrial/ periosteal cells, directly apposed to hypertrophic cells. These cells differentiate into osteoblasts and secrete a matrix that undergoes calcification, giving rise to the "bone collar." Because appositional ossification is morphologically similar to intramembranous ossification, it

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Type X Collagen

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has been thought to develop by the same mechanisms. Studies show, however, that different factors, to be reviewed below, are involved at least in the initial stages of appositional ossification. Although chondrocyte differentiation can be seen to progress longitudinally, the growth plate is spatially organized into morphologically distinct horizontal z o n e s ~ z o n e s of proliferation, maturation, and h y p e r t r o p h y ~ e a c h of which is heterogeneous, as evidenced by the genes expressed in each zone. For example, parathyroid hormone-related protein (PTHrP) mRNA is most intensely expressed in subarticular chondrocytes. H4 histone mRNA, a proliferation marker that identifies cells at the S phase of their cell cycle, is expressed in these cells, but also in more centrally located chondrocytes that express PTHrP at much lower levels. P T H / P T H r P receptor mRNA and protein, although expressed at low levels in proliferating chondrocytes, are most intensely localized to cells at the interface of columnar and hypertrophic cells in prehypertrophic and early hypertrophic chondrocytes. Indian hedgehog (Ihh) mRNA expression partially overlaps that of the P T H / P T H r P receptor, but extends to more mature hypertrophic cells as well. Type II collagen is expressed throughout the growth plate, except in hypertrophic cells. As cells become hypertrophic, they abruptly lower or cease their expression of type II collagen. All hypertrophic cells, except the maturest, express types VI and X collagen. Terminally differentiated chondrocytes in and near the zone of mineralization, most of which are apoptotic, intensely express osteopontin and vascular endothelial growth factor (VEGF) (4-6) (Fig. 2). This "spatial synchronization" integrates the individual chondrocyte clones within the PTH/PTHrP Receptor

FIG. 2 Differentiation of growth plate chondrocytes. (A) Distribution of proliferation/differentiation markers in the growth plate (fetal mouse femur, embryonic day 15.5). Growth plate chondrocytes, except for hypertrophic cells, express type II collagen. When chondrocytes become hypertrophic, they express type X collagen and exit from the cell cycle, as evidenced by the disappearance of H4histone expression, a marker for the S phase of the cell cycle. Transcripts for PTH/PTHrP receptors are most highly expressed at the boundary between the proliferating and hypertrophic zones. H & E, Hematoxylin and eosin. (B) Distribution of PTHrP mRNA (a, mouse fetal hindlimb, embryonic day 16.5). PTHrP is expressed in the subarticular perichondrium/chondrocytes and in tendon insertions (b, arrowheads). [Modified from Ref. (5); Lee K, Lanske B, Karaplis AC, Deeds JD, Kohno H, Nissenson RA, Kronenberg HM, Segre GV. Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 137: 5109-5118; 1996. © The Endocrine Society.]

ENDOCHONDRALBONE FORMATION / growth plate, suggesting that chondrocyte differentiation is regulated by enzymes, signaling, and, perhaps, matrix molecules that are secreted by cells within highly defined regions in the growth plate. Work from Werb's laboratory provides an excellent example of a synchronized program by which local factors drive critical events in the process by which bone replaces cartilage. They found that homozygous mice with a null mutation in the matrix metalloproteinase-9 (MMP-9)/gelatinase B gene have lengthening of the hypertrophic zone and exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive overall lengthening of the growth plate. Transplantation of wild-type bone

marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by cells of bone marrow origin that mature to become chondroclasts that secrete MMP-9/gelatinase B (7). In subsequent studies, Werb's group systemically administered a soluble VEGF receptor chimeric protein, Flt (1-3)-IgG, to young mice. This completely blocks blood vessel invasion of the cartilage, which concomitantly leads to expansion of the hypertrophic chondrocyte zone, impaired trabecular bone formation, and an overall shorter bone. Recruitment a n d / o r differentiation of chondroclasts and resorption of terminal chondrocyes decrease, although proliferation, differentiation, and maturation of chondrocytes are apparently normal (Fig. 3). VEGFmediated capillary invasion is an essential signal that

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FIG. 3 .MMP-9/gelatinase B and VEGF play crucial roles in the replacement of cartilage by bone. (A) Histologic analysis of the metatarsals of wild-type (upper panels) and MMP-9/gelatinase B ( - / - ) mice (lower panels) (sections of 3-week-old metatarsals). Magnified view (right lower panel) shows cortical bone surrounding the hypertrophic cartilage (hc) in MMP-9/gelatinase B ( - / - ) mice (boundaries are indicated by white arrowheads). Black arrowheads point to capillaries invading from the cortical bone; tb, trabecular bone. (B) Growth plates of MMP-9/gelatinase B ( - / - ) mice after bone marrow transplantation. Histologic sections of metatarsals from untransplanted and bone marrow (BM)-transplanted mice at 3 and 4 weeks after transplantation. Scale bar = 200 i~m. (C) Histologic sections of proximal tibia of mice treated for 2 weeks with control IgG (cont) (a,c) or a soluble VEGF receptor chimeric protein, mFIt(1-3)-IgG (mFIt) (hematoxylin and eosin staining) (b,d). hcz, Hypertrophic chondrocyte zone; stb, secondary trabecular bone; bm, bone marrow. Blood vessels are oriented parallel to the columns of hypertrophic zone in control mice, and fail to invade the hcz in mFIt-treated groups. Arrowheads indicate blood vessels. [Modified from Ref. (7), Cell 93; Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z; MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes, pp. 411-422. Copyright 1998, with permission from Elsevier Science; and from Gerber et aL (6), with permission.]

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regulates growth plate morphogenesis and triggers cartilage remodeling. VEGF, secreted by terminal hypertrophic chondrocytes, recruits endothelial cells and thus induces blood vessels. These blood vessels bring in not only nutrients, but also cells that become chondroclasts and osteoblasts. Because osteoblasts express Fltl, a VEGF receptor, and VEGF is chemotactic for osteoblasts in culture, it is reasonable to postulate an essential role for VEGF in recruiting osteoblasts to regions where terminal hypertrophic chondrocytes that were shown to express VEGF reside (6). Thus, VEGF is an essential coordinator of chondrocyte death, chondrocyte function, extracellular matrix remodeling, and angiogenesis, and it plays a vital role in the transition from cartilage to bone in the growth plate. Interestingly, mineralization of the matrix surrounding the maturest hypertrophic chondrocytes is unimpaired, indicating that cartilaginous mineralization is regulated by VEGF-independent mechanisms. Osteoblast precursors are attracted by VEGF secreted by hypertrophic cells, and after they mature into osteoblasts, they deposit bone matrix proteins, or osteoid, on the longitudinal septae, the vertical cartilaginous remnants left behind after apoptosis of the hypertrophic cells. This region of the primary ossification center is called the primary spongiosa. Resorption of the cartilaginous matrix by chondroclasts/osteoclasts continues, at least in part due to secretion by these cells of MMP-9/gelantinase B, and the bone matrix proteins elaborated by osteoblasts replace the cartilaginous matrix. When the longitudinal septae contain only bone matrix proteins from which all cartilaginous matrix has been removed, this area is called the secondary spongiosa. It consists of trabecular bone, which is remodeled by processes of bone resorption and subsequent formation throughout life to meet the demands of continued longitudinal growth, and in response to a variety of hormones, factors, and physical forces. The net result is that chondrocyte proliferation and hypertrophy largely drive longitudinal growth of endochondral bone. Interestingly, additional data suggest that at least postnatally, the activity of bone cells also may contribute to bone elongation (8). Many chondrodysplasias result from aberrant endochondral ossification. However, compared to chondrodysplasias caused by mutations in structural molecules, chondrodysplasias caused by mutations in regulatory molecules are relatively few (9). In this chapter, we concentrate on the roles of parathyroid hormone-related protein, parathyroid hormone, and Indian hedgehog, which by their dependent and independent actions play key roles in skeletal development. Our understanding of the roles these factors play in endochondral development has been greatly advanced by several novel approaches. These include determining the genetic basis of h u m a n chondrodysplasias and

deleting or overexpressing these genes in mice to study the development of skeletal abnormalities, and from studies conducted in vitro with fetal limbs from wildtype and mutant mice in which endochondral bone development can be manipulated. For those interested in roles of matrix molecules in endochondral bone development, excellent reviews (10,11) are available.

T H E A C T I O N S OF P T H r P A N D P T H

PTHrP was originally identified as a causative agent of hypercalcemia associated with malignancy (12). Both PTHrP and PTH bind to and activate the same receptor (13,14). PTH functions as a hormone to regulate the blood calcium concentration. PTHrP, however, functions mainly as a paracrine factor, although elevated circulating levels resulting from its synthesis and secretion by neoplastic tissue cause tumor-associated hypercalcemia. Its most well-defined physiologic roles are to regulate branching morphogenesis of the breast (15) and in endochondral bone formation. It is also thought to have more general developmental roles because its distribution and that of its cognate receptor are apposed across many epithelial-mesenchymal interfaces (5). Mutations in the P T H / P T H r P receptor cause two rare chondrodysplastic syndromes, thus providing insight into critical developmental roles played by this receptor. Jansen's metaphyseal chondrodysplasia is a rare form a short-limbed dwarfism characterized by hypercalcemia, normal or low circulating levels of PTH and PTHrP, and progressive metaphyseal changes, initially reminiscent of rickets or primary hyperparathyroidism (16). Elegant studies by Schipani et al. (17,18) linked this phenotype to mutant P T H / P T H r P receptors that were then showed to be constitutively active, when expressed in COS-7 cells. In these mutant receptors, histidine at position 223 is replaced by arginine, putatively at the border of the receptor's first intracellular loop and second transmembrane domain, or threonine at position 410 is replaced by proline in the receptor's sixth transmembrane helix. The phenotype of patients with Blomstrand chondrodysplasia, a lethal syndrome characterized by shortlimbed dwarfism, increased bone density, and remarkably advanced endochondral bone formation, is the mirror image of those in Jansen's chondrodysplasia (19). Blomstrand chondrodysplasia has been shown to be due to absence of functional P T H / P T H r P receptors. In their initial report, Jobert et al. (20) showed the syndrome to be due to a point mutation that leads to an l 1-amino acid deletion in the P T H / P T H r P receptor's fifth transmembrane domain. The same workers (21) and a second group (22) found the same phenotype to be caused by replacement of proline by leucine at position 132 of the receptor's amino-terminal extracellular

ENDOCHONDRAL BONE FORMATION / domain. Both mutations render the receptor unable to bind PTH or PTHrE The phenotypes of mice missing both copies of either the PTHrP or P T H / P T H r P receptor gene have many similarities, but phenotypic differences allow discrimination between characteristics due to absence of PTHrP from those due to failure to respond to either PTH or PTHrE Deletion of both copies of either gene leads to short-limbed dwarfism with an apparently accelerated differentiation of growth plate chondrocytes. Additionally, there is diminished polarity of hypertrophic chondrocytes, in which these cells are widely distributed throughout the cartilage (Fig. 4). This is particularly evident immediately before vascular invasion and the development of the primary ossification center of the developing bone (23-27). P T H / P T H r P receptor-deficient mice, however, exhibit two unique phenotypes not shared with the PTHrP-deficient mice. During appositional bone formation in the shafts of the long bones, only P T H / P T H r P receptor-deficient mice exhibit a dramatic increase in osteoblast n u m b e r and matrix accumulation (Fig. 5). Furthermore, the P T H / P T H r P receptor-deficient mice show a dramatic decrease in trabecular bone formation in the primary spongiosa

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and a delay in vascular invasion of the early cartilage model. The abnormal bone development is likely to result from the inability of the bone to respond to the actions of PTH, and is the expected opposite to that seen in clinical mild hyperparathyroidism. The delayed vascular invasion did not occur in PTHrP; P T H / P T H r P receptor doubly deficient mice, suggesting that PTHrP, perhaps through a distinct receptor or through its direct nuclear actions, slows vascular invasion (27). The role of PTHrP and its receptor in chondrocyte differentiation also has been explored in overexpression models: mice that express a transgene encoding either PTHrP or a constitutively active '~Jansen receptor" u n d e r the control of the rat collagen e~l (type II) promoter manifest an apparent delay of chondrocyte differentiation (28,29). Moreover, overexpression of the latter corrected at birth the growth plate abnormalities of the PTHrP-deficient mice (29). Treating murine limb in explant cultures with PTH or PTHrP mimics the apparent delay of chondrocyte differentiation (30). Cell-cell interactions in the developing endochondral bone have been elegantly addressed using chimeric mice that express various ratios of mutant and wild-type cells. In the growth plate of these

FIG. 4 Cartilage phenotype in PTHrP ( - / - ) and PTH/PTHrP receptor ( - / - ) mice. Hematoxylin and eosin staining (a-c) and type X collagen mRNA in situ hybridization (d-f) of wild-type (a, d), PTH/PTHrP receptor ( - / - ) (b, e), and PTHrP ( - / - ) (c, f) phalanges at embryonic day 16.5. There is a loss of polarity of the cartilage in both PTHrP ( - / - ) bone and PTH/PTHrP receptor ( - / - ) bones. In addition, PTH/PTHrP receptor ( - / - ) mice show a delay in chondrocytic differentiation. [Reprinted from Lanske et aL (27), with permission.]

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chimeric mice, chondrocytes missing the P T H / PTHrP receptor gene ectopically differentiate into hypertrophic cells and are surrounded by wild-type proliferating cells. Essentially all mutant chondrocytes became hypertrophic below a line approximately corresponding to the level at which the growth plate narrows, not at the level at which P T H / PTHrP receptor mRNA and protein are most intensely expressed (Fig. 6) (2). These ectopic cells express genes typical of hypertrophic chondrocytes; they also cease to proliferate and undergo apoptosis. Thus, the specific targets of PTHrP signaling are proliferative chondrocytes that express low levels of receptors. Differentiation of the PTH/PTHrPdeficient cells occurs independently of the surrounding normal proliferative cells and thus is cell autonomous. Interestingly, PTHrP protein localize most intensely to prehypertrophic cells that also express P T H / P T H r P receptors most abundantly (26). Cartilage mineralization, one of the final steps of chondrocyte differentiation, occurs ectopicially around hypertrophic cells at sites in the cartilage of chimeric mice with more than 60% contribution of mutant cells. Matrix mineralization, therefore, seems to require a critical mass of hypertrophic cells and thus is not cell autonomous.

FIG. 5 Increase in osteoblast number and cortical bone in PTH/PTHrP receptor ( - / - ) animals, von Kossa staining of methylmethacrylate sections at the level of the metaphyseal region of a tibia (a-c) and at the diaphyseal region of a phalanx (d-f)in wildtype (a, d), PTH/PTHrP receptor ( - / - ) (b, e), and PTHrP ( - / - ) (c, f) animals at embryonic day 18.5. PTH/PTHrP receptor mutant bones reveal an abnormal augmentation in osteoblast layers accompanied by an increased bone matrix (b, e) that does not mineralize, as demonstrated by the lack of von Kossa staining. In contrast, PTHrP ( - / - ) bones (c, f) look indistinguishable or somewhat advanced in terms of mineralization and replacement of cartilage by bone when compared with wild-type bones (a, d). [Reprinted from Lanske et aL, (27), with permission.]

THE ACTIONS OF INDIAN HEDGEHOG The hedgehog (hh) gene was first identified in

Drosophila as a segment polarity gene. Among the five reported vertebrate hh homologs, most attention has focused on Sonic hedgehog (Shh), which is absolutely critical to the patterning and development of many organs and organ systems in vertebrates. Shh is synthesized in cells in the notochord and controls development of the ventral neural tube (32-34). It also controls specification of sclerotomal cell fate in the somites (35), and the anteroposterior axis of the limb bud (36). It plays an important role in mediating epithelial-mesenchymal signaling in the gut (37), and in establishing left-fight asymmetry in the heart (38,39). The critical role of Shh in tissue patterning is made apparent by the multiple developmental defects in Shh-deficient mice; they die before birth and exhibit cyclopia, lack ventral cells in the neural tube, and have a degenerate notochord. They also lack a spinal column and most of the ribs and distal limb structures (40). Other members of the hedgehog protein family have also been implicated in tissue patterning: Desert hedgehog (Dhh) was shown to be essential for testes development, and zebra-fish-specific Echidna and Tiggywinkle hedgehog are involved in muscle cell specification and eye patterning, respectively (41-43).

ENDOClqOND~U~ BONE FORMATION /

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!i?iiiiiii ili:iiill iii! FI6. 6 Ectopic differentiation of PTH/PTHrP receptor ( - / - ) chondrocytes and elongation of chimeric growth plates. (A, B) Sections of the tibiae from embryonic day 17.5 wild-type embryos (A) and embryonic day 17.5 wild-type embryos containing cells with one 13-galactosidase transgene (stained dark) and cells with no transgene (B) were stained for 13-galactosidase activity and counterstained with nuclear fast red. Cells with and without the transgene behave indistinguishably, p, Proliferating layer; h, hypertrophic layer, including prehypertrophic chondrocytes. (C, D) Sections of the tibiae from embryonic day 17.5 chimeric embryos containing both wild-type and PTH/PTHrP receptor ( - / - ) cells were stained for 13-galactosidase activity and counterstained with nuclear fast red. PTH/PTHrP receptor ( - / - ) cells (stained dark) ectopically differentiate into hypertrophic-like cells in the middle of wild-type proliferating chondrocytes. All mutant cells below a line approximately corresponding to the level at which the growth plate narrows (arrowhead) appear hypertrophic. (E-H) Sections of the tibiae from wild-type (E) and chimeric embryos with various contributions of PTH/PTHrP receptor ( - / - ) cells (F-H) were stained for 13-galactosidase activity and counterstained with hematoxylin and eosin. Contributions of mutant cells are estimated 10% (F), 30% (G), and 60% (H). In proportion to the contribution of mutant cells, columns of wild-type proliferating chondrocytes elongate (two-headed arrows). Bar = 100 i~m. [From Chung et aL (2), Proc Natl Acad Sci USA 1998;95:13030-13035. Copyright 1998 National Academy of Sciences, U.S.A.]

The first clue to the functions of Indian hedgehog in endochondral bone development came from the work of Bitgood and McMahon (44), who localized expression of its gene to growth plate chondrocytes as well to the hindgut, tooth, hair, whiskers, vas deferens, urethra, and lung. Vortkamp et al. (30) then showed intense Ihh expression in a discrete portion of the growth plate in the chick. Cells expressing Ihh partially overlap with those that express P T H / P T H r P receptors, but expression also extends to early hypertrophic cells more

toward the center of the developing bone (Fig. 7). When Ihh is misexpressed in the developing chick using a replication-competent retroviral vector, the infected cartilage elements become broader and shorter and lack hypertrophic chondrocytes. Thus, Ihh misexpression results in a phenotype similar to that of mice overexpressing PTHrP under control of the collagen loL(II) promoter (28). The similar sites at which P T H / P T H r P receptor and Ihh are expressed, together with the effects of

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FIG. 7 Expression of Indian hedgehog (Ihh) and PTH/PTHrP receptors in developing endochondral bone (distal end of tibia, embryonic day 16.5). The expression domain of PTH/PTHrP receptor mRNA expression is different from that of Ihh, but the two domains overlap. [Modified with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

overexpression of Ihh, led to the hypothesis that Ihh and PTHrP might interact. Initial studies in fact showed that PTHrP expression in chick limbs was greatly increased by misexpressed Ihh. Functional relationships between PTHrP and Ihh were further explored in a model system in which fetal murine limbs are grown in explant culture u n d e r serum-free conditions. When PTHrP(1-34) or PTH(1-34) is added to the cultured normal limbs, formation of hypertrophic cells is completely suppressed, as evidenced by histologic examination and by the disappearance of cells that express type X collagen. As expected, addition of either peptide to limbs of PTHrP-deficient mice completely rescues the abnormal phenotype (30), but it does not affect limbs from P T H / P T H r P receptor-deficient mice (25). When the effect of synthetic Shh protein on endochondral bone development was tested (Ihh protein was not available, but its activities are indistinguishable from those of Shh), it did not affect limbs from PTHrPdeficient mice, whereas it repressed hypertrophic differentiation in limbs from normal mice (Fig. 8). Thus, PTHrP is required for the response to Shh to occur (30). Similar experiments were conducted with limbs of P T H / P T H r P receptor-deficient mice; neither PTHrP nor Shh was effective (25). Most importantly, both Ihh misexpression in chicks and Shh treatment of normal murine limbs markedly increased PTHrP expression in perichondrial/subarticular chondrocytes (Fig. 9). The receptor for h e d g e h o g proteins is a cell surface heterodimer, consisting of patched (ptc), which binds hedgehog, and s m o o t h e n e d (smo), which conducts the signal to inside the cell. In the unliganded state, ptc and smo are noncovalently linked and smo is inactive.

On binding hedgehog, smo is thought to dissociate from ptc, and smo then becomes constitutively active (45,46). By binding hedgehog, ptc also limits hedgehog diffusion and thus its range of direct actions. A transcription factor, gli-1, is activated by h e d g e h o g signaling. Both ptc and gli-1 are transcriptional targets of hedgehog. Therefore, high expression of their genes identifies cells that have responded to hedgehog (47,48). In the bones of normal mice fetuses at 13.5 days postcoitus (dpc), ptc and gli-1 are most intensely expressed in perichondrial cells next to hypertrophic cells that express Ihh. Later in fetal development, when the growth plate is more highly organized into discrete zones and the primary ossification center is formed, ptc continues to be expressed in perichondrial/subarticular cells, but it is now also expressed in the growth plate cartilage in a gradient; it is most intensely expressed in chondrocytes immediately distal to the site where Ihh is expressed, progressively less expressed in less mature chondrocytes, and is barely detectable in subarticular chondrocytes that most intensively express PTHrE Interestingly, it is also highly expressed in cells that reside in the primary spongiosa, at some distance from any obvious site of Ihh. Nonetheless, the high level of ptc expression in these cells raises the likelihood that these bone cells also are h e d g e h o g targets (Fig. 10) (2). Whether Ihh protein is stored in more mature hypertrophic chondrocytes where it is not expressed is currently not known. The lack of ptc expression in subarticular cells that express PTHrP and the relatively long distance between PTHrP-expressJng cells and hypertrophic cells that express Ihh make it very unlikely that subarticular cells are direct ta~rgets of Ihh.

ENI~OCI-IONI~RALBONE FORMATION /

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The mechanism by which Ihh regulates PTHrP expression has yet to be elucidated. Many of the roles of Ihh in skeletal development become clear from the phenotype of Ihh-deficient mice (49). Importantly, Ihh-deficient mice do not phenocopy either PTHrP- or P T H / P T H r P receptor-deficient mice. Therefore, a detailed comparative examination distinguishes which of the activities of Ihh are PTHrP d e p e n d e n t from those that are PTHrP independent. About half of the Ihh-deficient mice die between 10.5 and 12.5 days postcoitus. This is due most probably to circulatory anomalies in the yolk sac, which develops abnormally in Ihh-deficient mice and is a site where Ihh is normally expressed. Some mice die later in gestation, and the remainder die at birth. The perinatal lethality is likely due to extraordinary shortening of the ribs, which severely impairs respiration. The development of cartilage primordia is not affected, consistent with the lack of expression of Ihh in early cartilage condensates of normal mice. Although

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FIG. 8 Sonic hedgehog (Shh) requires PTHrP to block chondrocytic differentiation. Embryonic day 16.5 mouse hindlimbs from wild-type and PTHrP ( - / - ) mice were cultured for 4 days in control, PTHrP-, or Shhcontaining medium. (A) Col-X expression is repressed after PTHrP and Shh treatment in wild-type hindlimbs. In situ hybridization with a Col-X probe to sections of tibias from the control animals shows repression of the hypertrophic cartilage marker Col-X after PTHrP or Shh treatment, relative to untreated limbs. (B, C) PTHrP rescues the PTHrP ( - / - ) phenotype, whereas Shh has no effect on the cartilage. In PTHrR ( - / - ) animals the hypertrophy of the cartilage is advanced in all the bones, including the tibia (B) and the digits (C), as shown by Col-X expression (B) and hematoxylin and eosin staining (C). Treatment of the cultures with PTHrP not only rescues the wild-type phenotype but also induces the same repression of hypertrophic cartilage as observed in PTHrP treatment of normal limbs: CoI-X expression is repressed (B), and no hypertrophic chondrocytes form during culture (C). In contrast to the rescue of the PTHrP ( - / - ) phenotype by PTHrP, Shh treatment does not change the phenotype of the cartilage elements: Col-X expression is still advanced (B), and hematoxylin and eosin staining shows the premature hypertrophic cartilage (C). [Reprinted with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

the digits of Ihh-deficient mice do not appropriately segment, skeletal elements are for the most part present in the correct positions and numbers, thus demonstrafing that Ihh is not required for skeletal patterning. The most overt abnormality is failure of limb growth that is visible by 13.5 dpc, and which is so progressive that by birth forelimbs are only one-third as long as those of wild-type mice (Fig. 11). At birth, in addition to severe dwarfism, the Ihh-deficient mice show other stigmata of abnormal endochondral bone developmentmforeshortened snout and mandible and rounded skull. Most endochondral bones also are misshapen, whereas membranous bones are much less affected. BrdU labeling shows markedly reduced chondrocyte proliferation. The absence of ptc and gli-1 expression at any stage in the mutant skeleton confirms that Ihh signaling is required to induce expression of the genes. The high level of ptc expression in proliferating chondrocytes at sites adjacent to those of Ihh expression at all developmental stages in normal mice

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FIG. 9 Shh up-regulates PTHrP expression. Hindlimbs from wild-type mice (embryonic day 16.5) were cultured with control or Shh-containing medium for 4 days. PTHrP expression was increased in the subarticular perichondrium in Shh-treated hindlimbs. [Reprinted with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

suggests that proliferating chondrocytes are likely to be direct targets of Ihh signaling. Chondrocyte maturation also is abnormal in Ihh-deficient mice. Hypertrophic cells appear later than in normal mice and are fewer in number, smaller in size, and not well organized. There is an admixture of cells expressing type II and X collagens, similar to that seen in PTHrP- and PTH/PTHrP-receptor-deficient mice. Cartilaginous mineralization takes place in the Ihh-deficient mice, which because of the lack of proliferating cells and the

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presence of hypertrophic chondrocytes in regions where chondrocytes usually remain undifferentiated, extends closer to the articular ends of the bones than in normal mice. Vascular invasion occurs, although it is delayed and never extensive, but neither histologically identifiable bone nor mineralized bone collars develop. Importantly, there is no detectable expression of PTHrP anywhere in the developing bone, and PTH/PTHrP receptor expression, although present in the mutant chondrocytes, is absent from the perichon-

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drium/periosteum. Thus, it appears that Ihh is required for maintenance of PTHrP signaling and therefore Ihh indirectly regulates chondrocyte differentiation by controlling PTHrE The role of Ihh in skeletal development was further examined by assessing expression of transcripts typically associated with bone. In contrast to normal mice, OSF2/Cbfal, a transcriptional activator of bonespecific target genes (50) whose expression is essential for formation of osteoblasts (51,52), P T H / P T H r P receptors, and BMP3 are not expressed in the perichondrial/periosteal region of mutant mice, although the former two are expressed in chondrocytes. The absence of bone was further confirmed by the lack of expression of osteocalcin, currently considered the most specific marker of mature osteoblasts, in any endochondral bone in the mutant appendicular or axial skeleton. Interestingly, abundant osteocalcin expression takes place in mutant bones formed entirely or partially by intramembranous ossification, such as the flat bones of the skull, the mandible, and the clavicle. This indicates that the absence of Ihh signaling

255

affects osteoblast development only in endochondral, not membranous, bones, and provides clear evidence that the developmental programs directing intramembranous and appositional ossification differ. Further insights into the respective roles of PTHrP and Ihh in endochondral ossification and morphogenesis come from examining embryos generated by crosses between mice carrying various combinations of three alleles, Ihh- and PTHrP-null alleles and a transgene in which the constitutively active P T H / P T H r P receptor is expressed under control of the collagen oL1(II) promoter (53). At 18.5 dpc, the limb skeletons of I h h / P T H r P compound mutants are identical to Ihhnull mutants, suggesting that Ihh is necessary for PTHrP function. Although expression of the constitutively active P T H / P T H r P receptor in Ihh-null mice prevents premature chondrocyte hypertrophy, it does not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation (Fig. 12). As expected, expression of the constitutively active P T H / P T H r P receptor rescues the phenotype of PTHrP-null mice. These experiments confirm that molecular mechanisms controlling chondrocyte differentiation are distinct from those that drive proliferation. Ihh up-regulates PTHrP, which then prevents chondrocyte hypertrophy and maintains a population of cells competent to proliferate. In contrast, Ihh promotes chondrocyte proliferation via pathways that are independent of PTHrP and which, in the absence of Ihh, cannot be rescued by PTHrE The pivotal roles of Ihh on chondrocyte proliferation and osteogenesis are confirmed and extended by comparing the skeletal phenotypes of chimeric mice, resulting from experiments in which embryonic stem cells missing the P T H / P T H r P receptor gene alone or both the P T H / P T H r P receptor and Ihh genes were introduced into wild-type blastocysts (31,54). As mentioned above, chondrocytes deficient in P T H / P T H r P receptors hypertrophy at an ectopic location relatively near the articular end of the developing bone in these chimeric mice. These hypertrophic cells express Ihh, which induces expression of ptc in neighboring cells and up-regulates the expression of PTHrP in subarticular cells. Interestingly, higher degrees of chimerism result in proportional elongation of the wild-type proliferating chondrocytes. This elongation is not seen in chimeric animals, when doubly deficient, P T H / P T H r P receptor Ihh-null embryonic stem cells were introduced into wild-type blastocysts. Although doubly deficient chondrocytes hypertrophy ectopically in these mice, hypertrophy is not associated with either expression of ptc in adjacent cells or up-regulation of PTHrP in subarticular cells. Cartilage mineralization in areas of ectopic hypertrophic cells takes place when the chimerism is high, however, confirming that this process is not cell autonomous and is regulated by

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Fill. 12 Effects of manipulating PTHrP signaling in Ihh mutants. (A, F) Wild type; (B, G) PTHrP ( - / - ) ; (C, H) Ihh ( - / - ) ; (D, I)Ihh (-/-);PTHrP ( - / - ) ; (E, J)Ihh (-/-);PTHrPR* [PTHrPR*-constitutively active PTH/PTHrP receptor expression is driven by a collagen e~l (11)promoter]. (A-E) 18.5-dpc skeletal preparations taken at the same magnification, and (F-J) corresponding histology taken at the same magnification. Loss of PTHrP in Ihh ( - / - ) elements results in limbs that are identical by skeletal staining (C, D) and histology (H, I). Activation of PTHrP signaling in Ihh ( - / - ) using PTHrPR* significantly decreases red staining, which is entirely absent in the tibia (arrows in C and E). This is confirmed by histology (H, J), which demonstrates reduced chondrocyte hypertrophy. Arrows mark the regions of nonhypertrophic chondrocytes. [From Karp et aL (53), with permission from the Company of Biologists Ltd.]

neither PTHrP nor Ihh. Dams pregnant with chimeric fetuses in which PTH/PTHrP receptor-deficient embryonic stem cells were injected into the blastocyst were given BrdU at 17.5 dpc. The percentage of BrdUlabeled cells in growth plate chondrocytes differed little, if at all, from those in wild-type mice (U. Chung, personal communication, 2000). Thus, elongation of the wild-type proliferative zone in mice with a high degree of chimerism with PTH/PTHrP receptor-deficient cells appears to be due to Ihh-induced increases in PTHrP, which then inhibits the exit of proliferative cells to postmitotic hypertrophic chondrocytes, rather than to Ihh-induced chondrocyte proliferation. Some direct contribution of Ihh cannot, however, be excluded. Comparing osteogenesis in the same two chimeric mice models confirms the unique role of Ihh in osteogenesis suggested by the phenotype of Ihh-deficient mice. Whereas PTH/PTHrP receptor-deficient hypertrophic chondrocytes near the perichondium/periosteum induce an ectopic bone collar, I h h / P T H / P T H r P receptor doubly deficient hypertrophic chondrocytes do not.

Other lines of investigation support a major osteogenic role for Ihh. Hedgehog proteins have been shown to stimulate alkaline phosphatase in mesenchymal cells, primary osteoblasts, and osteogenic cell lines (55-57), and chicken fibroblasts engineered to overexpress Shh induce ectopic bone formation (55) when injected into nude mice. Hedgehog signaling also is modulated by a membrane-anchored glycoprotein, Hip (hedgehog interacting protein). Hip interacts with the amino-terminal portion of all three mammalian hedgehogs with an affinity similar to that of patched. Like ptc, hip is expressed next to cells that express hh and is a transcription target of hedgehog signaling. Cartilagespecific overexpression of hip phenocopies Ihhdeficient mouse, except that the bone collar forms normally in the periosteum where hip is not expressed, thus confirming the unique role played by Ihh in apposition ossification (31). Excessive signaling via the hedgehog pathway is responsible for some human disease. Many patients with nevoid basal cell carcinoma syndrome have ptc haploinsufficency (58,59). This syndrome is character-

ENDOCHONDRAL BONE FORMATION / ized by a high incidence of basal cell carcinomas and other cancers, and, of particular importance to this discussion, by skeletal overgrowth (60). The syndrome can be essentially phenocopied in mice by overexpressing Shh (61). Thus, perhaps not surprisingly, Shh is oncogenic and the gene for ptc functions as a tumor suppressor gene.

C U R R E N T M O D E L OF E N D O C H O N D R A L BONE DEVELOPMENT Knowledge acquired over the past few years concerning the actions of Ihh, PTHrP, and PTH make it possible to construct a far more detailed model than the one we proposed for regulation of cartilage differentiation by PTHrP and Ihh in 1996 (30). One model that takes all of the current data into account is as follows. In growth plate cartilage, Ihh up-regulates expression of PTHrP, whose major site of synthesis is in perichondrial/subarticular chondrocytes, and Ihh independently promotes chondrocyte proliferation. The proliferative actions of Ihh are likely to be direct, as evidenced by high expression of ptc and gli-1 in cells immediately adjacent to the hypertrophic chondrocytes that express Ihh. It is important to note that indirect actions of Ihh on chondrocyte proliferation via other signaling molecules cannot be excluded. The lack of ptc expression in subarticular cells and the relatively long distance between Ihh- and PTHrP-expressing cells make it highly likely that Ihh regulates PTHrP indirectly. The signaling molecules downstream of Ihh that control this action have not been defined. PTHrP determines the position relative to the articular ends of the developing bone at which chondrocytes exit the cell cycle and initiate the hypertrophic chondrocyte differentiation program. This would be consistent with PTHrP acting as a patterning molecule, or morphogen, and the periarticular perichondrium/subarticular chondrocytes as an organizing center. In this model, the periarticular perichondrium/subarticular chondrocytes are the major source of PTHrP, which then diffuses centrally through the growth plate, in which a PTHrP gradient is established. High concentrations of PTHrP inhibit hypertrophy, but a threshold exists below which PTHrP no longer prevents the cells from initiating the hypertrophic program. The distance from the articular end at which this threshold is attained depends on the level of PTHrP gene transcription. The high level of P T H / P T H r P receptors expressed in prehypertrophic cells probably serve as a sink to prevent further diffusion of PTHrP, and thus a sharp transition is established whereby PTHrP concentrations fall abruptly to below threshold levels. That prehypertrophic cells act as a sink for PTHrP is supported by the finding that most of the cell-bound PTHrP protein is

257

detected on these cells. This is consistent with the finding that the PTHrP acts mainly on cells that are relatively close to the articular end, rather than on prehypertrophic chondrocytes. It also predicts that chondrocytes undergo hypertrophy closer to the articular end in the absence of PTHrP, without necessarily affecting the rate of chondrocyte differentiation. Initiation of the hypertrophic chondrocyte program includes the expression of Ihh, but it is not dependent on Ihh. Because PTHrP has no or very small effects on chondrocyte proliferation in vivo, the absence of PTHrP results in a smaller population of proliferating cells, with hypertrophy and ossification displaced closer to the articular ends of the developing bone. This model is also consistent with the effects of PTHrP overexpression in the mouse where the PTHrP concentration is sufficiently high to markedly inhibit hypertrophy. Ihh-induced proliferation lengthens the distance between hypertrophic and subarticular cells, thus resulting in lowered PTHrP expression. The PTHrP gradient is altered, moving the threshold level needed to prevent proliferating cells from exiting the cell cycle closer to the articular end. The polarity of growth plate chondrocytes, namely, the columnar organization and orderly progression of chondrocytes from type II collagen-expressing cells to type X collagen-expressing cells, appears to be mainly controlled by PTHrE PTHrP-independent actions of Ihh on this process, however, cannot be excluded. Although hypertrophic chondrocytes undergo a defined differentiation program, the controlling factors, including those leading to apoptosis, are not well understood. Cartilage mineralization, however, occurs non-cell autonomously at sites where a sufficient number of late hypertrophic chondrocytes collect. These most mature hypertrophic chondrocytes control the process by which bone replaces cartilage. They express VEGF, attracting capillaries that bring chondroclast and osteoblast precursors to the region of dying chondrocytes. Chondroclasts, in part by secreting proteases, including MMP-9/gelatinase B, into their local environment and by phagocytosis, dispose of cell debris and cartilaginous matrix. Osteoblasts, attracted both by VEGF and perhaps also by matrix proteins, then lay down osteoid on the remnants of cartilaginous matrix, which, after further resorption and remodeling, form the trabeculae of the secondary spongiosa. Consideration of factors that mediate vascular invasion are beyond the scope of this chapter, other than to point out that PTHrP, by mechanisms apparently independent of the P T H / P T H r P receptor, is critical. Normal appositional ossification requires both PTH and Ihh that is secreted by hypertrophic chondrocytes abutting the perichondrium/periosteum, but neither alone is sufficient. Osteoblasts do not develop in the perichondrium/periosteum in the absence of Ihh,

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which obviously accounts for the lack of cortical bone matrix and mineral deposition. The periosteum of P T H / P T H r P receptor-deficient mice is dramatically different; it contains an excess of osteoblasts, and a b u n d a n t matrix that, however, does not mineralize. PTH is required to form a mineralized bone collar, but whether this is a due to direct or indirect mechanisms is currently not clear. The absence of bone cells in the core of skeletal elements in Ihh-deficient mice and the high level of ptc expression in the primary spongiosa of normal mice provide very strong evidence for an essential role for Ihh in the development of trabecular bone. The evidence, however, is not yet absolutely conclusive. A plausible counter a r g u m e n t is that the anomalous cartilage development, minimal vascular invasion, a n d / o r the early lethality of Ihh-deficient mice preclude the development of a primary spongiosa. The source of Ihh that potentially drives osteogenesis also is not clear; hypertrophic chondrocytes are the most plausible source, although osteoblastic cells, or other cells b r o u g h t in with vascularization, also may be sources. Experiments to resolve this issue definitively might be to eliminate Ihh expression selectively in hypertrophic chondrocytes or osteoblasts, or to block the putative actions of Ihh in osteoblasts by selectively overexpressing hip in these cells.

REFERENCES 1. Farnum CE, Wilsman NJ. Determination of proliferative characteristics of growth plate chondrocytes by labelling with bromodeoxyuridine. Calcif Tissue Int 1993;52:110-119. 2. Chung U, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad Sci USA 1998;95:13030-13035. 3. Gentili C, Bianco P, Neri M, Malpedi M, Campanile G, Castagnola P, Cancedda R, Cancedda FD. Cell proliferation, extracellular matrix mineralization and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast cells. J Cell Bio11993;122:703-712. 4. Lee K, Deeds JD, Chiba S, Un-no M, Bond AT, Segre GV. Parathyroid hormone induces c-fos expression in bone cells in vivo: In situ localization of its receptor and c-fos messenger ribonucleic acids. Endocrinology 1994;134:441-450. 5. Lee K, DeedsJD, Segre GV. Expression of parathyroid hormonerelated peptide and its messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453-463. 6. Gerber H-P, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat. Med 1999;5:623-628. 7. Vu TH, ShipleyJM, Bergers G, BergerJE, HelmsJA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411-422. 8. Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, Amling M, Karsenty G. A Cbfal-dependent genetic pathway con-

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ENDOCHONDRAL BONE FORMATION 25. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra A-B, Jfippner H, Segre GV, Kronenberg HM. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663-666. 26. Lee K, Lanske B, Karaplis AC, Deeds JD, Kohno H, Nissenson RA, Kronenberg HM, Segre GV. Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 1996;137:5109-5118. 27. Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999; 104:399-407. 28. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormonerelated peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 1996;93:10240-10245. 29. Schipani E, Lanske B, Hunzelman J, Luz A, Kovacs CS, Lee K, Pirro A, Kronenberg HM, Jfippner H. Targeted expression of constitutively active receptor for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormonerelated peptide. Proc Natl Acad Sci USA 1997;94:13689-13694. 30. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. 31. Chuang P-T, McMahon AP. Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein. Nature 1999;397:617-621. 32. Echelard Y, Epstein DJ, St-Jacques B, Shen L, MohlerJ, McMahon JA, McMahon AP. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 1993;75:1417-1430. 33. Krauss S, Concordet J-P, Ingham PW. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebraf'lsh embryos. Cell 1993; 75:1431-1444. 34. Roelink H, Augsburger A, Heemskerk J, Korzh V, Norlin S, Ruiz Altaba A, Tanabe Y, Placzek M, Edlund T, Jessell TM. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 1994;76:761-775. 35. Johnson RL, Laufer E, Riddle RD, Tabin C. Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell 1994;79:1165-1173. 36. Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 1993; 75:1401-1416. 37. Roberts DJ, Johnson RL, Burke AC, Nelson CE, Morgan BA, Tabin C. Sonic hedgehog is an endodermal signal inducing Bmp4 and Hox genes during induction and regionalization of the chick hindgut. Development 1995; 121:3163-3174. 38. Levin M, Johnson RL, Stern CD, Kuehn M, Tabin C. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 1995;82:803-814. 39. Pag~in-Westphal SM, Tabin CJ. The transfer of left-right positional information during chick embryogenesis. Cell 1998;93:25-35. 40. Chiang C, LitingtungY, Lee E, Young KE, CordenJL, Westphal H, Beachy PA Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 1996;383:407-413. 41. Bitgood MJ, Shen L, McMahon AP. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr Biol 1996;6:298-304. 42. Currie PD, Ingham PW. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 1996;382:452-455.

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43. Ekker SC, Ungar AR, Greenstein P, von Kessler DE Porter JA, Moon RT, Beachy PA. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 1995;5: 944-955. 44. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Bio11995;172:126-138. 45. Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 1996;384:129-134. 46. Marigo V, Davey RA, Zuo Y, Cunningham JM, Tabin CJ. Biochemical evidence that patched is the Hedgehog receptor. Nature 1996;384:176-179. 47. Goodrich LV,Johnson RL, Milenkovic L, McMahon JA, Scott ME Conservation of the hedgehog/patched signaling pathway from flies to mice: Induction of a mouse patched gene by hedgehog. Genes Dev 1996;10:310-312. 48. Marigo V, Scott ME Johnson RL, Goodrich LV, Tabin CJ. Conservation in hedgehog signaling: Induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 1996; 122:1225-1233. 49. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13:2072-2086. 50. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbal: A transcriptional activator of osteoblast differentiation. Cell 1997;89:747-754. 51. Komori T, Yagi H, Monura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755-764. 52. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765-771. 53. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP. Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone-related protein-dependent and -independent pathways. Development 2000;127:543-548. 54. Chung U, Wei W, Schipani E, McMahon AP, Kronenberg HM. Indian Hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Bone Miner Res 2000;15:$192. 55. Kinto N, Iwamoto M, Enomoto-Iwamoto M, Noji S, Ohuchi H, Yoshioka H, Kataoka H, Wada Y, Gao Y, Takahashi HE, Yoshiki S, Yamaguchi A. Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation. FEBS Lett 1997;404:319-323. 56. Nakamura T, Aikawa M, Iwamoto-Enomata M, Iwamoto M, Higuchi Y, Pacifici M, Kinto N, Yamguchi A, Noji S, Kurisu K, Matsya T, Maurizio E Induction of osteogenic differentiation by hedgehog proteins. Biochem Biophys Res Commun 1997;237: 465-469. 57. Jemtland R, Divieti P, Lee K, Segre GV. Recombinant sonic hedgehog promotes differentiation of primary mouse calvarial osteoblasts and increases PTHrP mRNA expression. J Bone Miner Res 1999;14:$293. 58. Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G, Wainwright B,

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Bale AE. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996;85: 841-851. 59. Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein EH, Jr, Scott ME Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 1996;272:1668-1171.

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CHAPTER16 Physiologic Actions of P T H and PTHrP IV. Vascular, Cardiovascular, and Neurologic Actions

THOMAS L. CLEMENS Division of Endocrinology and Metabolism, University of Cincinnati Collegeof Medicine, Cincinnati, Ohio 45267 ARTHUR E. BROADUS Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510

VASCULAR A N D C A R D I O V A S C U I A R ACTIONS

through different mechanisms. Finally, PTH exerts both ionotropic and chronotropic effects on the heart (6). Although the cardiovascular effects of PTH are undisputed, their physiologic significance has been frequently debated. This is in part because the concentrations of PTH required to produce vasodilation (10-100 nM) are substantially above those that normally circulate (low picomolar). Consequently, it has been difficult to conceptualize how physiologic levels of this systemic hormone, which is synthesized only in the parathyroid gland, could function in the local control of vascular tone. Also enigmatic is the fact that patients with primary hyperparathyroidism and elevated circulating PTH levels often have high (not low) blood pressure that sometimes returns to normal after parathyroidectomy. A plausible explanation for the seemingly enigmatic regulatory effects of PTH on the cardiovascular system emerged with the discovery of PTH-related protein (PTHrP) in 1987. As discussed in Chapter 13 of this volume, PTHrP was identified as the factor responsible for the paraneoplastic syndrome, humoral hypercalcemia of malignancy. Almost immediately after its cloning, expression of PTHrP was detected in many normal fetal and adult tissues but was undetectable in the circulation, suggesting that the protein functioned in an autocrine/paracrine mode. Although many functions have been ascribed for PTHrP, three main physiologic themes have emerged. Observations in gene knockout mice have demonstrated that PTHrP is required for development of cartilage, for morphogenesis of the

Historical Perspectives The origins of PTH as a putative cardiovascular regulatory factor date to the early 1900s, when the calcemic properties of the hormone were first identified. In classic studies, Collip and Clark (1) demonstrated that systemic injection into dogs of extracts of parathyroid glands lowered systemic blood pressure (Fig. 1). The first formal characterization of the cardiovascular activity of parathyroid hormone (PTH) was conducted by Charbon and colleagues in the early 1960s (2-4). These investigators quantified the vasodilatory effects of a purified parathyroid extract in the rabbit and cat and also showed that a synthetic N-terminal fragment displayed similar actions in the dog. The relaxant activity was not blocked by pharmacologic antagonists of other known vasoactive agents, suggesting a direct action of the hormone. Since then numerous studies have unequivocally established the hypotensive/vasodilatory and cardiac effects of PTH (5), and these can be summarized broadly as follows: First, the hypotensive and vasorelaxant actions of PTH occur in the absence of a change in blood calcium and are mediated by PTH activation of the type 1 PTH/PTH-related protein receptor (PTHIR) expressed in the smooth muscle layer of the vessel wall. Second, although all vascular beds are relaxed by PTH, resistance vessels appear to be more responsive than conduit vessels. Third, PTH can reduce the pressor effects of other vasoactive agents that exert their action

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FIG. 1 Effects of extracts of parathyroid glands on blood pressure in thyroparathyroidectomized dogs (from Collip JB, Clark EP. J. BioL Chem. 1925; 64:485-507, with permission) (1).

m a m m a r y gland, and for tooth eruption (7). PTHrP also appears to participate in materno-fetal calcium transfer across the placenta. The third physiologic role for PTHrP is in smooth muscle, in which the protein functions to regulate contractility and proliferation. Although this chapter focuses on the physiology of PTHrP in vascular smooth muscle, it is relevant to begin with a brief review of its physiologic effects in other smooth muscle-containing tissues. In all smooth muscle cell beds studied to date, induction of PTHrP expression occurs in close association with normal physiologic stimuli. In the smooth muscle layer of the chicken oviduct, induction of PTHrP expression coincides temporally with egg movement and its arrival in the shell gland (8). In the rat uterus, PTHrP expression is localized to the myometrium and is markedly up-regulated by fetal occupancy (9) or by mechanical distention of the uterine horn using a balloon catheter (10). PTHrP expression is increased in prelabor h u m a n amnion and abruptly decreases with the onset of labor and rupture of the amniotic sac (11). In the urinary bladder, induction of PTHrP mRNA occurs during filling in proportion to bladder distension (12). Finally, as discussed in detail below, PTHrP is also expressed in vascular smooth muscle, in which it is induced by vasoconstrictor agents and mechanical stimuli. In each of these smooth muscle beds, application of PTHrP to precontracted smooth muscle preparations induces relaxant activity, precisely mimicking the actions previously described for PTH. It would therefore appear that PTHrP rather than PTH represents the physiologically important regulator of smooth muscle tone. Consequently, the remainder of this chapter

focuses primarily on the physiology of PTHrP in the cardiovascular system.

P T H r P in the Vasculature Vascular Anatomy and Contractile Mechanisms Blood vessels are composed of three principal cellular layers: (1) the intima, which consists of a single epithelial cell layer, (2) the muscularis layer, made up of vascular smooth muscle cells embedded in a connective tissue matrix, and (3) an outer adventitial layer, which receives input from the cholinergic and adrenergic nervous system. The relative composition and contribution of each of the cell types to vascular growth and tone will vary during development and among different vascular beds. For example, during development, blood vessels form initially as simple tubular structures consisting entirely of endothelial cells into which smooth muscle cells migrate to form the vascular wall. In the mature mammal, the large conduit vessels (e.g., aorta) are highly elastic to accommodate high-capacity blood flow, whereas resistance vascular beds (e.g., mesentery) typically contain more smooth muscle cells and are densely innervated. Changes in the cellular and connective tissue constituents within the vasculature occur with normal aging and in particular during pathologic conditions such as athlerosclerosis. The regulation of vascular growth, remodeling, and smooth muscle cell tone is achieved through a coordinated network of both systemic and local factors as well as input from adrenergic, cholinergic, peptidegic, and sensory neurons.

PTHrP REGULATIONOF EXCITABLECELLS / The mechanisms regulating vascular smooth muscle cell contractility have been studied in detail (13). The intracellular free calcium concentration is the major d e t e r m i n a n t of vascular tone. Depolarization of vascular smooth muscle cells opens L-type voltage-sensitive calcium channels (L-VSCCs), enabling calcium to enter the cell. These events trigger the release of much larger quantities of calcium from the sarcoplasmic reticulum. Alternatively, pharmacologic or ligand activation of G protein receptors (e.g., angiotensin II) activate phospholipase (PLC), which catalyzes phosphoinositol hydrolysis and causes calcium release from intracellular stores. The increases in cytoplasmic calcium achieved by either of these mechanisms activate myosin lightchain kinase through the calcium-calmodulin complex and phosphorylation of the 20-kDa regulatory light chain of myosin, with subsequent cross-bridge cycling and force development. The mechanisms of vascular smooth muscle cell relaxation are less well understood. In the most simple scheme, a reduction of cytoplasmic calcium with a decrease in myosin light-chain kinase activity would suffice to account for dephosphorylation of the regulatory light chain and relaxation. However, other mechanisms have been implicated in cyclic nucleotide-dependent relaxation in vascular and other smooth muscle tissues (14). The demonstration of the calcium-sensing receptor in vascular smooth muscle with pharmacologic properties similar to those of the parathyroid calcium-sensing receptor (discussed in Chapter 3) has p r o m p t e d speculation that it might also participate in the regulation of contractile events (15). Alterations of extracellular calcium over the physiologic concentration range depress contractility of precontracted vascular smooth muscle. This effect of extracellular calcium has been shown to be mediated by activation of a calciumd e p e n d e n t potassium channel and is associated with alterations in myofilament calcium sensitivity. These activities were mimicked by gadolinium, neomycin, and lanthanum, all factors that activate the calciumsensing receptor. However, the structure of this putative calcium-sensing receptor is unknown and it remains unclear whether it bears homology to the renal or parathyroid or kidney calcium-sensing receptor.

expressed predominantly in the smooth muscle layer of the vessel, although its expression has also been reported in cultured endothelial cells (22,23). The regulation of PTHrP mRNA expression has been studied in detail using cultured vascular smooth muscle cells. In primary rat aortic vascular smooth muscle cells, expression of PTHrP is rapidly (2-4 hours) but transiently induced by exposure of quiescent cells to serum (24) (Fig. 2). This mode of tight regulation is reminiscent of the behavior of the cytokine mRNAs and would appear to constitute a mechanism that would restrict the activity of PTHrP to a narrow window of time. Among the most potent inducers of PTHrP are vasoconstrictors, including angiotensin II, seritonin, endothelin, norepinephrine, bradykinin, and thrombin, each of which induces PTHrP mRNA and protein levels over the same time course as that observed for serum (25). The induction of PTHrP mRNA by angiotensisn II is d e p e n d e n t on protein kinase C activation and is mediated by both transcriptional and posttranscriptional mechanisms (25). Prior addition of saralaysin and captopril, which inhibit angiotensin II action or generation, respectively, inhibits the serum-induced increase in PTHrP in vascular smooth muscle cells. This finding suggests that the angiotensin II present in serum represents a significant c o m p o n e n t of the serum induction of PTHrP. PTHrP is also induced in vascular smooth muscle in response to mechanical stimuli. PTHrP mRNA is transiently increased in rat aorta following distension with a balloon catheter (18). Flow motion-induced mechanical events induced by rocking or rotation of monolayer cultures of rat aortic vascular smooth muscle cells result

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Expression and Regulation of PTHrP PTHrP is expressed in blood vessels in essentially all vascular beds from a broad range of species, including rodent and h u m a n fetal blood vessels (16), adult rat aorta (17,18), vena cava (17), kidney afferent arterioles, artery, and microvasculature (19), the arterial and venous supply of the m a m m a r y gland (20), the serosal arterioles in avian egg shell gland (8), and blood vessels of the rat penis (21). The protein appears to be

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FIG. 2 Time course of serum induction of PTHrP mRNA in aortic vascular smooth muscle cells (from Hongo T, ot al. d. Glin. Invos. 1991; 88:1841-1847, with permission) (24).

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in increased PTHrP mRNA expression (26). The inductive effects of mechanical stretch and angiotensin II on PTHrP mRNA appear to be synergistic, suggesting that they occur through distinct mechanisms (27). PTHrP mRNA is also p r o d u c e d in capillaries of slow-twitch soleus and fast-twitch skeletal muscle, and its expression is increased in response to lowfrequency stimulation (28). This maneuver was associated with e n h a n c e d capillarization of the muscle, indicating that PTHrP might function to promote new capillary growth in response to increased contractile activity.

Vascular Actions of PTHrP Shortly after the identification of PTHrP, a n u m b e r of studies demonstrated that synthetic N-terminal fragments of the peptide replicated many of the vascular actions of PTH, including its vasorelaxant actions in aorta (29), portal vein (30), coronary artery (31 ), renal artery (32,33), placenta (34,35), and m a m m a r y gland (36). In general, the vasodilatory potency of PTHrP when examined in organ bath systems is comparable to that of PTH. By contrast, in mouse portal vein preparations, PTHrP(1-34) was shown to be a more potent vasorelaxant than PTH(1-34) (30). In perfused rabbit kidney (33) and in rat aorta (31) the vasorelaxant effects of PTHrP do not appear to require the presence of an intact endothelium. However, in mouse aortic rings, endothelium denudatation markedly attenuates the relaxant activity of PTHrP (37), possibly reflecting a species difference. In addition to its effects on vascular tone, PTHrP also modulates vascular smooth muscle cell proliferation. The peptide decreases serum and platelet-derived growth factor-activated DNA synthesis in primary arterial vascular smooth muscle cells (24,38) and in A-10 vascular smooth muscle cells stably expressing the PTH1R (39). In both cell types, the antimitogenic effects require the PTH-like N-terminal portion of the molecule and are mimicked by dibutyryl cAMP or forskolin. The mechanism for the antiproliferative effect of PTHrP involves the induction of the cyclind e p e n d e n t kinase inhibitor, p27Kipl, and impairment of the retinoblastoma gene product (Rb), which results in cell cycle arrest in mid-G 1 phase (40). On the other hand, Massfelder et al. (41) reported that overexpression of PTHrP in A-10 vascular smooth muscle cells was associated with an increase in DNA synthesis coincident with an increased nuclear localization of the protein. However, in these studies, exogenous application of PTHrP inhibited A-10 cell growth, in agreement with the studies cited above. A putative nuclear targeting motif was found to be required for nuclear import of PTHrP in vascular smooth muscle cells, in accordance

with previous studies in chondrocytes (42). Therefore, the ability of PTHrP to influence proliferation of vascular smooth muscle cells either positively or negatively appears to d e p e n d on where the protein is trafficked in the cell. Cellular levels of PTHrP fluctuate during the cell cycle and reach their highest levels in Gz/M (43). It is possible that the protein is directed to the nucleus in the later stages of the cell cycle to participate in mitotic events. PTHrP also inhibits platelet-derived growth factor (PDGF)-directed migration of vascular smooth muscle cells in vitro (44). The antimigratory effects of PTHrP are mediated through a cAMP-dependent mechanism that leads to diminished PDGF signaling through the PI3 kinase cascade. The effects on vascular smooth muscle cell (VSMC) growth and migration in vitro are likely to be physiologically relevant to conditions u n d e r which VSMC growth and migratory behavior are altered in vivo. For example, Ozeki et al. (45) have reported that PTHrP protein and mRNA expression were markedly up-regulated in neointimal smooth muscle in rat carotid arteries following experimental balloon injury. Moreover, immunoreactive PTHrP is increased in h u m a n arterial tissue removed from patients undergoing angioplasty. In light of the possibility of opposing effects of PTHrP on vascular smooth muscle cell growth cited above, these observations can be viewed in one of two ways: either up-regulation of PTHrP is a primary stimulus for growth under these conditions or, alternatively, it represents an antiproliferative signal. Consistent with the latter possibility, the local administration of 3',5'-cyclic AMP or the phosphodiesterase inhibitors aminophylline or amrinone inhibits neointimal formation following experimental balloon injury in rat carotid arteries in vivo (46). Moreover, other studies using a similar model of arterial injury showed high levels of p27Kipl expression in the media within 2 weeks after angioplasty (47). The ability of PTHrP to modulate vascular smooth muscle cell growth suggests that the protein might function during the development of the cardiovascular system. Although the cardiovascular system appears to develop normally in the PTHrP knockout mouse, homologous deletion of the PTH1R results in a higher incidence of early fetal death at approximately embryonic day 10, coincident with the development of the heart and major blood vessels (48). Furthermore, transgenic mice expressing high levels of PTHrP and its receptor in vascular smooth muscle, created by crossing the ligand- and receptor-overexpressing mice, die at day E9.5, with severe thinning of the ventricle and disruption of ventricular trabeculae (49) (Fig. 3). Additional anecdotal evidence for a role of PTHrP in heart and vascular development is evident from

PTHrP REGULATIONOF EXCITABLECELLS /

265

These patients die prenatally with coarctation of the aorta and hydrops fetalis, the latter condition typically caused by high-output heart failure.

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FIG. 3 Overexpression of PTHrP and the PTHrP receptor disrupts heart development. (A) Whole mounts at E9.5 of double-transgenic (left) and wild-type (right) embryos. The double transgenic exhibits a greatly enlarged heart with pericardial effusion and vascular pooling (arrows). (B) Histologic sections of double-transgenic (left) and wild-type (right) embryos at E9.5. The trabeculae within the ventricular cavity (v) of the wild-type embryo are prominent (large arrows), whereas in the double transgenic, trabeculae are severely reduced or absent (asterisks). Prominent gaps are also evident between the cardiomyocytes in the double-transgenic hearts (small arrowheads); a, atria; bar = 100 ~m. (C) Left panel shows the localization of SMP8 lacZ transgene in 9.5-day embryo. Staining is apparent in heart, hindgut, and somites. Right panel is an unstained control (from Qian J, et al, Endocrinology 1999;140:1826-1833, with permission) (49). (See color plates.)

the abnormalities seen in patients with the rare fatal condition known as Blomstrand chondrodysplasia, caused by an inactivating mutation of P T H I R (50).

PTHrP exerts its vasodilatory actions by activating the PTHIR. This receptor is expressed in rat vascular smooth muscle beds (51) and relaxation of aortic preparations is accompanied by an increased accumulation of cAMP (52). Cultured rat aortic smooth muscle cells also express the P T H I R and respond to N-terminal PTHrP peptide fragments with an increase in cAMP formation (53). Moreover, relaxation responses to PTH in aortic strip preparations are potentiated by phosphodiesterase inhibitors and forskolin (54). Although the P T H I R appears to be coupled primarily to adenylate cyclase, linkage to calcium-phosphoinositol pathways is suggested by studies by Nyby et al., who demonstrated a transient increase in cytosolic calcium and cAMP in response to PTHrP (1-34) in primary arterial rat vascular smooth muscle cells (55). However, other studies using similar preparations of primary rat aortic smooth muscle cells showed that PTHrP consistently stimulated cAMP accumulation but had no effect on intracellular calcium (53). Furthermore, in A-10 embryonic aortic vascular smooth muscle cells stably expressing recombinant PTH1Rs, PTHrP induced large increases in cAMP accumulation but did not increase cytoplasmic calcium (39), despite the presence of detectable levels of expression of Gq, known to be required for functional coupling of the receptor to the PLC-phosphoinositide calcium pathway. Gq was overexpressed in these cells, However, when PTHrP evoked a calcium transient. It therefore appears that u n d e r most conditions the PTH1R couples preferentially to G s and adenylate cyclase to elevate levels of intracellular cAMP, which would be consistent with the established vasodilatory properties of this cyclic nucleotide. This does not, however, preclude the possibility that u n d e r certain physiologic conditions (or in specific vascular smooth muscle cell beds), PTHrP might also activate PLC, which could mediate other as yet unidentified activities of the protein. Vasorelaxation induced by cyclic nucleotides in arterial smooth muscle has also been reported to be associated with a reduction in intracellular calcium. In addition, in rat tail artery, PTH relaxes KCl-induced contraction; this effect is inhibited by nifedipine, suggesting an inhibition of the L-VSCC (56). Subsequent patch-clamp experiments (57) confirmed a decrease in L-type voltage-dependent calcium currents in vascular smooth muscle cells in response to PTH. Although not yet formally tested, it is likely that

266

/

CI-IAPTWR16

PTHrP also inhibits the L-VSCC activity in vascular smooth muscle, as is the case in cultured neuroblastoma cells (see later). As discussed in detail in Chapter 3, PTHrP is subject to posttranslational processing to produce both N-terminal peptides, midregion PTHrP fragments, and possibly also C-terminal forms. PTHrP peptides, which lack the PTH-like N-terminal region, likely activate receptors distinct from the P T H I R and would be expected to exhibit a biologic profile different from N-terminal PTHrP peptides. To date, however, there is no evidence that these midregion or C-terminal forms of PTHrP are biologically active either in cultured vascular smooth muscle cells (53) or in intact vessel preparations (37). Although PTHrP is capable of relaxing vascular preparations devoid of endothelium, studies in mouse aortic preparations suggest that the endothelial layer may serve to amplify relaxant effects of PTHrP and PTH (37). The mechanism accounting for the endotheliumd e p e n d e n t relaxant effects of PTH and PTHrP remains unclear but does not appear to require nitric oxide formation. The demonstration of expression of a novel PTH2 receptor (PTH2R; see below) in endothelial and smooth muscle cells in blood vessels and heart (58) suggests an additional pathway through which PTHrelated peptides could alter vascular reactivity. As with other G-coupled receptors, prolonged exposure of vessel preparations (55) or cultured aortic smooth muscle cells (59) to PTHrP is associated with desensitization. Angiotensin II, which induces PTHrP expression in cultured aortic vascular smooth muscle cells, also rapidly desensitizes cells to PTHrP and downregulates PTH1R mRNA expression (59), indicating cross-talk in the signaling circuitry a m o n g these vasoactive peptides. From the studies summarized above it is possible to construct a simple model for the mode of PTHrP action in vascular smooth muscle (Fig. 4). In response to mitogenic, vasoconstrictor, or mechanical signals, PTHrP is

Vasoconstrictors ~ , , ~ _ , i - - - ~ Mechanical Stimuli ~ , g P ~

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released and acts locally via a short feedback loop to activate the PTH1R and stimulate adenylate cyclase in adjacent cells. Effector pathways downstream of cAMP impact on specific sets of genes that function to oppose the pressor (contraction coupling) and mitogenic (cell cycle) events. As m e n t i o n e d above, induction of p27Kipl with consequent inhibition of Rb phosphorylation would represent one such target for cAMP-induced cell cycle arrest. With regard to relaxant activity, stimulation of cAMP-dependent protein kinase A (PKA) is associated with a reduction in cytoplasmic calcium and attenuated myosin lightchain kinase activity (14). If PTH and PTHrP activate the same receptor, how does the smooth muscle P T H I R distinguish between these two ligands? A likely possibility is that the sensitivity of the tissues to PTH or PTHrP is governed by the relative abundance of each ligand and the n u m b e r of PTH1R. For example, in tissues such as vascular smooth muscle, which express high levels of PTHrP but relatively low numbers of the PTH1R, the fraction of receptor occupancy must be high in order to achieve a response, thus favoring the local (PTHrP) regulator. By contrast, in bone cells, PTHrP expression is low and the receptor expression is high, enabling preferential receptor activation by PTH arriving from the systemic circulation.

P T H r P in the Heart PTHrP and the PTH1R are expressed in fetal and adult heart from a n u m b e r of different species (17). PTHrP has been immunolocalized to atrial natriuretic peptide-containing granules of rat atria. One interpretation of this finding is that PTHrP, like atrial natriuretic peptide, is released in response to stretch, but this concept has yet to be tested. Both PTH and PTHrP exert p r o n o u n c e d effects on cardiac function (61 ).

PTH.PTHrPReceptor

~~~----. ~ d k ~ ~ ~-"q~:---~-"Zllr~"icAMP" ';"" ~__...L'~--~ ImFt,4p"~dt L-VSCC i n h i b i t i o n

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FIG. 4 Model for PTHrP production and action in vascular smooth muscle cells (see text for description). (See color plates.)

PTHrP REGULATIONOF EXCITABLECELLS / Infusion of physiologic levels of N-terminal fragments of PTH and PTHrP induces hypotension and tachycardia in intact rats (5). In isolated perfused hearts, PTHrP induces chronotropic and ionotropic effects that are i n d e p e n d e n t of perfusion pressure (31). It has been established that the inotropic activity of PTHrP occurs indirectly in response to increased coronary blood flow (6). The mechanisms responsible for the chronotropic effects of PTH and PTHrP have been examined in cultured cardiomyocytes (60). In neonatal cardiomyocytes, PTH increases beating frequency through a cAMP-dependent pathway. These effects are associated with increased L-type calcium currents, precisely the opposite of what is observed in vascular smooth muscle cells. By contrast, in adult rat cardiac myocytes, both PTH (1-34) and PTHrP (1-34) increase the rate of spontaneous contraction, but only PTHrP was found to stimulate cAMP accumulation. The reason for this difference is unclear but may relate to the coupling of the P T H I R to different G proteins. PTH has also been shown to elicit a hypertrophic response in adult rat cardiomyocytes characterized by increases in protein synthesis, cell mass, and the reexpression of embryonic cardiac proteins. These effects, together with clinical observations of patients with elevated PTH and increased left ventricular mass, have been interpreted as evidence for a pathogenic role of PTH in ventricular hypertrophy. Finally, as discussed above, the timing (El0) of the embryonic death occurring in the PTHIR-null mice raises the possibility that PTHrP functions during heart development. Insight into the global actions of PTHrP in the cardiovascular system have come from studies in genetically manipulated mice. Transgenic mice overexpressing either PTHrP or the PTH1R in smooth muscle have reduced systemic blood pressure, consistent with the prediction that PTHrP acts as a local vasodilator (49). In aortic ring preparations from the PTHrP-overexpressing mice the relaxant effects of both PTHrP and acetylcholine seen in the nontransgenic mice were markedly attenuated in aortas from PTHrPoverexpressing mice. This finding suggests that local overexpression of PTHrP not only desensitizes the vasculature to PTHrP but also dampens relaxation to acetylcholine and perhaps other vasorelaxants. Thus it appears that prolonged stimulation of the PTH1R and the consequent increase in cAMP converge on signaling circuitry used by acetylcholine.

PTH-Related Proteins and Hypertensive States Several lines of circumstantial evidence suggest that PTH and PTHrP alter vascular tone in hypertensive humans and animals. For example, primary hyperparathyroidism is commonly associated with hyperten-

267

sion that may be corrected on removal of the parathyroid lesion (61). However, because alterations in circulating PTH also influence other regulators of vascular tone (e.g., ionized calcium), it is probable that the hypertension seen in long-term hyperparathyroidism is a secondary event. Alternatively, prolonged exposure to elevated PTH concentrations in these patients could desensitize vascular tissue to PTH or PTHrP and thereby increase vascular tone (55). A similar scenario appears to occur in two rat models of hypertension. For example, removal of the parathyroid glands in the spontaneously hypertensive (SH) rat and the DOCAsalt hypertensive rat attenuates the development of hypertension (62). Moreover, the PTH-induced changes in urinary cAME magnesium, calcium, and phosphorus responses are blunted in the SH rats, again suggesting a desensitization of the PTH1R. The apparent resistance to PTH and PTHrP in humans and rats with hypertension as described above p r o m p t e d Pang and co-workers to propose the existence of an additional "hypertensive" factor made in the parathyroid gland (63). This group has undertaken an extensive analysis of this factor, which they originally isolated from the serum of SH rats. Cross-transplantation of the parathyroid glands between SH rats and controls implicated the parathyroid gland as the source of the hypertensive factor. Furthermore, a polyclonal antibody raised against a partially purified factor reduced blood pressure in SH rats. However, despite over a decade of work on this putative hypertensive factor, its precise structure is still unknown.

NEUROLOGIC ACTIONS Introduction As already noted, interest in potential regulation of excitable cells by P T H / P T H r P began with Collip's demonstration in 1925 that parathyroid extracts had hypotensive effects in the dog. For the next 60 years, PTH was the focus of work in both vascular and nonvascular smooth muscle and in neurons. In smooth muscle, it now seems quite clear that the physiologic regulator is actually PTHrP, acting on the PTH1R. In the central nervous system (CNS), the best functional evidence also involves PTHrP acting on the PTH1R. In addition, there is evidence that PTH may influence pituitary function, and the description of TIP39 acting on the PTH2R may prove to be an important CNS regulatory system (see the following discussion). There are two aspects of the P T H - s m o o t h / c a r d i a c muscle literature that are relevant to PTHrP function in the CNS. The first is that the L-type voltage-sensitive calcium channel seems to be the pivotal target

268

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CI-IAPTV.k16

of P T H / P T H r P regulation. The second is that PTH a n d / o r PTHrP appear to be capable of either inhibiting or stimulating L-VSCC-mediated C a 2+ influx, d e p e n d i n g on the cell/tissue in question (56).

PTH/PTHrP Gene Family Expression in the CNS The CNS was one of the first sites to be examined in detail for PTHrP gene expression, and the gene was found to be widely expressed in neurons of the cerebral cortex, hippocampus, and cerebellum (64) (Fig. 5). This work was extended by a second survey that included the P T H I R as well as PTHrP (65). Both were found to be widely expressed, and they colocalized in a n u m b e r of sites (65). It was noted at the time that the hot spots for PTHrP gene expression are neuronal populations that have a n u m b e r of features in common, including high-density L-VSCC expression as well as high-density expression of excita-

ANTISENSE

tory amino acid receptors and a known susceptibility to excitotoxicity. T h o u g h there were early histochemical studies suggesting that PTH might be present in a n u m b e r of neuronal populations, the most careful and reproducible work has localized PTH to nuclei on the hypothalamus, with projections into the portal system (66,67). The implication is that PTH may regulate pituitary function, specifically including prolactin secretion (67). Usdin and colleagues identified the PTH2R in a cerebral cortical cDNA library by homology screening in 1995 (68). This receptor is highly sensitive to PTH(1-34) (EC50 about 1 nM) but is unresponsive to PTHrP(1-36). The PTH2R is most abundantly expressed in several basal forebrain nuclei and hypothalamic nuclei (58). Usdin et al. have also succeeded in purifying the natural ligand for this receptor using a staggering 50 pounds of bovine hypothalamus as starting material (69). This ligand is a small unmodified peptide of 39 amino acids, referred

SENSE

FIG. 5 In situ hybridization histochemistry of PTHrP mRNA in the CNS of the rat. A wide range of hybridization intensities was observed, with many positive neurons throughout all regions of the nervous system. In the hippocampus (hip; x35), dentate granule (g) cells, pyramidal (p) neurons, and large dentate interneurons (arrowhead) were densely labeled. In the section of cerebral cortex (ctx; cingulate cortex; x90) the intercerebral surface (s) is to the left. Many large pyramidal (p) neurons in the deep layers and granule (g) cells in layer II exhibited strong hybridization, in contrast to small neuronlike horizontal cells in the molecular (m) layer. In the cerebellum (crb; x35), granule (g) cell perikarya hybridized intensely, in contrast to weak signals observed for Purkinje cells (arrowheads) and basket and stellate neurons in the molecular (m) layer. Glia did not appear to stain positively (from Weir EC, et aL Proc Natl Acad Sci USA 1990;87:108, Copyright 1990 National Academy of Sciences, U.S.A.) (64).

PTHrP REGULATIONOF EXCITABLECELLS / to as tuberoinfundibular peptide 39 (TIP39), and it bears only nine of 39 amino acids that are identical to those of bovine PTH. Only limited structure-function work has been done, but TIP39 is at least as potent as PTH(1-34) at the PTH2R and may be one or two orders of magnitude more potent than PTH depending on the species of origin of the PTH2R (69). The sites of PTH2R expression imply potential TIP39 function in regulating the pituitary and in modulating pain sensitivity. Thus, three ligands and at least two receptors of the P T H / P T H r P gene family are expressed in the CNS. Two of the ligands (PTH and TIP39) are expressed in highly discrete locations, whereas PTHrP is widely expressed in neuronal populations throughout the brain.

Calcium Channels, Neuromodulation, and Signaling Microdomains Calcium Channels Calcium channels are heteromeric associations of four or five subunits (70). The oL1 subunit is the poreforming structure that is responsible for permeation as well as the gating function of the channel. There are a half-dozen classes of calcium channels, each defined by a specific e~1 gene. Given the n u m b e r of different genes for each subunit and alternate splicing of these gene products, the combinatorial possibilities are enormous (perhaps 1000). In brief, calcium channels are either L-type or nonL-type (e.g., N, P / Q , T, and R channels) (70). The Ltype channels mediate large and long-lasting (L) Ca 2+ fluxes and are composed of three subclasses, defined by their oL1 subunits as well as by the locations in which they were initially identified. These are S (skeletal; OLlS), C (cardiac; oL~c), and D (neuroendocrine; OLld). The L-channels are dihydropyridine sensitive, and there are a n u m b e r of classes of these widely used drugs (nifedipine, diltiazem). Virtually every class of calcium channel is expressed in the CNS (70). The N and P / Q channels are expressed in both pre- and postsynaptic locations and are involved in regulation of synaptic transmission. The L-channels are widely expressed in neurons t h r o u g h o u t the brain and are found only in postsynaptic locations, specifically on cell bodies and proximal dendrites (71). This localization is crucial to L-channel function. These channels appear to regulate cytosolic Ca 2+ levels in the soma and proximal dendrites of neurons as a function of the integrated excitatory synaptic input into these locations (71). Given the location and gating of these channels, it is quite clear that their C a 2+ c u r r e n t s are not involved in neurotransmission, but rather with fundamental aspects of neuronal cell biology such as regu-

269

lation of cellular signaling pathways and regulation of gene expression. Neuromodulation

The clustering of L-VSCCs on neuronal cell bodies is characteristic also of the location of n e u r o p e p t i d e / growth factor receptors. This clustering of receptors is strategically convenient to the nucleus as well as to the regulation of channels of all sorts and the capacity of peptides and growth factors to cross-talk with each other (72). This kind of short-range a u t o c r i n e / paracrine signaling to the soma and proximal processes of neurons is referred to as "neuromodulation" to emphasize that the regulation and signaling involved are very different from neurotransmission (72).

Signaling Microdomains Even a generation ago, it was clear that signal transduction corresponded to more than cells simply serving as bags of rising and falling tides of cyclic nucleotides a n d Ca 2+, but the biochemical details that account for the exquisite specificity of signal transduction have become clear only in the past decade. The work of Greenberg and co-workers has provided great insight into the specificity of neuronal C a 2+ signaling. Depending on the specific route of entry into a n e u r o n , C a 2+ has highly specific and differential effects on a wide variety of neuronal processes, such as gene expression, learning, and memory; modulation of synaptic strength; and CaZ+-mediated cell death (73). For example, C a 2+ entry via L-VSCCs elicits an entirely different response in terms of gene expression than does Ca 2+ entry mediated via NMDA receptors (73). Clearly, every calcium ion entering the cytosol of a n e u r o n is not perceived in the same way. Equally clearly, cAMP generated in a n e u r o n by a voltage-sensitive adenylate cyclase as opposed to a G protein coupled to a h o r m o n e receptor is not perceived by the cell in the same way. A major advance in understanding the specificity of signaling has come from the recognition that microdomains that exist at the cell surface cluster together the r e c e p t o r / c h a n n e l in question, the PKA a n d / o r PKC transducers, and the target to be modified. The key recent players that account for this clustering of specific signaling components are the A kinase anchoring proteins (AKAPs) and the receptors for activated C kinase (RACKs) (74). In certain cases, a single AKAP is capable of binding both PKA and PKC, thus serving as a scaffold that brings together all of the early components of a complex regulatory system. The net result of this tethering of signaling receptor, transducer, and target into a microdomain is a tremendous resolution in terms of specificity and speed.

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Cr~AeTF~k16

PTHrP Is Neuroprotective

KA

PTHrP Gene Expression in Neurons Is Regulated by L-VSCC Ca 2+ Influx

It turns out that the regulation of PTHrP gene expression in cerebellar granule cells is a classic example of the kind of specificity of C a 2+ signaling described in the previous section. Cerebellar granule cells are a hot spot of PTHrP and PTH1R expression in vivo (64, 65), and cultured cerebellar granule cells are a commonly used neuronal model system in vitro. PTHrP gene expression in these cells is a direct function of depolarization, which triggers L-VSCC C a 2+ influx that tracks to the PTHrP gene via the calmodulin/CaM kinase cascade (75, 76). C a 2+ entry into granule cells by any other means (e.g., veratridine treatment) has no influence whatsoever on the PTHrP gene. In granule cells, as in most other cells that express the PTHrP gene, PTHrP is a constitutive secretory product, so that the quantity of PTHrP secreted by the cell is a linear function of the level of PTHrP mRNA expression. PTHrP immunolocalizes principally to the granule cell soma (75), so that it is presumably secreted by the cell bodies, acting in the autocrine/paracrine fashion typical of a n e u r o m o d u l a t o r y peptide. PTHrP Inhibits L-VSCC Ca 2+ Influx, Defining a Protective Feedback Loop

Overstimulation can lead to neuronal injury or death, a process referred to as excitotoxicity. Excitotoxicity comes in two flavors. High concentrations of the excitory amino acid, glutamate, cause a generalized influx of cations and a collapse in mitochondrial function, leading to almost immediate necrosis (77). Lower concentrations of glutamate or exposure to other excitotoxins such as kainic acid trigger C a 2+ entry via L-VSCCs, and this leads to excitotoxicity characterized by a long latency (6-24 hours to death) (77,78). The granule cell system is subject to both the immediate and the latent forms of excitotoxicity. A low concentration of kainic acid produces about 50% granule cell death at 24 hours, and the calcium channel blocker, nitrendipine, is capable of fully protecting these cells, thereby defining the central importance of L-VSCC Ca '~+ influx in long-latency excitotoxicity (79). It will be recalled that PTH has been shown to inhibit

Neuron

EAA, K+, etc. ~

Depolarization

KA & PTHrP & antagonist

KA & PTHrP

FIG. 6 Cell death assessed by propidium iodide staining. Propidium iodide can bind to nuclear DNA only when the cell membrane is not intact; each bright dot therefore represents the nucleus of a dead cell. The left panel shows kainic acid (KA) alone, the middle panel shows KA plus PTHrP, and the right panel shows KA together with PTHrP and a 10-fold molar excess of a competitive antagonist of PTHrP binding. The percent kill (+_ SEM) under these three conditions was 23 _+ 3% (n = 10), 2 _ 2% (n = 11, P < 0.001 with respect to KA alone), and 23 +_ 2% (n = 10), respectively; bar = 25 IJ.M (Reprinted from Neurosci Lett, Vol. 274; ML Brines, Z Ling, AE Broadus. Parathyroid hormone-related protein protects against kainic acid excitotoxicity in rat cerebellar granule cells by regulating L-type channel calcium flux, pp. 13-16. © 1999, with permission from Elsevier Science) (79).

L-VSCCs in smooth muscle and neuroblastoma cells (56,80). This led to the working hypothesis that PTHrP might be capable of inhibiting L-VSCC C a 2+ influx in cerebellar granular cells, and this proved to be the case. PTHrP was found to be fully neuroprotective in kainic acid-treated granule cells (Fig. 6) and was as effective as nitrendipine in reducing kainic acid-induced L-VSCC C a 2+ influx (79). Pang's group used whole-cell patchclamp techniques to demonstrate that PTH is capable of inhibiting L-VSCC Ca 2+ influx in mouse neuroblastoma cells (80), and one of us (A.E.B.) has used patchclamp techniques to demonstrate the same findings with PTHrP in these cells. This effect is mediated by the PTH1R, but nothing is yet known of the mechanism by which the channel is actually regulated. Taken together, these findings indicate that PTHrP serves as an endogenous L-VSCC regulator that functions in a neuroprotective feedback loop of the sort depicted in Fig. 7. As shown, the L-VSCC is the fulcrum of this loop, and the rheostat is L-VSCC C a 2+ entry. This loop would provide neuroprotection to individual (autocrine) and neighboring (paracrine) neurons. The calcium hypothesis of neurodegeneration and neuronal aging holds that abnormalities in C a 2+ regu-

= VSCC ~ l t

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fl" PTHrP

1

Neuroprotection

FIG. 7 Scheme of autofeedback look in which PTHrP, triggered by L-VCSS Ca 2+ influx, feeds back via the PTH1R to dampen L-VCSS Ca 2÷ currents.

PTHrP REGULATION OF EXCITABLE CELLS / lation contribute to neuronal death and degenerative disorders such as Alzheimer and Parkinson diseases as well as to age-related vulnerability of neurons to such disorders (81). This hypothesis would predict that the absence of an endogenous neuroprotective p r o d u c t with the effects described above would predispose to excitotoxic neuronal damage and that this would likely be progressive. As described in Chapter 13, the PTHrP knockout mouse dies at birth as a result of a systemic chondrodystrophy. This mouse has been rescued by a genetic strategy, generating a mouse that is PTHrP-sufficient in chondrocytes but PTHrP-null in all other sites (82). There are no CNS abnormalities in the rescued mice before 4 months of age, but at 4 months and beyond, progressive neurodegenerative changes are seen in many regions of the cerebral cortex and in the posterior hippocampus (these data are noted only briefly because the work is yet unpublished). All of the involved areas are regions of known expression of L-VSCCs as well as of sensitivity to excitotoxic injury.

Other Potential Calcium Channel Effects PTHrP increases L-VSCC activity and thereby enhances d o p a m i n e secretion in PC-12 cells (83). PTH a n d / o r PTHrP have been reported to increase calcium channel-like activity in snail neurons (84) and in rat hippocampal neurons (85,86), but these effects are slow and perhaps involve channels other than the classic L-VSCC. UMR-106 osteoblast-like cells contain L-VSCCs that are stimulated by PTH treatment (87). L-VSCCs are also widely expressed in a great many other excitable and nonexcitable cells that have thus far not been examined with respect to PTHrP regulation.

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CI4AeI:F17 Physiolo gic Actions of PTH and PTHrP V. Epidermal, Mammary, Reproductive, and Pancreatic Tissues J O H N j. WYSOLMERSKI Yale University School of Medicine, New Haven, Connecticut 06520 ANDREW E STEWART University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

JOHN T. MARTIN st.

Vincent's Institute of Medical Research, Fitzroy, VIC3065 Australia

INTRODUCTION

ple studies have confirmed that in tissue culture, rodent and h u m a n keratinocytes express the PTHrP gene and secrete bioactive PTHrP (2). PTHrP expression has also been examined in skin in vivo using both immunohistochemistry and in situ hybridization. During fetal development in rats and mice, PTHrP is principally expressed within the epithelial cells of developing hair follicles (3,4). In mature skin, PTHrP has been found at low levels throughout the epidermis from the basal layer to the granular layer. Some studies have suggested that PTHrP is more highly expressed in the superbasal keratinocytes (5,6), although not all studies have reported this pattern (7,8). A variety of factors have been reported to regulate PTHrP production by cultured keratinocytes (2). For example, glucocorticoids and 1,25(OH)2D have been shown to downregulate PTHrP production, whereas fetal bovine serum, matrigel, and as yet unidentified factors secreted from cultured fibroblasts have been shown to up-regulate PTHrP production. The up-regulation of PTHrP production by fibroblast-conditioned media is particularly interesting, because PTHrP, in turn, acts on dermal fibroblasts, suggesting that it may function in a short regulatory loop between keratinocytes and dermal fibroblasts (9,10). Finally, in vivo, PTHrP expression has been shown to be up-regulated at the margins of healing wounds in guinea pigs (11). Interestingly, in this study, PTHrP was also detected in myofibroblasts and macrophages, suggesting that keratinocytes may not be the only source of PTHrP in skin. It is now clear that keratinocytes do not express the type 1 P T H / P T H r P receptor (PTH1R), but dermal

Documentation of the skeletal abnormalities in mice that either overexpressed parathyroid hormone-related protein (PTHrP) in their skeletons or had the genes for PTHrP and the PTH receptor ablated by the techniques of homologous recombination provided an exciting impetus for the rapid accumulation of knowledge regarding the mechanisms by which PTHrP regulates bone and cartlilage development and physiology. These findings are reviewed in Chapters 13 and 15. Over the past 5 years, increasing evidence has accumulated that PTHrP and the P T H / P T H r P receptor family also contribute to the development and functioning of several nonskeletal organs. The data regarding actions of PTHrP in the vascular system and the central nervous system are reviewed in Chapter 16. Here, the last of the series of chapters on the physiologic actions of PTHrP, we review the data regarding the functions of PTHrP in several other nonskeletal sites. We first consider functions of PTHrP in skin. Next, we review its functions in the mammary gland, placenta, and other reproductive tissues. Finally, we examine its role in the endocrine pancreas.

SKIN PTHrP and PTHrP Receptor Expression in Skin Normal human keratinocytes were the first nonmalignant cells shown to produce PTHrP (1), and multiThe Parathyroids, Second Edition

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fibroblasts do (12,13). PTHrP has been shown to bind to skin fibroblasts and to elicit biochemical and biologic responses in these cells (9,10,14). In addition, studies utilizing in situ hybridization have demonstrated that in fetal skin, P T H I R mRNA is absent from the epidermis, yet abundant in the fetal dermis, especially in those cells adjacent to the fetal keratinocytes (3,4,15). There are fewer data concerning the expression patterns of the P T H I R in more mature skin, but in mice, it appears that the relative amount of PTH1R mRNA in dermal fibroblasts is reduced in adult as compared to fetal skin (J.E Zhang a n d J J . Wysolmerski, unpublished data). Although keratinocytes do not express the classical P T H / P T H r P receptor, studies have shown that these cells bind and respond to PTHrP by inducing calcium transients, suggesting that there may be other receptors for PTHrP expressed on these cells (13,16). However, to date, no such receptors have been isolated, so their existence remains uncertain.

Biochemistry of PTHrP in Skin As described in Chapters 3 and 4, during transcription, the PTHrP gene undergoes alternative splicing to generate multiple mRNAs, which in human cells give rise to three main protein isoforms. In addition, each of these isoforms is subject to posttranslational processing to generate a variety of peptides of varying length. H u m a n keratinocytes have been shown to contain mRNA encoding for each of the three main isoforms, although, as in other systems, no clearly defined or unique role has yet emerged for any of the three individual isoforms (2). Keratinocytes have also been shown to process full-length PTHrP into a variety of smaller peptides including PTHrP(1-36) and a midregion fragment beginning at amino acid 38 (17). These cells have also been shown to secrete a large (---10 kDa) aminoterminal form that is glycosylated (18). There is currently no specific information regarding the secretion of COOH-terminal peptides of PTHrP in skin, but keratinocytes are also likely to produce these peptides.

Function of PTHrP in Skin There have now been several studies suggesting that PTHrP is involved in the regulation of hair growth. As noted above, in embryonic skin the PTHrP gene is most prominently expressed in developing hair follicles and overexpression of PTHrP in the basal keratinocytes of skin in transgenic mice leads to a severe inhibition of hair follicle morphogenesis during fetal development (19). The effects of PTHrP overexpression appear to act early during hair follicle induction, implicating PTHrP in the regulation of epidermal patterning dur-

ing embryogenesis. However, any such function of PTHrP during hair follicle morphogenesis is not critical, because disruption of the PTHrP or P T H I R genes does not seem to affect hair follicle formation or patterning in mice (20-22). In addition to effects on hair follicle morphogenesis, it has also been suggested that PTHrP may participate in the regulation of the hair cycle. It has been reported that the systemic administration of PTH1R antagonists to young mice perturbs the hair cycle by prematurely terminating telogen, prolonging anagen growth, and inhibiting catagen (23). These findings imply that PTHrP acts to inhibit hair follicle growth by pushing growing hair follicles into the growth-arrested or catagen/telogen phase of the hair cycle. If this hypothesis were correct, one would expect PTHrP knockout mice to exhibit findings similar to the PTH1R antagonisttreated mice. However, this does not appear to be the case. In mice that lack PTHrP in their skin, the hair cycle appears to be normal (22). In fact, rather than a promotion of hair growth, these mice demonstrate a thinning of their coat over time. These conflicting results are difficult to rationalize at this point, but they raise the intriguing possibility that the PTH1R antagonist might be inhibiting the function of another m e m b e r of the PTH receptor family and that there may be ligands other than PTHrP for such a receptor in skin. In addition to its effects on hair follicles, PTHrP has been implicated in the regulation of keratinocyte proliferation a n d / o r differentiation. Although there have been studies in cultured cells suggesting that PTHrP either enhances or inhibits keratinocyte proliferation, data from studies in vitro have suggested that PTHrP promotes the differentiation of keratinocytes (2). Studies in vivo have also supported a role for PTHrP in regulating keratinocyte differentiation, but have suggested that it inhibits their differentiation (22). A careful comparison of the histology of PTHrP-null and PTHrP-overexpressing skin demonstrated reciprocal changes in keratinocyte differentiation. In the absence of PTHrP, it appeared that keratinocyte differentiation was accelerated, whereas in skin exposed to PTHrP overexpression, keratinocyte differentiation appeared to be retarded (22). Therefore, in a physiologic context, PTHrP appears to slow the rate of keratinocyte differentiation and to preserve the proliferative, basal compartment. Remarkably, these changes in the rate of keratinocyte differentiation are exactly analogous to those noted for chondrocyte differentiation in the growth plates of mice overexpressing PTHrP as compared to PTHrP-null and PTH1R-null mice (2) (see Chapter 15). Again, at present, it is difficult to rationalize the conflicting data regarding the effects of PTHrP

PHYSIOLOGYOF PTH AND PTHrP on keratinocyte proliferation and differentiation, but the studies in genetically altered mice clearly indicate that PTHrP participates in the complex regulation of these processes in vivo. Further research will be needed to understand its exact role. An important but still unresolved question is whether the effects of PTHrP on keratinocyte proliferation, differentiation, and hair follicle growth are the result of its effects on keratinocytes directly or via its effects on dermal fibroblasts. At present there are more data to support the paracrine possibility. The P T H I R is expressed on dermal fibroblasts in vivo and in culture (4,12). Dermal fibroblasts have been demonstrated to show biochemical and biologic responses to PTHrP (9,10,14). Furthermore, PTHrP has been shown to induce changes in growth factor and extracellular matrix production that could, in turn, lead to changes in keratinocyte proliferation a n d / o r differentiation and hair follicle growth (9,10,24). Of course, the autocrine and paracrine signaling pathways are not mutually exclusive, but any direct autocrine effects of PTHrP on keratinocytes, as discussed above, would require the presence of PTHrP receptors other than the P T H I R on these cells. Although preliminary biochemical evidence has suggested that this possibility exists, no such receptors have been identified on keratinocytes (13,16). An alternative possibility by which PTHrP might have cell autonomous effects on keratinocytes is via an intracrine pathway involving its translocation to the nucleus (2). Clearly, much research is needed to define the receptors and signaling pathways by which PTHrP acts in skin. Only when this information is available will we be able to understand the mechanisms leading to the skin phenotypes that have been observed in the various transgenic models discussed above.

Pathophysiology of PTHrP in Skin To date, PTHrP has not been implicated in any diseases of the skin. It has been suggested that the skin and skin appendage findings in the rescued PTHrPnull mice are reminiscent of a series of disorders collectively known as the ectodermal dysplasias (22), but PTHrP has not been formally linked to any of these diseases. The most c o m m o n tumors causing humoral hypercalcemia of malignancy (HHM) are those of squamous histology, but these tumors rarely arise from skin keratinocytes. In fact, the most c o m m o n skin tumors, basal cell carcinomas, do not overexpress PTHrP and are not associated with hypercalcemia (2). Although PTHrP appears to participate in the normal physiology of the skin it is not clear at this juncture if it will be involved in skin pathophysiology.

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MAMMARY GLAND Very soon after its discovery, PTHrP was reported to be expressed in mammary tissue and to be secreted into milk (25,26). It is now known that PTHrP is critically important for the proper development and functioning of the mammary gland throughout life. In addition, it has been implicated as an important modulator of the biologic behavior of breast cancer. The mammary gland develops in several discrete stages and only reaches its fully differentiated state during pregnancy and lactation. PTHrP appears to serve different functions during these different stages of m a m m a r y development and, therefore, we will organize our discussion around three principal stages of mammary development: embryonic development, adolescent growth, and pregnancy and lactation. For each stage, we will first outline the pertinent developmental events in rodents, because the data regarding the function(s) of PTHrP largely come from studies in mice and rats. Next, we will discuss the localization of PTHrP and PTHrP receptors and the regulation of the expression of PTHrP and its receptors. Finally we will address the function of PTHrE

Embryonic Mammary Development In mice, there are two phases of embryonic mammary development. The first involves the formation of five pairs of m a m m a r y buds, each of which consists of a lightbulb-shaped collection of epithelial cells surr o u n d e d by several layers of fibroblasts known as the m a m m a r y mesenchyme (27). After the formation of these buds, mouse m a m m a r y development displays a characteristic pattern of sexual dimorphism. In male embryos, in response to androgens, the m a m m a r y mesenchyme destroys the epithelial bud and male mice are left without m a m m a r y glands or nipples (27). In female embryos, however, the m a m m a r y buds remain quiescent until embryonic day 16 (E16), when they u n d e r g o a transition into the second step of embryonic development, the formation of the rudimentary ductal tree. This process involves the elongation of the m a m m a r y bud, its penetration out of the dermis and into a specialized stromal c o m p a r t m e n t known as the m a m m a r y fat pad, and the initiation of ductal branching morphogenesis. At the time of birth, the gland consists of a simple epithelial ductal tree consisting of 15-20 branched tubes within a fatty stroma (27). This initial pattern persists until puberty, at which time the mature virgin gland is formed through a second r o u n d of branching morphogenesis, regulated by circulating h o r m o n e s (discussed below). The PTHrP gene is expressed exclusively within epithelial cells of the mammary bud, soon after it begins

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to form. PTHrP mRNA continues to be localized to mammary epithelial cells during the initial round of branching morphogenesis, as the bud grows out into the presumptive mammary fat pad and begins to branch (15,28,29). At some point after birth, PTHrP gene expression is down-regulated, and in the adult virgin gland PTHrP mRNA is found only within specific portions of the duct system (discussed below) (29). In contrast to the PTHrP gene, the PTH1R gene appears to be expressed within the mesenchyme but its expression is widespread and not limited to the developing mammary structures. Transcripts for the P T H I R gene are found within the mammary mesenchyme but also throughout the developing dermis (15,28). It is not clear when the receptor gene is first expressed within the subepidermal mesenchyme. However, it appears already to be present

when the mammary bud begins to form and it continues to be expressed within fibroblasts surrounding the mammary ducts as they begin to grow out and branch (28,29). The epithelial expression of PTHrP and the mesenchymal expression of the PTH1R are not unique to the developing mammary gland, and this pattern has long led to speculation that PTHrP and its receptor might contribute to the regulation of epithelialmesenchymal interactions during organogenesis. There is now solid evidence that this is the case during embryonic mammary development, when PTHrP appears to serve as an epithelial signal that influences cell fate decisions within the developing mammary mesenchyme. The data supporting this notion come from studies in several genetically altered mouse models. First, in PTHrP or PTH1R knockout mice, there is a failure of the normal

FI6. 1 Failure of sexual dimorphism during embryonic mammary development. Mammary buds from PTHrP knockout (A and B) and wild-type (B and D) embryos at E15. Note the destruction of the mammary bud in normal male embryos (B) as evidenced by the mesenchymal condensation that has obliterated the bud stalk (arrowheads) and the degenerating epithelial remnant (arrows). In PTHrP knockout males (A), this process is completely absent. The destruction of the mammary bud is an androgen-dependent phenomenon and the mesenchymal cells are the targets for androgen action, as evidenced by the positive staining for androgen receptors seen in the mammary mesenchyme surrounding a normal female mammary bud in D. In PTHrP knockout mammary buds, this process fails due to the failure of androgen receptor expression in the mammary mesenchyme, as shown in C (modified from Ref. 15 with permission).

PHYSIOLOGY OF P T H AND P T H r P

androgen-mediated destruction of the mammary bud, due to the failure of the mammary mesenchyme to differentiate properly and to express androgen receptors (15) (see Fig. 1). Second, in PTHrP or PTH1R knockout mice, the mammary buds fail to grow out into the fat pad and initiate branching morphogenesis, again due to defects in the mammary mesenchyme (28,29). Finally, in keratin 14 (K14)-PTHrP transgenic mice that ectopically overexpress PTHrP within all the basal keratinocytes of the developing embryo, subepidermal mesenchymal cells that should acquire a dermal fate instead become mammary mesenchyme (15). As demonstrated by these studies, PTHrP signaling is essential for mammary gland formation in rodents. When the mammary gland begins to form, the PTH1R is expressed in all the mesenchymal cells underneath the epidermis, but PTHrP is expressed only within the mammary epithelial buds and not within the epidermis itself (3,25,28). As the mammary bud grows down into the mesenchyme, the PTHrP produced by the mammary epithelial cells interacts over short distances with the PTH1R on the immature mesenchymal cells closest to the epithelial bud, and triggers these cells to differentiate into mammary mesenchyme. In this way, PTHrP acts as a patterning molecule contributing to the formation of small patches of mammary-specific stroma around the mammary buds and within the surrounding sea of presumptive dermis. The process of differentiation set in motion by PTHrP signaling is critical to the ability of the mammary-specific stroma to direct further morphogenesis of the epithelium. In the absence of this signaling, the mesenchyme can neither destroy the epithelial bud in response to androgens nor trigger the outgrowth of the bud and the initiation of branching morphogenesis (15,28,29). Although the above model was developed from studies in mice, it appears that PTHrP is also critical to the formation of breast tissues in human fetuses. It has been demonstrated that a fatal form of dwarfism known as Blomstrand chondrodyplasia is a result of null mutations of the P T H I R gene (30) (see Chapter 44). Affected fetuses have skeletal abnormalities similar to those caused by deletion of the PTHrP and P T H I R genes in mice (see Chapter 15), and, in addition, lack breast tissue or nipples (31). In normal human fetuses, the PTHrP gene is expressed within the mammary epithelial bud, and the PTH1R gene is expressed in surrounding mesenchyme (31). Therefore, in humans, as in mice, epithelial-to-mesenchymal PTHrP-PTH1R signaling is essential to the formation of the embryonic mammary gland.

Adolescent Mammary Development Following birth, the murine mammary gland undergoes little development until the onset of puberty. At

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that point, in response to hormonal changes, the distal ends of the mammary ducts form specialized structures called terminal end buds. These structures serve as the sites of cellular proliferation and differentiation for a period of active growth that gives rise to the typical branched duct system of the mature virgin gland (32). Once formed, the ductal tree remains relatively unchanged until another round of hormonal stimulation during pregnancy induces the formation of the lobuloalveolar units that produce milk. Similar to the findings in the embryonic mammary gland, during puberty, PTHrP appears to be a product of mammary epithelial cells and the P T H I R appears to be expressed in stromal cells (29). However, the structure of the pubertal gland is more complex than that of the embryonic gland and, here, there are conflicting data regarding the localization of PTHrP and the PTH receptor. Although there is general agreement that PTHrP is expressed in epithelial cells in the postnatal gland, there is some disagreement regarding the specific epithelial compartments in which PTHrP is found. Studies employing in situ hybridization in mice have suggested that, after birth, the overall levels of PTHrP gene expression in mammary ducts were reduced except for in the terminal end buds during puberty (29). In these structures, there were appreciable amounts of PTHrP mRNA detected in the peripherally located cap cells. In other parts of the gland there was little, if any, specific hybridization for PTHrP. In contrast, studies looking at mature h u m a n and canine mammary glands using immunohistochemical techniques have suggested that PTHrP can be found in both the luminal epithelial and myoepithelial cells throughout the ducts (8,33). Furthermore, studies using cultured cells have suggested that PTHrP is produced by luminal and myoepithelial cells isolated from normal glands (34-36). There have been fewer reports looking at the localization of P T H I R expression in the postnatal mammary gland, but as is the case during embryologic development, it is expressed in the mammary stroma (29). As with PTHrP gene expression, in situ hybridization studies have found the highest concentration of P T H I R mRNA in the stroma immediately surrounding terminal end buds during puberty (29). This same study found lower levels of P T H I R mRNA distributed generally within the fat pad stroma, but very little expression in the dense stroma surrounding the more mature ducts. In addition, these investigators found no evidence of PTH1R mRNA in freshly isolated epithelial cells (29). However, there have been reports suggesting that this receptor is expressed in cultured luminal epithelial and myoepithelial cells (35,36) as well as in cultured breast cancer cell lines (37). In summary, it is clear that during puberty, the expression of PTHrP and its receptor appear to be localized

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predominantly to the terminal end buds, with PTHrP found in the epithelium and the P T H I R found in the stroma. It remains an open and interesting question whether at some time during mammary ductal development epithelial cells express low levels of PTH1R. Studies in transgenic mice have demonstrated that PTHrP is an important regulator of mammary morphogenesis during puberty. Overexpression of PTHrP in mammary epithelial cells using the K14 promoter results in an impairment of ductal branching morphogenesis (38). There are two aspects to the defect. First, the terminal end buds advance through the mammary fat pad at a significantly slower rate than normal. Second, there is a severe reduction in the branching complexity of the ductal tree. As seen in Fig. 2, this results in a spare and stunted epithelial duct system. Experiments altering the timing and duration of PTHrP overexpression in the mammary gland by using a tetracycline-regulated K14-PTHrP transgene have demonstrated that the two aspects of this pubertal phenotype appear to represent separate functions of PTHrE The branching (or patterning) defect results from embryonic overexpression of PTHrP, whereas the

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ductal elongation defect is a function of overexpression of PTHrP during puberty (39). These effects on ductal patterning provide further evidence of the importance of PTHrP as a regulator of embryonic mammary development. In addition, the localization patterns for PTHrP and the P T H I R during puberty combined with the effects of pubertal overexpression of PTHrP on ductal growth suggest that PTHrP also functions later in mammary development. During puberty it appears to modulate epithelial-mesenchymal interactions that govern ductal elongation at the terminal end buds.

Pregnancy and Lactation Mammary epithelial cells reach their fully differentiated state only during lactation. Under hormonal stimulation during pregnancy, there is a massive wave of epithelial proliferation and morphogenesis, giving rise to terminal ductules and lobuloalveolar units. During the later stages of pregnancy the epithelial cells fully differentiate and then begin to secrete milk during lactation. By the time lactation commences, the fatty stroma of the mammary gland is completely replaced by

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FIG. 2 Overexpression of PTHrP in the mammary gland of K14-PTHrP transgenic mice antagonizes ductal elongation and branching morphogenesis during puberty. (A, B) Typical whole-mount analyses of the fourth inguinal mammary glands from wild-type (A) and K14-PTHrP transgenic mice (B) at 6 weeks of age. The dark oval in the center of each gland is a lymph node. Growth of the ducts during puberty is directional and each gland is arranged so that the primary duct (the origin of the duct system) is toward the center of the figure. Note that overexpression of PTHrP results in an impairment of the elongation of the ducts through the fat pad as well as a dramatic reduction of the branching complexity of the ductal tree. (C, D) Higher magnifications of a portion of the ducts from the wild-type (C) and transgenic (D) glands, demonstrating the reduction in side branching caused by overexpression of PTHrP (modified from Ref. 38 with permission)

PHYSIOLOGYOF PTH AND PTHrP actively secreting lobuloalveoli. On the completion of lactation, there is widespread apoptosis of the differentiated epithelial cells and the gland remodels itself into a duct system similar to that of the virgin animal (32). Localization studies in humans, rodents, and cows have all noted epithelial cells to be the source of PTHrP in the m a m m a r y gland during pregnancy and lactation (33,36,40,41). Based on the assessment of whole-gland RNA, PTHrP expression appears to be up-regulated at the start of lactation u n d e r the control of both local and systemic factors (2,25,42-44). Thiede and Rodan originally reported that in rats, PTHrP expression is d e p e n d e n t on suckling and on serum prolactin concentrations (25,42). However, prolactin must serve only as a permissive factor, for additional studies have shown that the suckling response is a local one and that PTHrP levels increase only in the milked gland (43). Furthermore, PTHrP expression gradually increases over the course of lactation, and in the later stages its expression becomes i n d e p e n d e n t of serum prolactin levels (44). It is clear that much of the PTHrP made during lactation ends up in milk, in which levels of PTHrP are up to 10,000-fold higher than in the circulation of normal individuals and 1000-fold higher than in patients suffering from humoral hypercalcemia of malignancy (2). PTHrP concentrations in milk have generally been found to mirror RNA levels in the gland, increasing over the duration of lactation, and rising acutely with suckling (43,45-47). In addition, there is evidence that PTHrP levels correlate with the calcium content of milk in humans and cows, but not in rodents (45-49). Finally, in mice, PTHrP mRNA levels are promptly down-regulated during the early stages of involution and then increase to prelactation levels about a week into the remodeling process, (M.E. Dunbar and J.J. Wysolmerski, unpublished data). In contrast to PTHrP, there has been little study of the expression or regulation of PTHrP receptors during pregnancy and lactation. In early pregnancy, the P T H / P T H r P receptor is expressed at low levels in the stroma surrounding the developing lobuloalveolar units (29). Studies using whole-gland RNA demonstrate a reciprocal relationship between PTH1R and PTHrP mRNA levels. That is, as PTHrP expression rises during lactation, PTH1R mRNA levels decrease, and as PTHrP mRNA levels fall during early involution, PTH1R expression increases to its former level (M.E. Dunbar and JJ. Wysolmerski, unpublished data). This may represent active down-regulation of the receptor by PTHrP or may simply reflect the changing a m o u n t of stroma within the gland at these different stages. However, in a study of cells isolated from lactating rats, it was suggested that epithelial cells as well as stromal cells express this receptor (36), so the regulation of the expression of this receptor during pregnancy and lactation may be complex.

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The initial reports of the presence of PTHrP in the mammary gland and in milk p r o m p t e d a great deal of speculation regarding its functions in breast tissue during lactation. These proposals have revolved around four general hypotheses: (1) PTHrP may be involved in maternal calcium homeostasis and the mobilization of calcium from the maternal skeleton; (2) PTHrP may be involved in regulating vascular a n d / o r myoepithelial tone in the lactating m a m m a r y gland; (3) PTHrP may be involved in transepithelial calcium transport into milk; a n d / o r (4) PTHrP may be involved in neonatal calcium homeostasis or neonatal gut physiology. Although the true functions of PTHrP during lactation remain obscure, there is some experimental evidence addressing the first two of these possibilities. These data are considered in the following discussions. However, at this point, the latter two ideas remain simple speculation and will not be discussed further. The control of maternal mineral metabolism and the mobilization of skeletal calcium for milk production remain enigmatic. Although a significant proportion of the calcium transported into milk is derived from the maternal skeleton, neither of the established calciumregulating hormones, PTH nor 1,25(OH)2D, seem to be necessary or sufficient to account for this p h e n o m e n o n (49). Therefore, the finding that PTHrP was produced in the lactating breast aroused interest in this protein as the missing factor acting to mobilize calcium during lactation. Although not every study has concurred, in support of such a role the weight of evidence across species now suggests that PTHrP levels in the systemic circulation are elevated during lactation (49). In addition, circulating PTHrP levels have been shown to correlate with bone density changes in lactating humans (50) and it appears that suckling leads to transient increases in circulating PTHrP levels (51). Suckling has also been shown to lead to increases in urinary phosphate and cAMP excretion in rodents and in cows (52,53), changes that might be expected if PTHrP released from the mammary gland was acting in a systemic fashion. Of course, none of these data actually prove that the source of the PTHrP in the circulation of lactating humans or animals is the mammary gland. More significantly, passive immunization of lactating mice with anti-PTHrP antibodies has not been found to influence maternal calcium homeostasis or the calcium content of milk (54). Therefore, although PTHrP remains an appealing candidate regulator of maternal calcium homeostasis during lactation, such an action remains unproved. The second potential function of PTHrP during lactation concerns the regulation of vascular a n d / o r myoepithelial cell tone. As discussed in Chapter 16, PTHrP has been shown to modulate smooth muscle cell tone in a variety of organs, including the vascular tree, where it acts as a vasodilator. Consistent with these effects, two studies have shown that PTHrP increases

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m a m m a r y blood flow during lactation (55,56). The injection of amino-terminal fragments of PTHrP into the m a m m a r y artery of dried ewes was shown to increase m a m m a r y blood flow and to override the vasoconstrictive effects of endothelin (55). Thiede and colleagues have demonstrated that the nutrient arteries of the inguinal m a m m a r y glands of rats make PTHrP and that its production is responsive to suckling and prolactin (56). Myoepithelial cells in the breast are similar, in some ways, to vascular smooth muscle cells and are thought to participate in the control of milk ejection by contracting in response to oxytocin (32). Therefore, it is interesting that myoepithelial cells in culture have been shown to express the PTH1R, and to respond to PTHrP by elevating intracellular cAMP (35,36). Furthermore, mirroring the effects of PTHrP on endothelin-induced contraction of vascular smooth muscle, PTHrP has been shown to block the rise in intracellular calcium normally induced in response to oxytocin in myoepithelial cells (35). Although much more study is needed, the current data support speculation that PTHrP might have effects on m a m m a r y blood flow a n d / o r milk ejection.

Pathophysiology of PTHrP in the Mammary Gland Although PTHrP has not been directly implicated in the pathogenesis of any specific disease of the mammary gland, there are now several instances in which it appears to contribute to pathophysiology in the h u m a n breast. First, as noted previously, fetuses afflicted with Blomstrand chondrodystrophy lack nipples and breast tissue (31). Second, there have been two case reports in which lactational hypercalcemia was noted to be related to elevations in circulating levels of PTHrP (57,58). One of these cases was caused by massive breast hyperplasia, and the patient required reduction mammoplasty in order to ameliorate her hypercalcemia (57). Finally, the area with the most potential impact on h u m a n health is the relationship of PTHrP production to breast cancer. This is evolving into a complicated topic and will be addressed only briefly here. However, it will be reviewed in more depth in Chapter 43. It is well documented that PTHrP is produced by a n u m b e r of primary breast carcinomas and that this sometimes leads to classic humoral hypercalcemia of malignancy (59). A potentially more widespread role may be the involvement of PTHrP in the osteotrophism of breast cancer (60,61). Animal models have suggested that PTHrP production by breast tumor cells is important to their ability to form skeletal metastases (60,61). However, there is conflicting evidence as to whether PTHrP production by a primary breast tumor is predictive of bone metastases in patients (62,63). The largest and most carefully controlled study to date suggested

that PTHrP production by the primary tumor is actually a negative predictor, not a positive predictor, of skeletal metastases (63). It may be that PTHrP production does not enable a tumor cell to get into the skeleton, but once there, the ability of tumor cells to up-regulate PTHrP production within the bone microenvironment becomes important to their ability to grow in the skeleton (61). These are important issues and ongoing studies should provide us more information in the near future.

REPRODUCTIVE TISSUES PTHrP and Placental Calcium Transport Nearly all of the calcium, and a large proportion of the inorganic phosphate (85%) and magnesium (70%), transferred from the mother to the fetus is associated with development and mineralization of the fetal skeleton (64). The concentrations of both total and ionized Ca in all mammalian fetuses studied during late gestation have been observed to be higher than maternal levels. As a result of studies in which sheep were used extensively for the study of fetal calcium control, one of the first suggested physiologic roles of PTHrP was that of regulating the transport of calcium from m o t h e r to fetus in the mammal, thereby making calcium available for the growing fetal skeleton (65). Immunoreactive PTH levels were found to be low in fetal lambs, whereas PTH-like biological activity in serum was high (66), suggesting the presence of another PTH-like substance. Parathyroidectomy in the fetal lamb resulted in loss of the calcium gradient that exists between m o t h e r and fetus as well as impairment of bone mineralization, implicating the parathyroids as the source of the regulatory agent. Crude, partially purified, or recombinant PTHrP, but neither PTH nor maternal parathyroid extract that contained no immunoreactive PTHrP, restored the gradient (65). Thus, PTHrP appeared to be the active c o m p o n e n t of the fetal parathyroid glands responsible for maintaining fetal calcium levels and suppressing fetal PTH levels. In support of this hypothesis, immunoreactive PTHrP was found to be readily detectable in sheep fetal parathyroids from the time they form (67) and also was found in early placenta, suggesting that the latter tissue may be a source of PTHrP for calcium transport early in gestation. The portion of PTHrP that appears to be responsible for regulating placental calcium transport lies between residues 67 and 86 (68), but the responsible receptor has not yet been identified. T h o u g h the syncytiotrophoblasts are believed to be central in the transport of calcium to the fetus, the cytotrophoblasts (which differentiate to form the syncytium) are believed to be the calcium-sensing cells, and elevating the extracellu-

PHYSIOLOGYOF PTH AND PTHrP lar calcium concentration has been shown to inhibit PTHrP release from these cells (69). The calciumsensing receptor (CaSR) has been localized to cytotrophoblasts of h u m a n placenta (70) and the work of Kovacs et al. (71) has implicated it in placental calcium transport. Furthermore, a calreticulin-like calciumbinding protein has been isolated from trophoblast cells and its expression is increased by treatment with PTHrP(67-84) but not with N-terminal PTHrP (72). A working hypothesis for how the CaSR, midregion PTHrP, and a midregion PTHrP receptor might interact to regulate transplacental calcium transport is presented in Fig. 3. Although these observations are strongly suggestive of involvement of PTHrP and the CaSR, the mechanisms of placental calcium transport are still not fully understood. Support for the role of PTHrP also comes from the PTHrP gene knockout mouse in which placental calcium transport is severely impaired (73). In mice homozygous for deletion of the PTHrP gene, fetal plasma calcium and the maternal-fetal calcium gradient were significantly reduced. When fetuses were injected in utero with fragments of PTHrP or PTH, cal-

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FIG, 3 A potential model for the interactions of PTHrP and the calcium-sensing receptor in regulating placental calcium transport. A decrease in circulating calcium levels in the fetus activates the calcium-sensing receptor on the cytotrophoblast. This leads to PTHrP secretion by the cytotrophoblast, which may then act via the putative midmolecule PTHrP receptor to promote calcium transport from maternal to fetal circulation. The nature and cellular location of the midmolecule PTHrP receptor are unknown, but are likely to be on the syncytiotrophoblast implicated in calcium transport across the placenta. Midmolecule PTHrP signaling could involve several mechanisms as indicated and may include participation of a calcium-binding protein (CaBP) (modified from Bradbury, Ph.D. Thesis, University of Sydney 1999, with permission).

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cium transport was significantly restored only by treatment with a midmolecular region of PTHrP that does not act via the PTH1R. Thus, the conclusions from the murine studies are similar to those in the sheep, namely, that PTHrP contributes to fetal skeleton calcium supply by controlling maternal-fetal calcium transport through actions mediated by a midmolecule portion of the PTHrP molecule.

Uterus and Extraembryonic Tissues The uterus, both pregnant and nonpregnant, is another of the many sites of production and action of PTHrE The relaxing effect of PTH on uterine smooth muscle has been long recognized (74), and it is not surprising that PTHrP has the same effect (75). The finding that expression of mRNA for PTHrP in the myometrium during late gestation in the rat was controlled by intrauterine occupancy by the fetoplacental unit raised the possibility of a role for PTHrP in regulating uterine muscle tone (76). In studies in rats with or without estrogen treatment, protein and mRNA for PTHrP were localized not only in the myometrium, as had been shown in pregnancy (76), but also in the epithelial cells lining the e n d o m e t r i u m and endometrial glands. Indeed, the strongest PTHrP production appeared to be in these sites (77), suggesting that the e n d o m e t r i u m and endometrial glands might be the major uterine site of PTHrP production, and that PTHrP might be a local regulator of endometrial function and myometrial contractility. Estrogen treatment enhanced uterine production of PTHrP, but most significantly, the relaxing effect of PTHrP on uterine contractility in vitro was greatly enhanced by pretreatment of noncycling rats with estrogen. In keeping with this observation, uterine horns from cycling rats in proestrous and estrous phases of the cycle showed a greater responsiveness to PTHrP than did those from noncycling rats. These findings are consistent with a role for PTHrP as an autocrine a n d / o r paracrine regulator of uterine motility and function. Furthermore, they suggest that PTHrP belongs to a class of other locally acting peptides such as oxytocin, vasoactive intestinal peptide, and relaxin, for which pretreatment of animals with estrogen increases the response of the uterus (78-80). Further evidence for a specific and regulated role of PTHrP in the uterus during gestation comes from the observation of a temporal pattern in the relaxation response to PTHrP by longitudinal uterine muscle during pregnancy in the rat, with maximal responses at times when estrogen levels would be high. In contrast, the circular muscle did not respond at any stage during gestation (81). The inability of PTHrP to relax uterine muscle in the last stages of gestation does not support a direct role in the onset of parturition. It has been

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hypothesized that PTHrP may be involved in keeping the uterine muscle relaxed to accommodate the fetus during pregnancy, with the demonstration (76) that expression of mRNA was dependent on the presence of the fetus and that levels increased throughout pregnancy and decreased sharply after delivery. It seems likely, therefore, that the observed decrease in PTHrP level reflects the recontracted state of the uterine muscle, consistent with the observation in the bladder (82), and that the level of expression is functionally related to contractility. The temporal expression of PTHrP in the endometrial glands and blood vessels (81) also supports roles in other regulated functions that might include uterine growth during pregnancy and the regulation of uterine and placental blood flow (83). Placenta and Fetal Membranes

PTHrP mRNA and protein have been detected in rat and human placenta in various cell types (69,84,85). In addition, neoplastic cells of placental origin secrete PTHrP, including hydatidiform moles and choriocarcinomas in vitro (86). The presence of P T H / P T H r P receptor mRNA has been demonstrated in rat (87) and human (88) placenta, and infusion of PTHrP(1-34) into isolated h u m a n placental lobules stimulates cyclic AMP production (89). Three further sets of observations lend support to the hypothesis that PTHrP is involved in placental/uterine interactions and that its most likely role in the placenta and placental membranes is related to the growth and maintenance of the placenta during pregnancy. First, PTHrP production by cultured amniotic cells has been shown to be regulated by prolactin, h u m a n placental lactogen, transforming growth factor-J3 (TGF-[3), insulin, insulin-like growth factor, and epidermal growth factor (90). Second, PTHrP has been shown to regulate epidermal growth factor receptor expression in cytotrophoblast cultures (91), an event associated with placental development. Third, studies of vascular reactivity in isolated human placental cotyledons preconstricted with a thromboxane A2 mimetic showed PTHrP to be a very effective vasodilator (92). The narrow concentration range to which the tissue responded, together with the desensitization in response to repeated PTHrP infusions, seemed consistent with a paracrine a n d / o r autocrine action of PTHrP in human gestational tissues. Adequacy of the fetoplacental circulation is essential for the nutritional demands of the growing fetus, and both humoral and local factors are likely to be important in its control. It is possible that alterations of the expression, localization, a n d / o r action of PTHrP might contribute to the genesis of conditions such as preeclampsia and intrauterine growth retardation, in

which placental vascular resistance is increased (93). Another related and potentially interacting influence is angiotensin II, known to be a powerful enhancer of PTHrP production in the vasculature and in human placental explants (94). The ability of angiotensin II to stimulate estradiol production in h u m a n placental explants through actions on its AT 1 receptor (95) provides a further link with PTHrP control. The most likely source of the increased amniotic fluid PTHrP concentrations during pregnancy is the amnion, because PTHrP mRNA expression is also highest at term and greater in the amnion than in choriodecidua or placenta (96-98). In tissue from women with full-term pregnancies and not in labor, the concentration of N-terminal PTHrP has been found to be higher in amnion covering the placenta than in the reflected amnion covering the decidua parietalis (96). Nevertheless, the concentration of N-terminal PTHrP in reflected amnion (the layer apposed to the uterus) was inversely related to the interval between rupture of the membranes and delivery. The observation that PTHrP levels in the amnion decrease after rupture of the fetal membranes has led to the proposal that PTHrP derived from the membranes may inhibit uterine contraction, and that labor may occur following loss of this inhibition. Plasma levels of PTHrP increase during pregnancy and at 6 weeks postpartum (99,100), with the likely sources being placenta and breast, respectively. Human fetal membranes have been shown to inhibit contractions of the rat uterus in vitro (101), so this tissue does appear to produce factors that can modulate uterine activity. Furthermore, primary cultures of human amniotic cells secrete PTHrP into the medium (84). Thus, though the physiological functions of amnion-derived PTHrP are currently unknown, the preliminary evidence suggests that it may play a role in the regulation of the onset of labor. It is also possible that it is a source of PTHrP ingested by the fetus, with a growth factor role in lung a n d / o r gut development. In summary, although many functional studies remain to be completed, potential roles for PTHrP produced by fetal membranes and placenta include transport of calcium across the placenta, accommodation of stretch of membranes, growth and differentiation of fetal a n d / o r maternal tissues, vasoregulation, and the regulation of labor.

Implantation and Early Pregnancy Some physiologic functions other than control of myometrial activity were suggested by findings of Beck et al. (102), who identified PTHrP mRNA as being limited to epithelial cells of implantation sites. This pregnancy-related expression appeared at day 5. 5 in

PHYSIOLOGYOF PTH AND PTHrP the rat fetus in the antimesometrial uterine epithelium of implantation sites, raising the possibility of a further function of PTHrP, playing a part in the localization of implantation or initial decidualization. Decidual cells produced mRNA for PTHrP both in normal gestation and after the induction of deciduomata. Expression of the gene in these cells followed epithelial expression by 48 hours. It was concluded from this work that the location of PTHrP gene expression in the uterus, together with the time of its expression, suggests that it plays a part in implantation of the blastocyst. Further evidence for a function of PTHrP in the implantation process came from Nowak et al. (103), who showed that PTHrP and TGF-[3 were potent stimulators of trophoblast outgrowth by mouse blastocysts cultured in vitro. The TGF-[3 effect appeared to be mediated by PTHrP, which was acting through a mechanism distinct from the PTHIR. Thus, both the timing and localization of PTHrP gene expression suggested that it might play a part in the implantation of the blastocyst (102). On finding substantial levels of immunoreactive PTHrP in uterine luminal fluid of estrogen-treated immature rats, and because the P T H / P T H r P receptor was known to be expressed in rat uterus (87), Williams et al. (104) investigated the role of PTHrP acting through this receptor, in influencing early pregnancy in the rat. Infusion of either a PTHrP antagonist peptide or a monoclonal anti-PTHrP antibody into the uterine lumen during pregnancy resulted in excessive decidualization. The latter appeared to be the result of a decrease in the n u m b e r of apoptotic decidual cells in the antagonist infused horn. In p s e u d o p r e g n a n t rats, infusion of receptor antagonist into the uterine lumen resulted in increases in wet weight of the infused h o r n compared with the control side, indicating an effect on decidu o m a formation. These observations suggest that activation of the P T H / P T H r P receptor by locally produced PTHrP might be crucial for normal decidualization during pregnancy in rats, probably not by being involved in the initiation of the decidual reaction, but rather in the maintenance of the decidual cell mass. Summary The multiple roles of PTHrP in the reproductive tissues and cycle and in the placenta largely reflect its roles as a p a r a c r i n e / a u t o c r i n e / i n t r a c r i n e regulator. Of the many functions it exerts in these systems, probably the only endocrine role is that in which PTHrP in the fetal circulation regulates placental calcium transport. There remains much to be learned of the role of PTHrP in reproductive and placental physiology and pathology.

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THE END O CRINE PANCREAS PTHrP and Its Receptors in the Endocrine Pancreas The presence of PTHrP in the pancreatic islet became apparent shortly following the identification of PTHrP in 1987. Drucker, Asa, and colleagues (105) demonstrated that PTHrP was present in islet cells, and demonstrated that it was present in all four cell types of the islet, including the alpha, beta, delta, and PP cells. PTHrP mRNA was demonstrated to be present in isolated islet RNA as well (106), demonstrating that the peptide could be p r o d u c e d within the islet. Gaich and collaborators (107) confirmed these findings, demonstrating that PTHrP was indeed present in islet cells of all four types, and that it was also present in pancreatic ductular epithelial cells. The peptide is not present in adult pancreatic exocrine cells. Plawner and colleagues (108) demonstrated that PTHrP is present in individual beta cells in culture, and showed that PTHrP colocalized with insulin in the Golgi apparatus, as well as in insulin secretory granules. Interestingly, in a perifusion system employing a beta cell line, PTHrP was shown to be cosecreted with insulin from beta cells following depolarization of the cell (108). The secreted forms of PTHrP included amino-terminal, midregion, and carboxy-terminal forms of PTHrP (see below). With regard to receptors for PTHrP on beta cells, little direct evidence has been provided for the presence of the PTHIR, although its presence has not been rigorously sought. On the other hand, there can be no question as to the presence of some type of PTHrP receptor on the pancreatic beta cell, because it is clear that PTHrP(1-36) elicits p r o m p t and vigorous responses in intracellular calcium in cultured beta cell lines. For example, Gaich et al. have demonstrated that PTHrP(1-36) in doses as low a s 1 0 -12 M stimulates calcium release from intracellular stores (107). Interestingly, unlike events observed in bone and renal cell types in which PTHrP receptor activation is associated with activation of both the cAMP/PKA and the PKC/intracellular calcium pathways, only the latter is observed in cultured beta cells in response to PTH or PTHrP(1-36) (107). W h e t h e r this reflects the presence of a different type of receptor on beta cells or differential coupling of the P T H I R to subsets of specific G proteins or catalytic subunits in beta cells, as compared to bone and renal cells, has not been studied.

Regulation of PTHrP and PTHrP Receptors There is little information describing how or to what degree PTHrP or the PTH receptor family is regulated in the pancreatic islet. As will become clear from the

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following discussions, there are physiologic reasons why such regulation might occur u n d e r normal circumstances, but this area remains unexplored.

Biochemistry of PTHrP in the Endocrine Pancreas PTHrP undergoes extensive posttranslational processing as described in Chapters 3 and 4. Most of what is known or inferred regarding PTHrP processing is derived from studies in the rat insulinoma line, RIN1038 cells (17,109,110). These cells have served as a model of PTHrP processing because they have been shown to serve as a model for authentic processing of other h u m a n neuroendocrine peptides such as insulin, proopiomelanocortin, glucagon, and calcitonin. Using a combination of untransfected RIN-1038 cells, RIN-1038 cells overexpressing hPTHrP(1-139), hPTHrP(1-141), or hPTHrP(1-173), and a panel of region-specific radioimmunoassays and immunoradiometric assays, RIN cells have been shown to secrete PTHrP(1-36), PTHrP(38-94), PTHrP(38-95), and PTHrP(38-101) (17,109,110). In addition, RIN-1038 cells have been shown to secrete a form of PTHrP that is recognized by a PTHrP (109-138) radioimmunoassay (109), and another form that is recognized by a PTHrP (139-173) radioimmunoassay ( 111 ). As described above, PTHrP(1-36) stimulates intracellular calcium increments in cultured beta cells (107). PTHrP(38-94) has also been shown to stimulate intracellular calcium release in these cells (110). PTHrP(38-94) does not activate adenylyl cyclase in cultured beta cells, and other PTHrP species have not been explored in beta cells in functional terms.

Function of PTHrP in the Endocrine Pancreas Pancreas development in rodents begins at approximately days E9-E10, and by days E18-E19, clusters of beta cells have begun to coalesce and form immature islets (112). These islet cell clusters continue to increase in number, in size, and in density of beta cells in the week following delivery and then decline abruptly in n u m b e r through a wave of beta cell apoptosis (113). The role of PTHrP in beta cell development and function is poorly understood at present. The pancreas of PTHrP-null mice (20) develops normally in anatomic terms (R.C. Vasavada and A.E Stewart, unpublished observations), but nothing is known about the function of these islets. PTHrP-null mice die immediately after delivery, so nothing is known of islet function or development following birth in the absence of PTHrP. "Rescued" PTHrP mice do exist (28) and they survive to adulthood. These mice have normal-

appearing pancreata and islets (R.C. Vasavada and A.E Stewart, unpublished observations), but they have dental abnormalities, are undernourished, and they grow poorly. Therefore, it is difficult to characterize their islets in functional terms, because islet mass, proliferation, and function are heavily d e p e n d e n t on fuel availability. Steuker and Drucker have suggested that PTHrP may play a role in beta cell differentiation, because it is up-regulated in beta cell lines in the presence of the islet-differentiating agent, butyrate (114). In an effort to understand the role of PTHrP in the pancreatic islet, Vasavada and collaborators have developed transgenic mice that overexpress PTHrP under the control of the rat insulin-II p r o m o t e r (RIP) (115,116). RIP-PTHrP mice display striking degrees of islet hyperplasia and an increase in islet n u m b e r as well as in the size of individual islets. This increased islet mass is associated with increased function: RIP-PTHrP mice are hyperinsulinemic and hypoglycemic as compared to their littermates (115,116). They become profoundly and symptomatically hypoglycemic with fasting. Interestingly, RIP-PTHrP mice are also resistant to the diabetogenic effects of the beta cell toxin, streptozotocin. Following the administration of streptozotocin, normal mice readily develop diabetes, but RIP-PTHrP mice either fail to become diabetic or develop only mild hyperglycemia (116). The mechanisms responsible for the increase in islet mass in the RIP-PTHrP mouse remain undefined. There are two levels at which this question can be addressed: identification of the source of the cells responsible for the increase in islet mass and the signaling mechanisms that are responsible for the increase. With respect to the first, islet mass can, in theory, be increased by three pathways: (1) the recruitment of new islets from the pancreatic duct or its branches distributed t h r o u g h o u t the exocrine pancreas, in a process referred to as "islet neogenesis"; (2) induction of proliferation of existing beta cells within islets; a n d / o r (3) prolongation of the life span of existing beta cells. Of these options, there is clear evidence against the second possibility (115), suggesting that islet neogenesis [e.g., PTHrP is present in the normal pancreatic duct and is up-regulated during ductular differentiation into beta cells (107,117)] or inhibition of islet cell death (as occurs in the presence of PTHrP in other cell types) is the likely explanation. These processes are u n d e r active study. At the signaling level, little is known regarding the mechanism of action of PTHrP on beta cells. Though it is known that PTHrP can stimulate intracellular calcium in cultured beta cell lines (107,110), it is not known whether this occurs in vivo in normal, nontransformed beta cells within intact islets. Nor is it known if PTHrP stimulates adenylyl cyclase in normal beta cells in vivo, or if it participates in nuclear or intracrine sig-

PHYSIOLOGYOF PTH AND PTHrP naling in beta cells as it appears to in chondrocytes, osteoblasts, vascular smooth muscle cells, or other cell types (118-120) (see Chapter 6). These processes, too, are u n d e r study. Finally, and importantly, the results of overexpression studies do not demonstrate that PTHrP plays beta cell mass-enhancing roles in vivo u n d e r normal circumstances. In the absence of meaningful data from knockout or rescued knockout mice, it is difficult to be sure if PTHrP is important in normal islet biology. This question, too, must await further studies, such as examination of the conditional or islet-specific deletion of the PTHrP gene.

Pathophysiology of PTHrP in the Endocrine Pancreas From the preceding discussion, it is clear that the normal physiologic role of PTHrP in the pancreatic islet remains undefined. In contrast, PTHrP plays clear pathophysiologic roles in at least some pancreatic islet neoplasms. PTHrP overexpression with resultant development of humoral hypercalcemia of malignancy has been demonstrated on multiple occasions in multiple investigators' hands (105,121-123). In the only large series of studies of malignancy-associated hypercalcemia in which tumors have been fully subdivided based on histology (123), islet cell carcinomas, which are not particularly common, produce humoral hypercalcemia of malignancy fully as often as pancreatic adenocarcinomas, a very c o m m o n neoplasm. Historically, islet tumors were among the first in which PTHrP bioactivity was identified (121,122). Furthermore, patients with islet carcinomas regularly demonstrate increases in circulating PTHrP as determined by radioimmunoassay or immunoradiometric assay (21). When assessed by immunohistochemistry, these tumors also demonstrate increased staining for PTHrP (105,106). The significance of these findings for islet tumor oncogenesis is not known. Is this simply a r a n d o m derepression of the PTHrP gene? Or is it a specific up-regulation of the PTHrP gene? Is there a pathologic role for PTHrP in the development of pancreatic islet tumors, corresponding to the mass enhancing effects of PTHrP in the islets of the RIP-PTHrP mouse? These questions remain interesting but unanswered at present.

CONCLUSION Advances in mouse genetics and in transgenic technology have been a b o o n to the study of physiology. This has certainly been the case for the PTHrP field, wherein studies in genetically altered mice have provided a starting place for the study of the physiology of

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a protein that was discovered out of its natural context. In this chapter we have outlined the current state of knowledge regarding the physiologic roles of PTHrP in skin, the m a m m a r y gland, placenta, uterus, and pancreas. Much of this information (although not all) has come from studies performed in a variety of transgenic mice. These studies have shown that PTHrP is important to both the development and the physiologic functioning of these organs. However, at this point, we have only the rudiments of an understanding of the functions of PTHrP at these sites. Thus, we continue to have more questions than answers. There will be many challenges to be overcome before we truly c o m p r e h e n d all the nuances of the functions of PTHrP at these sites. There will also be new tools with which to investigate these questions (as this chapter is being written, several eagerly anticipated experiments ablating the PTHrP and P T H I R genes in organ-specific fashion are in the pipeline). The next several years promise to be an exciting time for the investigation of the nonskeletal effects of this remarkable molecule, and the next edition of this volume will certainly d o c u m e n t some upcoming surprises.

ACKNOWLEDGMENTS The authors would like to thank Dr. Jane Moseley for valuable discussions and advice. This work was supported by the National Health and Medical Research Council of Australia (to J.T.M.), the National Institutes of Health (DK47168 and DK55023 to A.ES.; DK55501 to j j . w . ) , the U.S. Department of Defense (DAMD17-96-16198 to j.j.w.), and the Juvenile Diabetes Foundation (to A.ES.).

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CHAPTER18 Parathyroid Growth Normal and Abnormal

A. M I C H A E L

PARFITT

Little Rock, Arkansas 72205

Division of Endocrinology and Centerfor Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Sciences,

INTRODUCTION AND BACKGROUND

parathyroid gland. Yet another reason is that people are attracted to endocrinology by their fascination with the h o r m o n e s and hormonal mechanisms of secretion and action, rather with the morphology of their glands of origin. A neglect of gland size may be justified pragmatically if the aim is restricted to understanding normal physiology, but it involves neglect also of several interesting scientific questions. Why is the parathyroid gland so much smaller than many other endocrine glands? Why is total parathyroid weight a m u c h larger fraction of body weight in the chick (---10 m g / k g ) than in the rat (---1 m g / k g ) (3)? Such questions lie within the realm of evolutionary biology, but their answers might well provide information relevant to medical science. At a more practical level, short-term changes in h o r m o n e secretion are of lesser magnitude than those in other glands and are not sustainable indefinitely; long-term changes, whether they are expressions of adaptation or of disease, invariably require changes in cell n u m b e r as well as in individual cell function. Every parathyroid disorder considered in this book is a reflection of, or at least is associated with, characteristic changes in the n u m b e r of functioning parathyroid cells. Understanding the mechanisms whereby parathyroid cells are able to change their n u m b e r is essential to a full understanding of pathogenesis and is also relevant to both medical and surgical treatment. However, before these clinically important issues are approached, it is necessary to review concepts of normal organ and tissue growth, the regulation of cell n u m b e r as the balance between cell proliferation and cell death, and the normal growth

Why Study Parathyroid Growth? The functional performance of every endocrine gland requires delivery of the right n u m b e r of horm o n e molecules into the circulation during each successive interval of the appropriate time scale. Total h o r m o n e secretion comprises the aggregate contribution of each cell and so depends not only on the average secretion per cell, but also on the n u m b e r of contributing cells. The regulation of cell number, it seems, should receive as much attention as the regulation of individual cell behavior, but in practice it is almost entirely ignored. For example, in a 3000-page textbook of endocrinology (1), less than 1% of the material is concerned with the attainment and maintenance of gland size. Whether there are 100 or 1 billion cells in a gland appears not to matter, and cell n u m b e r is never considered explicitly in the description of feedback relationships. There are several reasons for this neglect. The rules of development normally ensure that each organ grows to the right size (2), so that adult cell n u m b e r varies only over a three- to fourfold range. By contrast, h o r m o n e secretion by many endocrine glands can vary acutely over as much as a 100-fold range, which must reflect changes in the performance of individual cells. Another reason is that the endocrine glands are among the smallest of organs. Only the thyroid and the gonads can be palpated; the other glands are inaccessible clinically, and estimation of their size by noninvasive methods is of varying precision, which is least for the The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER18

and turnover of parathyroid tissue. A central theme will be the close interrelationship between the regulation of h o r m o n e secretion and the regulation of cell n u m b e r in the parathyroid glands.

Concepts of Growth There is more to biologic growth than increase in size; growth has been described as "the study of change in an organism not yet mature" (4), which includes the processes of development and the determination of form, both external and internal. Relative growth is most rapid in utero, although for some tissues and organs absolute growth is more rapid after birth. It is useful to extend the scope of growth to cover also maintenance by turnover, repair, and regeneration (5). Growth can be studied at different levels of organization, both functional and structural. A functional unit, the smallest structure that can carry out a specific function by itself, may correspond to any structural level: whole organisms, organ, tissue, cell, or subcellular entity. In the endocrine system, functional units may be either multicellular, such as thyroid and ovarian follicles, or unicellular, as in the anterior pituitary and the testis. All functional units can increase or decrease in size (hypertrophy or hypotrophy), but only some can increase or decrease in n u m b e r (hyperplasia or hypoplasia) (3,5,6). During early development, hyperplasia, whether of individual cells or of functional units, precedes hypertrophy (2,7). After birth there is overlap between these different modes of growth, although in most organs significant hypertrophy is absent after the first 2 weeks, and at all ages hyperplasia is the main determinant of increase in size (3). The basic instrument of growth is the cyclical process of cell replication and division. Despite spectacular advances in the understanding of this process (8), along with understanding of its stimulation by an ever larger n u m b e r of growth factors (8-10), knowledge concerning growth regulation at higher levels of organization remains incomplete (11,12). In species of finite life span and determinate size, a growth target for the whole body and for each organ has to be specified very early in development (2,5), and approach toward the target has to be monitored and controlled in accordance with a characteristic growth curve. Precisely how such a "sizostat" (13) could function remains a mystery, but its rules of operation must be encoded somewhere in the g e n o m e (11). Many general theories of growth have been proposed, but none has become established. Almost 200 years ago, J o h n H u n t e r concluded that growth was regulated by functional d e m a n d (3). This remains a sound principle of physiologic adaptation in mature organisms but cannot be the main explanation for developmental growth. During functional adapta-

tion, hypertrophy is the initial response and may remain the only response if the work of the cell is mainly physical or if the functional unit is multicellular and of sufficient complexity that new ones cannot develop after birth, as for n e p h r o n s or lung alveoli (5). However, if the work of the cell is mainly biochemical, as in the endocrine glands, eventually hyperplasia will also develop. It has been proposed that the default state of all normal cells is proliferation rather than quiescence (12). In accordance with this notion, each organ or tissue may secrete, in proportion to its size, an inhibitor of its own growth, referred to as its chalone (3,14). Much evidence for the existence of chalones has been assembled (15), including feedback regulation of intestinal crypt cell proliferation in a m a n n e r consistent with the chalone concept (16), but no chalone has been fully characterized (14). A currently more popular theme is external control by means of tissue-specific stimulators, but most of these remain unidentified, and most known growth factors are ubiquitous in their distribution and sites of action (8-12). In some organs the same growth factors may be involved in developmental growth, regeneration after injury, and functional adaptation (17,18). In the pituitary and probably also in the glands u n d e r its control, the same h o r m o n e s stimulate growth as well as h o r m o n e secretion (19), but at the appointed time growth stops, yet h o r m o n e secretion continues. There is evidence for central regulation of targeted whole-body growth by a non-growth-hormone-dependent mechanism in the brain (3,13), but individual organs are more likely to be controlled locally by some widely distributed system, such as cells derived from the neural crest, lymphoid tissue (3), or vascular endothelium (20). The same growth factor could be used by different tissues, target specificity depending on autocrine or paracrine mechanisms (9), or on the constraints of the local microcirculation, but this would not explain how a predetermined size was chosen and reached. The possible role of parathyroid endothelium (21) is mentioned below.

Patterns of Cell Renewal All cells can be classified, on the basis of their current relationship to the cell cycle (Fig. 1), as cycling or noncycling. Cycling cells are in one of the four stages of the cycle: G 1, S, Gz, or M. Noncycling cells may be in a resting state (Go) between one cycle and the next or no longer dividing because of terminal differentiation (14,22). Cells in G o have withdrawn from the cycle and differ from cells in G 1 in several respects (14), most obviously in that they are not enlarging in preparation for cytokinesis. From the standpoint of cell kinetics, all adult tissues have traditionally been classified into one

PARATHYROID GkOWrH: N O I ~ . L AND ABNORMAL /

295

G2 ¸

M

DIFF (NC)

S

" ~ Death

"1~ (shedding)

Discontinuous (low turnover) (GO) G2

M

v

DIFF (Go)

y

S

.,d

"1~ Death (apoptosis)

8

FI6. 1 Cell cycle in two types of tissue with respect to replication. The cell cycle comprises two periods of executionmDNA synthesis (S) and mitosis (M)--interrupted by two periods of preparationmgaps (G) 1 and 2. Durations (in hours) are chosen partly for convenience of illustration but are broadly representative of human nonneoplastic tissue (14). As well as duplication of the nucleus, the cycle involves duplication of all other constituents of the cell. The interrupted line between M and early G is traversed only by continuously cycling cells. Other cells enter a quiescent state with respect to proliferation (Go), the duration of which varies from days to decades in different tissues. Tissues in which replication is continuous (top) have high turnover, and a separate population of stem cells (ST). Periodically, a stem cell switches from G o to G1, and on average the mitosis leads to one new stem cell and one cell committed to differentiate. The commitment step is amplified by continuous cycling in a dividing transit (DT) compartment for a variable number of generations (two in this case). The differentiated cells (DIFF) are noncycling (NC) and are eventually lost by shedding, or by sequestration, as in bone. Tissues in which replication is discontinuous (bottom) have low turnover and no stem cell or dividing transit compartments. Periodically, a differentiated cell switches from G o to G1, and the result of the mitosis is two new differentiated cells. The addition of each new cell is balanced by loss of one old cell by apoptosis; it is this type of replication that occurs in the parathyroid gland.

of three major types (14,22). Tissues with a high rate of cell turnover (continuous replicators) have a high rate of mitosis, which occurs initially in a functionally (and often anatomically) separate population of stem cells (Table 1). These cells, usually in G 0, repeatedly traverse the cell cycle, each complete cycle on the average producing one new cell committed to differentiate in a particular direction and one new stem cell. Renewal of stem cells probably depends on a stochastic balance between two types of symmetric cell division rather than on each stem cell division being asymmetric (23). The committed daughter cells undergo clonal expansion in a transit c o m p a r t m e n t for a variable n u m b e r of generations (22,24). The n u m b e r of terminally differentiated, nondividing, but functionally active cells is maintained approximately constant by continuous shedding, either from the body (as in the skin or the intestinal mucosa) or into the circulation (as in the bone marrow).

TABLE 1

Contrasting Characteristics of Two Mechanisms for Maintenance of Adult Tissue Mass Type of replication

Aspect Cell function Cell life span Mitosis Potential Location Rate Cell loss Mechanism Rate

Continuous

Discontinuous

Poststem cells only Short

All cells Long

Stem cells and transit cells only Often segregated High

All cells

External b shedding High

Internal b deletion Low

Scattered LOWa

aCan increase sharply in response to various stimuli. bwith respect to the tissue.

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CHAPTF~k18

By contrast, tissues with low cell turnover (discontinuous replicators or conditional renewal tissues) have a low rate of mitosis and no separate stem cell population (14,22) (Table 1). All cells of the tissue spend most of their time in G o carrying out normal function, but all have the potential for undergoing cell division. Periodically, a functioning cell enters the cell cycle (G o + G1), and the result of the mitosis is two new functioning cells. Cell balance is maintained by a few cells losing the capacity for division, eventually dying, and being removed in some way. The low basal rate of mitosis can be increased by the need to regenerate (as in the liver) or by increased functional d e m a n d (as in endocrine glands) and occurs at r a n d o m t h r o u g h o u t the tissue. Tissues such as brain and cardiac muscle used to be regarded as nonreplicators, the cells of which had permanently lost the capacity to divide, but it is now evident that at least some cells retain this capacity (25,26), although the great majority of adult cells in these tissues will not divide during a normal lifetime. Continuously and discontinuously replicating tissues differ in their relationship to the control of cell cycle progression, which depends on the sequential synthesis and destruction of different members of the cyclin family (27), and on passing several checkpoints, at which are verified successful completion of earlier stages and maintenance of genomic integrity (28). In the former, the duration of the cycle may be rate limitingma reduction from 36 to 24 hours could increase the rate of proliferation by 50%. But if the interval between successive divisions is more than 3 months, shortening the cycle can have only a trivial effect; tissue turnover depends only on the frequency with which quiescent cells enter the cycle; very little is known about how the switch from G o to G 1 is regulated (29), although protooncogenes such as c-myc may promote reassembly of the cell cycle machinery (8). What is the mechanism of cell loss in tissues with a low rate of turnover and no obvious means of shedding cells? Except in pathologic situations in which the blood supply is jeopardized, it does not occur by necrosis. In recent years a nonnecrotic mechanism of cell deletion has been identified, referred to as apoptosis (30). This process normally affects scattered single cells and has characteristic histologic and ultrastructural features, beginning with condensation and disintegration of the nucleus, followed by breaking up of the whole cell into m e m b r a n e - b o u n d fragments of varying size, which are rapidly subjected to phagocytosis by adjacent cells. The remaining cells simply close ranks so that no gap is left by the deleted cell. The distinction between necrosis and apoptosis may be likened to the distinction between m u r d e r and suicide. Apoptosis is an active process d e p e n d e n t on altered gene expression, of which the molecular mechanisms are u n d e r intense

investigation (31). Apoptosis is a convenient way of eliminating cycling cells with irreparable DNA damage (32) but more commonly occurs in noncycling cells that have outlived their usefulness (33). Particularly relevant to the present discussion is that whenever tissue that is hyperplastic as the result of endocrine stimulation undergoes involution, the process of cell loss occurs by apoptosis (3,30). For regulating the n u m b e r of functioning cells in tissues and organs with normally low turnover, apoptosis is as important as mitosis (30).

NORMAL PARATHYROID GROWTH

AND TURNOVER Methods of Study

The most straightforward m e t h o d of studying growth is to express some index of size, either weight or volume, as a function of age. Weight is preferred in that it is easier to measure accurately and is in general use in anatomy and pathology. Because of variation in tissue density, weight is a more accurate index of cell n u m b e r than is volume (= weight/density). A commonly used approximation is that 1 g of tissue contains 2 "~° (1.094 × 109) cells (34). This disregards variation in cell size but in the parathyroid gland gives results very similar to total DNA content (35). Parathyroid tissue contains fat cells and a vascular connective tissue stroma as well as the parenchyma consisting of chief cells and their derivatives (see Chapter 1). Parenchymal volume as a fraction of total volume can be measured by point counting on histologic sections (36), and parenchymal weight as a fraction of total weight can be derived from measurement of wholegland density, assuming a constant value for fat cell density (37). Estimated parenchymal weight provides a more accurate value for parenchymal cell n u m b e r than does total weight. Regrettably, even though "the best histological criterion (of diagnosis) is the weight of the gland" (3), weighing of parathyroid glands is frequently omitted, and only linear dimensions are recorded. The product of three dimensions (rectangular volume; RV) is significantly correlated with weight (r = 0.81) and can be used to estimate weight from the regression, Wt (g) = 0.585RV (cm ~) + 0.134 (38). The product of only two dimensions, measured in a representative section, also correlates quite well (r = 0.76) with weight (39). If the parathyroid glands are very small, as in the rat, measurement of weight is less accurate because complete removal of extraneous tissue is more difficult, and measurement of volume is a good alternative. This can be obtained by means of the Cavalieri principle from serial sections that cover the entire length of the e m b e d d e d gland. Volume can be calculated from the n u m b e r of sections, the average distance between the

PARATHYROIDGROWTH: NORMALAND ABNORMAL / sections, and the average cross-sectional area in the sections (36,40). For an unbiased estimate, it is necessary that the location of the first section with respect to the pole of the gland is selected randomly, but this precaution has usually been omitted. In the past, section area was measured by various planimetric and projection methods, but today point counting with systematic random sampling and an unbiased counting frame (36), digitization, or automated image analysis (41) would be used. The estimation of gland volume in this m a n n e r is essential for the most accurate distinction between hyperplasia (increase in cell number) and hypertrophy (increase in cell size). Average cell profile area can be measured in sections and average cell volume calculated on the basis of reasonable assumptions (42). More accurately, the n u m b e r of cells or cell nuclei per unit volume of tissue can be obtained through the disector m e t h o d (42). The distinction can also be approached biochemically, using total DNA as an index of cell number, and total protein as an index of total cell mass; the p r o t e i n / D N A ratio remains u n c h a n g e d in hyperplasia but increases in hypertrophy (3,14). Cell number, cell size, and tissue mass represent the "bottom line" of growth but give no information concerning mechanisms. For a complete description of cell cycle kinetics, the proportion of cycling cells (or growth fraction) and the durations of each phase of the cell cycle must be determined (14,22). Together these give the birth rate of new cells, from which can be calculated the rate of increase in tissue volume, usually expressed as potential doubling time (34). Comparison of this with the actual rate of tissue growth provides an indirect estimate of the rate of cell loss (22,30,34). The necessary methods are complex, are most appropriate for rapidly growing tissues, and have never been applied to the parathyroid gland. More generally useful is determination of the proportion of cells in one or more stages of the cell cycle (Table 2). The durations of S, G2,

TABLE 2

Cell Cycle Markers a

Aspect

Marker

Feature

Need either administration in vivo or cell survival ex vivo or in vitro Information already in nucleus

[3H]Thymidine BrDu

Accepted standard Non radioactive

Mitosis Ki-67 Cyclin/PCNA

First to be used Short half-life DNA repair as well as synthesis

aExcept for mitosis, these markers mainly label S phase but Ki-67 and cyclin/proliferating cell nuclear antigen (PCNA) label a greater fraction of the cycle.

297

and M phases (Fig. 1) vary between fairly narrow limits among different tissues (14,22), and, if a representative value is assumed, the birth rate of new cells can be estimated, provided the rate of proliferation is stable and growth is slow (34,38,40). The first cell cycle marker to be used was the change in nuclear morphology during mitosis (43). Prompt fixation for an adequate duration in fluid of the right pH is n e e d e d (44), and positive cells are much less frequent than with other cell cycle markers because the duration of mitosis is m u c h shorter (Fig. 1). If mitosis is arrested, mitotic figures will be more frequent, but cell birth rate can no longer be estimated. The most well established of such methods is identification of S-phase cells by autoradiography after administration of tritiated thymidine (14,22,34). Much less satisfactory is m e a s u r e m e n t of thymidine incorporation into acid-insoluble macromolecules, the results of which are influenced not only by the rate of DNA synthesis but also by thymidine pool size, activity of thymidine kinase, utilization of the salvage pathway for pyrimidine biosynthesis, and diffusion of thymidine into dead or dying cells (3,17,45). These pitfalls are m u c h more significant with acute in vitro experiments than with steady-state in vivo measurements, and in parathyroid adenomas there is a good correlation between thymidine incorporation and the proportion of S-phase cells (35,46). The S-phase cells can also be labeled by bromodeoxyuridine, a nonradioactive analog of thymidine that can be detected by immunostaining (3). Results can be obtained more quickly, but the m e t h o d is less useful for following the migration of labeled cells or the recognition of subsequent division by the dilution of label intensity. More widely applicable are methods for the immunologic identification of other cell cycle markers (43); because the information is already present in the nucleus, nothing has to be administered, and the methods can be applied to paraffin-embedded sections and so to archival material. Of those most commonly used, the MIB-1 antibody to the Ki-67 antigen has several advantages over proliferating cell nuclear antigen (PCNA). The latter has a long halflife so that expression persists for some time after the end of the cycle, expression can be induced in adjacent noncycling cells, and expression can occur in cells undergoing DNA repair as well as in cycling cells (40,47). Whatever m e t h o d is used, it is essential that an appropriately randomized sampling procedure is adopted, because biased sampling, which is often deliberate in diagnostic studies, can increase the a p p a r e n t prevalence of labeled cells by up to 50-fold (47). Cells can be sorted according to their stage in the cycle by flow cytometry based on their content of DNA, which doubles between G0/G 1 and Gz/M phases and is intermediate in S phase (48). Despite the very large n u m b e r of cells that can be counted, it is impossible to

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Cell Number, Proliferation, and Loss as

get accurate results when cell turnover is low and the growth fraction very small, because of cellular debris and other artifacts (49). In this circumstance, changes in DNA content are m u c h more likely to reflect changes in ploidy than changes related to the cell cycle. Cells with a normal n u m b e r of chromosomes are referred to as diploid. Cells with a diploid n u m b e r increased exactly by a power of 2 are polyploid, most commonly tetraploid, and occasionally octaploid or of higher ploidy (50). Polyploidy is a normal occurrence in the liver and the heart. The mechanism is unknown but presumably involves normal duplication of each chromosome, with failure of mitosis and cytokinesis. Such cells are, in effect, arrested in G2, a condition that may also represent a response to a growth stimulus that shares features of both hyperplasia and hypertrophy (14). Euploid cells can be either diploid or polyploid. Cells with an abnormal c h r o m o s o m e n u m b e r that does not differ exactly by a power of 2 from the diploid number are aneuploid, either h y p o - o r hyperdiploid. This is the result of loss or duplication of individual chromosomes, a cytogenetic abnormality that is not related to normal cell replication (50,51).

Cell No. (10 6)

Wt. (mg)

19.3

18 -

16.1

15 -

12.9

12 -

9.7

9-

6.6

6-

3.2

3-

0

0-

PRENATAL

_

0

8

-

F u n c t i o n s o f Age The parathyroid glands appear abruptly during the fifth week of gestation (52). Based on the very small a m o u n t of fat present during growth and a mean parenchymal cell density of 1.065 (37), the glands grow to a total parenchymal weight of---3 lxg at a crownr u m p length of---30 m m , corresponding to a gestational age o f - - - 8 weeks (3). Weight increases quite slowly from 3 to 6 txg between 8 and 12 weeks, but then increases m u c h more rapidly, growing to ---300 Ixg (0.3 mg) between 12 and 18 weeks, and ---4 mg, corresponding to ---4.3 × 1 0 6 cells at birth (3). Based on interpolation and smoothing of quite sparse data (3), parathyroid growth appears to follow a sigmoid curve through the end of the first year of life, or up to 92 weeks of conceptional age (Fig. 2). The conversion of weight to cell n u m b e r is only approximate, because in the rat parathyroid cell volume increases ---50% in the first 10 weeks of life (53). Similar sigmoid growth curves have been found for the adrenal, thyroid, and pituitary glands (54), but with the inflections occurring before

=B

•

I

•

I I

POSTNATAL

w

!

r

!

!

f

I

I

i

i

!

16

24

32

40

48

56

64

72

80

88

96

Age (weeks since conception)

FIG. 2 Parathyroid growth curve during the first 2 years of life. Total parenchymal weight and calculated cell number are plotted as a function of age (in weeks) since conception. The cell number scale assumes that 1 mg of tissue contains 1.074 x 106 cells (34) and disregards modest changes in parathyroid cell volume during growth (53). A continuous curve w a s constructed based on data in Refs. 3 and 52. The dashed line denotes time of birth (B); the dotted line indicates predicted outcome of continued exponential growth at the peak rate found at 16 weeks.

PARATHYROIDGROWTH: NORMALAND ABNORMAL / rather than after birth. If the interpretation given in Fig. 2 is correct, absolute weight gain is most rapid at "--20 weeks after birth, although relative weight gain is most rapid at ---16 weeks of gestational age. Parathyroid growth continues at the rate, established toward the end of the first year, of ---2.5-3.0 mg/year. The data are too few to demonstrate a second parathyroid growth spurt during adolescence, but such a spurt has been found in every organ and tissue for which sufficient data are available (54), and it seems reasonable to assume that such a spurt would also occur in the parathyroid gland (Fig. 3). Growth then occurs progressively more slowly until the mature parenchymal weight is reached at age ~-30 years (3,55). Total mature weight in white subjects has varied from "-85 mg (3,55,56) to "--95 mg (57,58), corresponding to a total cell n u m b e r of (90-100) × 10 6, with a coefficient of variation [CV; = (SD/mean)100] of---30% (55), but there is no significant further change with increasing age (3,56,57). In different series, parathyroid weight in m e n has been less than (3), the same as (55), or more than (56,58) that in women. In three series (56,58,59), parathyroid weight has been significantly greater in black than in white subjects of both sexes, to a greater extent than could be accounted for by differences in body weight. Also in blacks, in contrast to whites, there appear to be significant changes in parathyroid weight after attainment of maturity, with peak values in both sexes between the ages of 41 and 60 years (58).

Cell No. (106)

During embryonic development, in organs and tissues that change in location or shape, as well as in size, or in which provisional structures must be replaced, growth is partly offset by apoptotic deletion of unwanted cells (30), but there is neither evidence nor apparent need for this process in the developing parathyroid gland. Assuming the absence of apoptosis, the first derivative of the growth curve, or growth velocity, provides an estimate of the absolute frequency of mitoses associated with growth, which is about five times higher during the first year of life than at any other time (Fig. 3), presumably with a second, much smaller peak during the adolescent growth spurt. The absolute frequency of cell division depends on the n u m b e r of cells present at a particular time and on the relative rate of cell division, or specific cell birth rate; the latter rate determines how frequently successive divisions occur in the same cell line. Disregarding turnover and considering only growth, this rate is most rapid during the second trimester of embryonic development, which is the only time of life when the interval between successive divisions is 2 SD above the normal mean, whether arithmetic or geometric, although the highest values were far outside the normal range. Replacement of fat is an early stage of hyperplasia (213), but, even taking this into account, mean parenchymal weight could not have been increased by more than two- to threefold. In a more recent series, total parathyroid parenchymal weight was positively correlated with serum creatinine and was inversely correlated with renal weight, but hyperplasia could be detected histologically in patients without clinically manifest renal dysfunction (217). In patients with rickets or osteomalacia as the main skeletal abnormality, the geometric mean parathyroid weight was similar to, and individual values were in the same range as, that in the unselected cases (216). The data suggest that mild parathyroid hyperplasia occurs in most patients with chronic renal failure (87) and in the predialysis era the aggregate increase in cell number was equivalent to a small adenoma. In some dialysis patients PTH levels continue to increase, but the PTH assays commonly used probably exaggerate the degree of PTH hypersecretion in renal failure (218). In patients with osteitis fibrosa as the main skeletal abnormality, there was very little overlap with the unselected cases; the geometric mean weight was increased >30-fold, and both the mean and range were much the same as for single adenomas causing osteitis fibrosa in primary hyperparathyroidism (Table 5). In later years, parathyroid weights were published mainly for patients who were selected for surgical treatment, usually because of osteitis fibrosa or hypercalcemia or both (219-223) (Table 9). The values for total excised weight obviously reflect both historic changes and local variation in surgical policy but are in general quite similar to the total weights previously found at autopsy in patients with osteitis fibrosa. In patients with hyperparathyroidism that was of severity sufficient to merit either individual publication of autopsy findings or selection for surgical treatment, the increase in parathyroid weight was so much greater than in unselected patients with chronic renal failure or in those with defective bone mineralization alone (Table 9) that the difference is likely to be qualitative as well as quantitative. Although precise figures are not available, it seems reasonably certain that such severe hyperparathyroidism remains more frequent than in the prehemodialysis era, mainly because longer survival provides more time for the necessary parathyroid growth to occur. A good candidate for a factor that could lead to much more severe parathyroid growth in a minority of patients is an increase in PTH secretory set point; abnormal growth would then be driven by the same

mechanism as in primary parathyroid adenomas with a set-point dysfunction, with the difference that many cells would be affected, not just a single cell. Conceptually, the set point is a property of individual cells (75) and was originally determined by manipulation of PTH secretion in vitro (100). Studies of set point in vivo in patients with normal renal function have given consistent and physiologically intelligible results (75,101,189). However, in patients with renal failure the results have been inconsistent and often uninterpretable partly because of differences in experimental protocol (224) and perhaps partly because of unrecognized defects of current PTH assays in uremic patients (218). For example, intravenous calcitriol has been reported both to decrease (225) and to increase (226) the set point. With this reservation in mind, in unselected dialysis patients the secretory set point appears to be normal and not to increase with the severity of hyperparathyroidism (227). But in many patients needing surgical removal of parathyroid tissue, an increase in secretory set point has been found in dispersed cells obtained at surgery (228), an abnormality even more evident in cells harvested from nodular regions within the enlarged gland (229). More recently, reduced expression of the CaSR, the probable regulator of the set point, has been found in such patients (76,178). However, because of the many factors involved in chronic renal failure that impair the effectiveness of PTH in raising plasma calcium (see Chapter 39), the increase in total h o r m o n e secretion necessary to attain the new set point can be achieved only with an enormous increase in the n u m b e r of secretory cells. Consequently, the asymptotic value for total gland size is much more difficult to reach than in primary hyperparathyroidism. An increase in secretory set point due to reduced calcium receptor expression (76,178) also provides a plausible explanation for the development of hypercalcemia in a minority of patients with severe secondary hyperparathyroidism (223,230). This is often attributed to autonomy of h o r m o n e secretion, but in such patients, both parathyroid weight (220,230) and the rate of DNA synthesis (223) are positively correlated with both PTH levels and plasma calcium; indicating that it is not h o r m o n e secretion but rather growth that has become autonomous, because plasma calcium is behaving as the dependent, rather than as the independent, variable (75), a conclusion supported by the sustained restoration of normocalcemia after surgery (223,230). The birth rate of new cells is higher than in primary parathyroid adenomas, and does not slow down as in primary hyperparathyroidism, but remains as rapid as when the glands first began to enlarge soon after the onset of renal failure (223). Calcitriol increases CaSR expression in rat parathyroid tissue

PARATHYROID GROWTH: NORMALAND ABNORMAL /

(231 ), so that calcitriol deficiency could account for the increase in set point (223) as well as for the progressive resistance to the hypercalcemic effect of PTH. If so, the target of the parathyroid cells would continually recede instead of remaining stable, accounting for the difference in growth behavior between the two disorders (223). This p h e n o m e n o n would be augmented by the lower levels of calcitriol receptors in parathyroid cells from uremic patients (232), which would blunt the suppressive effect of calcitriol on both hormone secretion and cell proliferation, and possibly contribute further to CaSR underexpression (180). Further insight into the mechanism of the growth disorder is provided by the histologic distinction between diffuse and nodular hyperplasia (see Chapter 1). The nodules consist of cells that are more closely packed together with larger nuclei and a greater prevalence of cell cycle markers (221,233), more cycling cells by flow cytometry (234), higher expression of cyclin D1 (233), greater depletion of calcitriol receptors (232), higher secretory set point (229), and greater reduction of CaSR expression (76,178). These characteristics account for the significantly greater increase in total parathyroid weight (Table 9) and for the continued growth, often rapid, of many parathyroid autografts (235). Detailed histochemical and immunocytochemical studies indicate similarity in gene expression between the cells in each nodule but differences between nodules (236). Each nodule could have arisen from a different single cell or, more likely, from a group of adjacent cells that were the clonal descendants of a founder cell present during embryonic development and so constituted a patch (153). In this sense, nodular hyperplasia is multiclonal, in contrast to the polyclonality of diffuse hyperplasia and the monoclonality of adenomas. There can be as many as 100 nodules in four enlarged glands, so the likelihood that each nodule arose from a separate mutation is infinitesimally small. In some cases, parathyroid glands removed from such patients have been reported as monoclonal, implying an origin from a single mutant cell (237). As previously discussed, the biochemical determination of clonality, based on polymorphism for X-linked DNA sequences, is less rigorous than the morphologic determination of clonality, based on studying gene expression in individual cells (153,154). Consequently, the proportion of the original hyperplastic gland that contributed to the tumor, and the relationship between such a tumor and the hyperplastic nodules previously described, are unclear. The parathyroid growth response to chronic renal failure progresses through several stages. Diffuse secondary hyperplasia (polyclonal) is initiated by hypocalcemia, becomes more severe as the result of hyperphosphatemia and calcitriol deficiency, and leads

317

eventually to osteitis fibrosa in some patients (216). Because of the additional effects of CaSR underexpression to increase the parathyroid secretory set point and VDR underexpression to decrease responsiveness to calcitriol, continued growth can lead to hypercalcemia (223); the hyperplasia becomes nodular (multiclonal) and the glandular enlargement asymmetric (238). The next stage is the emergence of an adenoma in one, or occasionally more than one, of the nodules, as the expression of a genetic dysfunction in one of the cells undergoing the most rapid proliferation; the accumulation of 1.0 g of additional parathyroid tissue in 5 years requires a 50-fold increase in the frequency of mitosis. This sequence is referred to as tertiary hyperparathyroidism (3,239), a term often misapplied to patients with hypercalcemic secondary hyperparathyroidism but more accurately reserved for the disorder that combines in its etiology the hyperplasia of secondary hyperparathyroidism with the monoclonality of primary hyperparathyroidism (211,239). In addition to the usual histologic features of adenoma, nuclear diameter is increased to the same extent as in primary hyperparathyroidism (240) and a variety of allelic losses have been found in several different chromosomes, as in primary hyperparathyroidism (241-243) (see also Chapter 19). In other cases the dysfunction could lead to a further increase in secretory set point. The final and least common stage is malignant transformation leading to parathyroid carcinoma, an event that seems to occur with unusual frequency in patients on longterm hemodialysis (3,87,244,245), possibly because of defective DNA repair (246) as well as increased cell proliferation. Because long-term continued stimulation of parathyroid growth by chronic renal failure can culminate in neoplasia, either benign or malignant, the same would be expected in other circumstances. The occurrence of unusually large parathyroid adenomas in India was mentioned earlier (204). Hyperplasia in the nonadenomatous glands was reported in some cases but has not been sought systematically, so that the distinction between primary and tertiary hyperparathyroidism may be difficult (3,211). Hypercalcemic hyperparathyroidism developed in some vitamin D-deficient migrants from India to England (247) but its anatomic basis remains unclear. A parathyroid adenoma was found in two such patients (248), but whether the incidence is increased in this population has not been established. In intestinal malabsorption, secondary hyperparathyroidism has been demonstrated biochemically (211), but the histologic data are fragmentary (249). The apparent association between intestinal malabsorption and parathyroid adenomas has been described as tertiary hyperparathyroidism (3), but there was no evidence for hyperplasia of the other

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glands (250), as is found invariably in tertiary hyperparathyroidism due to renal failure. In the early stages of calcium malabsorption, serum calcitriol levels tend to be increased rather than decreased (211). This would delay the rise in secretory set point, which is probably necessary for the emergence of multiple foci of rapidly proliferating cells and increased mutagenic risk. Consequently, as has been suggested (250), growth would be stimulated only in the most responsive cells. A similar mechanism may underlie the rare case of parathyroid carcinoma in patients with intestinal malabsorption (245). There is also evidence for tertiary hyperparathyroidism in patients with hypophosphatemic osteomalacia given long-term treatment with supplemental phosphate, which induces intermittent slight decreases in plasma calcium and stimulation of PTH secretion (251). Hypercalcemic hyperparathyroidism has developed in at least 25 such patients (3,252-254), in whom parathyroid pathology encompassed the same spectrum of diffuse hyperplasia, nodular hyperplasia, and adenoma as in patients with chronic renal failure. Another similarity is that in three cases the secretory set point was increased (253); in contrast to renal failure, the serum calcitriol levels were normal, but they tend to be low in the disease and to be affected little by treatment with calciferol (211,251). One of the three cases was treated only with calcitriol, but this compound seems to be more effective than calciferol in preventing the progression of parathyroid growth (251). Another therapeutic regimen causing short-term stimulation of PTH secretion is sodium fluoride administration (255). The author has observed the development of hypercalcemic hyperparathyroidism during prolonged treatment with sodium fluoride and its cure by removal of a parathyroid adenoma, but whether this was more than a coincidence is not clear. In summary, tertiary hyperparathyroidism definitely occurs in chronic renal failure and probably also in chronic vitamin D deficiency and in phosphate-treated osteomalacia.

Therapeutic Implications of Parathyroid Growth In primary hyperparathyroidism, a sustained decrease in PTH secretion can at present be achieved only by ablation of surplus parathyroid tissue, usually by surgical resection (see Chapter 31). Short-term control of hormone secretion is possible with calcimimetic drugs (256) and their suitability for long-term management is currently under investigation, but there is currently no parathyroid counterpart to bromocriptine, which can produce shrinkage as well as reduced hormone secretion in many pituitary tumors (257). The rate of growth influences the timing of surgery, which is urgently needed in acute primary hyperparathyroidism,

is mandatory but not immediately required in many patients with progressive disease, and can be delayed, sometimes indefinitely, in mild nonprogressive disease (117). Regarding secondary hyperparathyroidism, however, there is a long-standing and pervasive assumption that, if the initial stimulus to cell proliferation could be removed, the parathyroid glands would eventually return to their previous size. There are several bases for this assumption. First, a noninvasive reduction in size has for many years been easy to observe in the thyroid gland (1). Second, classic nutritional experiments that demonstrated cross-sectional differences in parathyroid growth (79) have often been misquoted as evidence for parathyroid involution. Third, the ultrastructural changes that accompany suppression of hormone synthesis and release by hypercalcemia (258) have been misinterpreted as evidence for suppression of growth. Nevertheless, it is far from certain that a nondestructive increase in cell loss is possible in human parathyroid glands. The absence of a parathyrotrophic hormone may limit the capacity for involution as well as for growth, but even in the thyroid gland regression of hyperplasia is less frequent and less complete than is commonly assumed (167). The first indication that hyperplastic parathyroid glands regressed very slowly, if at all, came from the frequent persistence of PTH hypersecretion after renal transplantation (6). There are several causes for hypercalcemia in this situation, but in current practice the most common is hyperparathyroidism. Although sometimes this is described as tertiary hyperparathyroidism, in most patients only hyperplasia is found (259). Nevertheless, hypercalcemic patients had higher PTH levels before transplantation and their secondary set point is increased (260). Mild hypercalcemia can persist unchanged, with no tendency to resolve; if parathyroid involution is occurring in such patients, it is at a rate too low to be reflected by detectable changes in plasma calcium for up to 10 years (6). In primary hyperparathyroidism, the nonadenomatous parathyroid glands may show ultrastructural evidence of functional suppression (258), and a 50% reduction in cell proliferation (164), but the reduction in parenchymal cell mass is only--~10% (261), consistent with regression of hypertrophy without cell loss (40). In patients with nonparathyroid hypercalcemia, the parathyroid glands at autopsy are indistinguishable from normal glands in histologic appearance, in relative proportions of fat and parenchyma, and in size (39). For these reasons, if stable hypercalcemia lasts for more than 1 year after renal transplantation, its permanence should be assumed and surgery r e c o m m e n d e d unless there is a strong contraindication (87). The therapeutic implications of disorders in growth in untransplanted patients are more complex. In

PARATHYROIDGROWTH: NORMALAND ABNORMAL / primary hyperparathyroidism, cell birth rate in nonadenomatous glands falls only by about 50% (164). Even if, as a result of medical treatment, cell birth rate in patients with renal failure fell to zero, and apoptosis continued at the normal rate of 5%/year, parathyroid cell n u m b e r would fall by about 40% in 10 years and 3.0 g of surplus parathyroid tissue would shrink to 0.5 g in about 35 years. These extremely pessimistic calculations have several important implications. First, much more emphasis needs to be placed on the prevention of parathyroid hyperplasia in early chronic renal failure. Because parathyroid hyperplasia is always a later response than PTH hypersecretion to the same stimuli, if PTH levels are increased more than twofold for more than 6 months, it is safe to assume that some parathyroid hyperplasia has already occurred. Prevention of PTH hypersecretion, which is easy to monitor, will prevent parathyroid hyperplasia, which at present is impossible to monitor in its early stages. Second, once severe hyperparathyroidism has been allowed to occur, then however successful medical treatment may be in reducing PTH hypersecretion, then as after transplantation, in the absence of evidence to the contrary it should be assumed that there will be no significant reduction in parathyroid cell number. In one of the few prospective controlled clinical trials in this field, there was no significant difference in the response to oral and intravenous calcitriol given in the same dose, and after 36 weeks there was no change in ultrasonographically determined parathyroid size or dynamic indices of PTH hypersecretion, regardless of the route of administration (262). Implicit in the preceding discussion is that apoptosis of parathyroid cells continues at the normal rate of about 5%/year; an important unresolved issue is whether it is possible to stimulate apoptosis and so accelerate parathyroid involution. If calcitriol is a negative parathyroid growth factor, then a large abrupt rise in its concentration might have the same effect as a large abrupt fall in the concentration of a positive growth factor, which induces apoptosis in many endocrine tissues (30). There have been several reports that pulse administration of calcitriol in high dose (8 txg) may be followed by substantial reduction (about 40%) in parathyroid volume determined ultrasonographically; most of the shrinkage occurred in the first 4 weeks (263,264). Calcitriol may induce parathyroid apoptosis in rats (265), but no histologic verification has been reported in the clinical studies, and the shrinkage could reflect reductions in cell size and in vascularity, rather than in cell number. Nevertheless, the latter interpretation is supported by experiments in 3-month-old vitamin D-deficient chicks that received vitamin D replacement. There was good indirect evidence for a reduction in parathyroid cell number,

319

based on similar reductions in weight, protein, and DNA content (266); the change was detectable within 4 days and could have resulted only from apoptosis. In the controlled trial previously referred to (262), calcitriol was administered in pulse fashion, although the maximum dose of 4 txg may have been too small to achieve the effects reported with a higher dose. If induction of apoptosis by calcitriol can be confirmed in patients, a bromocriptine for the parathyroid glands may be at hand. Disordered parathyroid growth also has implications for the surgical m a n a g e m e n t of hyperparathyroidism in untransplanted patients (87). The first procedure carried out was subtotal parathyroidectomy, leaving only a portion of the smallest gland. Much later an alternative procedure was introduced, namely, total parathyroidectomy and autotransplantation of tissue into the forearm. The advantages claimed were that monitoring of the function of the autograft was easier because its venous drainage was accessible, and if the r e m n a n t grew rapidly e n o u g h for severe hyperparathyroidism to recur, reexploration would be easier (267). More recently, total parathyroidectomy without autotransplantation has been r e c o m m e n d e d (268). The prolonged maintenance of normocalcemia that sometimes follows this procedure is presumed to d e p e n d on parathyroid embryonic rests that were too small or too aberrant in location to be removed. Each of these procedures has its proponents and opponents, but more important than the choice between them is the timing, regardless of which procedure is adopted. Before the development of nodular hyperplasia, subtotal parathyroidectomy, followed by calcitriol to control the secretion and growth of the parathyroid remnant, should be effective and is rarely followed by recurrence of severe hyperparathyroidism. Conversely, if parathyroidectomy is delayed until the patient has severe osteitis fibrosa or hypercalcemia, then nodular hyperplasia or a d e n o m a formation can be presumed. If total parathyroidectomy is chosen, it makes sense to transplant only tissue harvested from the smallest and least nodular gland, or, if all glands are nodular, from an internodular region (267,269), because indiscriminate choice of transplanted tissue is more likely to be followed by aggressive and even invasive growth (270).

Parathyroid Growth in Hypoparathyroidism The parathyroid glands are difficult to find at autopsy even when they are normal in size, and there is only fragmentary information about their pathology when they are small. The available data in the various forms of idiopathic hypoparathyroidism are described in Chapters 1, 47, and 50. In surgical hypoparathyroidism, there has been almost no histologic examination, but an

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interesting paradox can be formulated on the basis of the course of parathyroid function in the absence of treatment. In many patients, the level of plasma calcium is higher than expected for the complete absence of PTH (271), there is a low but detectable level of intact PTH (272), and some recovery from EDTA-induced hypocalcemia is possible (273). Evidently some cells capable of secreting PTH remain, but depressed function persists. According to the model depicted in Figs. 7 and 8, because the relationship between cell number and plasma calcium is curvilinear, the decrease in plasma calcium is proportionally less than the decrease in cell number. Hormone secretion per cell is increased, and as cell number declines, each cell operates ever more closely to its upper limit with respect to hormone secretion (75). In these circumstances, why do not the remaining cells proliferate until normal function is restored? As mentioned earlier the effect of calcium on parathyroid cell proliferation is biphasic, and severe hypocalcemia is less effective than mild hypocalcemia. But this cannot be the main explanation for the paradox, because even mild hypoparathyroidism can remain unchanged in severity for many years (271). Except when all four glands are removed, which almost never happens with neck surgery for nonmalignant disease, there is no relationship between the development of hypocalcemia and the amount of parathyroid tissue in the surgical specimen (274). Rather than simple removal of some parathyroid tissue, there is interference with the blood supply of all four glands as the result of the surgical dissection and hemostasis (275). This occurs only in a small minority of patients, presumably because of individual differences in the precise location of the parathyroid glands and in the detailed anatomy of their blood supply. The parathyroid glands are also susceptible to fibrosis (276,277), and the progression of fibrosis is a likely explanation for the late occurrence of surgical hypoparathyroidism after a period of apparent recovery with normocalcemia (278). Parathyroid ischemia is a likely explanation for the development of apparently idiopathic hypoparathyroidism late in life (279); the susceptibility of parathyroid tissue to ischemia is indicated by uncertain survival after transplantation (235), and by spontaneous infarction in rapidly growing tumors (147). A subnormal degree of parathyroid function can be maintained by small islands of cells, but if separated by bands of fibrous tissue their viability will be precarious. Regeneration depends on neoangiogenesis as well as on chief cell proliferation (95), and both processes would be compromised by vascular injury, impaired circulation, and persistent fibrosis.

I N T E G R A T I O N OF PARATHYROID GROWTH A N D H O R M O N E SECRETION Increases in Set Point and in Cell P r o l i f e r a t i o n ~ Further Examples Both in primary and in hypercalcemic secondary hyperparathyroidism, cell proliferation is driven in large part by an increase in secretory set point, due to CaSR under expression for one or other reason. Three additional examples of this relationship merit brief discussion--normal embryonic development (280), familial hypocalciuric hypercalcemia (FHH) (see Chapter 38), and lithium-induced hypercalcemia (see Chapter 55). The fetal parathyroid gland secretes PTH-related peptide (PTHrP) rather than PTH, but its secretory activity is regulated by calcium in the same manner as the adult parathyroid gland, except that the secretory set point is higher than in the adult (280) most probably because of CaSR underexpression (281). The rapidity of embryonic parathyroid growth is consonant with a need to prevent fetal hypocalcemia (52) by supplying PTHrP to maintain normal placental calcium transport (282), a process dependent on CaSR expression (281). This is probably an evolutionary rather than an individual functional adaptation, but proliferation could be driven in part by the increase in set point. In FHH there is an inactivating mutation of the CaSR gene (174). As a result the parathyroid secretory set point determined in vivo is increased (101,189,283) and there is mild and nonprogressive hypercalcemia. Because of ineffective renal tubular CaSR, tubular resorption of calcium is increased; urinary calcium excretion is normal, but is low relative to the plasma level. Whether bone lining cells express the CaSR is unknown, but the blood-bone equilibrium is probably set at a higher level in FHH (75). Serum PTH levels are usually normal in childhood but increase slowly with time and are often above normal after age 30 (284). The geometric mean for parathyroid parenchymal area in histologic sections was increased approximately three fold, compared to a fivefold increase in other forms of primary hyperplasia, whether familial or sporadic (285). Parathyroid weights have been reported only in a few patients; the mean was increased about twofold, but few individual glands weighed more than 75 mg (286). The mild hyperplasia represents the polyclonal (or multiclonal) expression of a heterozygous germinal mutation, for which homozygosity leads to severe neonatal hyperparathyroidism (174). In terms of the set-point hypothesis, a lesser degree of parathyroid enlargement is needed to satisfy the increased secretory set point because the elevated renal tubular set point does not depend on increased PTH secretion (75).

PARATHYROID GROWTH: NORMALAND ABNORMAL / A double dose of the mutant gene, or a d o m i n a n t negative mutant, causes a higher set point (111,287) and a consequent greater drive to cell proliferation. Administration of lithium salts, usually for bipolar affective disorder, is in most patients followed within a few weeks by modest increases in plasma PTH and calcium levels, occasionally of sufficient magnitude to constitute hypercalcemia (288,289). Even a single dose of lithium carbonate increases the intact PTH level after 2 hours, with no change in ionized calcium (290). The early changes are reversible, but mild persistent hypercalcemia is found in about 6% of patients given prolonged treatment (291). The features of lithiuminduced hypercalcemia are similar to those of FHH (289,292). Serum PTH levels are nonsuppressed or moderately raised, and tubular reabsorption of calcium is increased. Both short-term and long-term clinical studies indicate that lithium administration increases the PTH secretory set point (292,293), a conclusion supported by the in vitro effects of lithium on cultured parathyroid cells, both bovine and h u m a n (293,294). As predicted by the set point hypothesis, lithium administration stimulates parathyroid growth. Exposure to lithium increases tritiated thymidine uptake by cells cultured from parathyroid adenomas (294), and there is a significant increase in parathyroid volume measured by ultrasound in patients on long-term treatment (295). In lithium-induced hypercalcemia of sufficient severity to need surgical treatment, both hyperplasia and aden o m a have been found (3,288,289,296). A d e n o m a tends to occur earlier than hyperplasia in the course of lithium treatment (297), suggesting that lithium promotes hyperplasia of normal parathyroid tissue and stimulates the growth of small adenomas already present (292).

A New Concept of Calcium Homeostasis The consistency of the association between increased secretory set point and increased cell proliferation suggests a new way of looking at plasma calcium homeostasis. According to this view, the controlled variable is not the plasma calcium, but rather the difference between the parathyroid secretory set point (using the physiologic definition given earlier) and the prevailing plasma calcium level, with the target value for this variable being zero (Fig. 11). If plasma calcium is below the set point, the well-known short-term feedback loop is initiated; based on the hierarchy of mechanisms described earlier (see above, Physiologic Influences on Parathyroid Growth, and Ref. 75), total horm o n e secretion by all glands is increased. Depending on the response of target cells in bone and in the renal tubule to higher PTH levels (75), plasma calcium rises

321

[Set Pt-P.Ca]

(I)I(~~

(6) (~ Cell Number (~. ~-(~)PTHSecretion

,(~ Target cells

(4) ® GO-----~ G~ FIG. 11 Short-term and long-term loops in calcium homeostasis. The controlled variable is the difference between the parathyroid secretory set point and the current level of plasma calcium, with a target value of zero. The signs indicate, for each step, the directional effect on the dependent variable of an increase in the independent variable. Steps 1, 2, and 3 constitute the short-term loop, and steps 4, 5, and 6 constitute the long-term loop. Step 4 in initiated when PTH hypersecretion by individual cells is prolonged because a positive value of the controlled variable persists, but the mechanism is unknown. (For further details, see text.)

and the deviation from the set point is eliminated. If this process is incompletely effective, there will be a sustained increase in d e m a n d for PTH, bringing into play a long-term feedback loop, whereby some parathyroid cells are triggered from the quiescent G o state to the G 1 stage of the cell cycle. The initiating signal may be a fall in PTH concentration in some critical intracellular compartment, augmented by the autocrine mechanism described earlier (see Physiologic Influences on Parathyroid Growth and Ref. 21). The result will be an increase in the n u m b e r of parathyroid cells, a consequent increase in total h o r m o n e secretion in response to the same stimulus, and a rise in plasma calcium toward the set point. The cycle will be repeated until cell n u m b e r has increased sufficiently to reduce the disturbing signal to trivial magnitude. As m e n t i o n e d earlier, the long-term loop adds integral control to the proportional and derivative controls that suffice for the short-term loop (75). This ensures that stimulated parathyroid cells need not long sustain m a x i m u m rates of h o r m o n e synthesis and secretion, in which state they cannot respond to an additional demand, but are returned to the steep central portion of the sigmoid curve where they can respond with greatest efficiency in either direction (75). The sustained increase in the controlled variable needed to initiate the long-term feedback loop can arise either because the plasma calcium is reduced or because the set point is increased. Chronic hypocalcemia (if not due to hypoparathyroidism) is the result of decreased efficacy of PTH in maintaining normocalcemia, for one of the reasons previously given (see above, Parathyroid

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CHAPTWR18 TABLE 10

Comparison of Different Mechanisms of Increase in Set Point

Disorder

P rim a ry adenoma

Seco nda ry hyperplasia

FHH a

Lith iu m therapy

Set-point mechanism Affected cells PTH efficacy ~

Genetic dysfunction One clone Normal

Calcitriol deficiency Nodules Reduced c

Germinal mutation All Increased ~

Direct effects on cell All Increased a

Cell increase

Moderate

Severe

Mild

Mild

aFamilial hypocalciuric hypercalcemia: the situation in other forms of genetic hyperplasia is less clear. °In raising plasma calcium. CBecause of calcitriol deficiency and other effects of renal failure dBecause of increase in tubular reabsorption of calcium.

Growth in Secondary and Tertiary Hyperparathyroidism), leading to diffuse secondary parathyroid hyperplasia. Depending on its cause, an increase in secretory set point will affect a different population of cells and have different morphologic consequences (Table 10). If due to an epimutation, then only a single clone of cells will be affected, leading to primary or tertiary hyperparathyroidism (Fig. 10). If due to renal failure and calcitriol deficiency, some groups of cells may be affected more than others, leading to nodular secondary hyperplasia. If due to a germinal mutation or to an external agent such as lithium, all cells will be affected equally, leading to primary hyperplasia (Table 10). There are significant differences between individuals in mean plasma calcium level, reflecting small individual differences in parathyroid secretory set point (75), due to polymorphism of the CaSR gene (298), which also accounts for significant differences between families (75). According to the set-point hypothesis, there should be corresponding differences in cell number, and parathyroid weight should be positively correlated with plasma calcium level. But, in fact, the correlation is not positive but negative (56,58)! The patients available for sampling at autopsy probably encompassed a wide range of calcium and vitamin D nutrition, so that in this multiethnic population the variation in target cell responsiveness to PTH (107) was large enough to dominate the reciprocal feedback relationship, allowing the observations to be reconciled with the set-point hypothesis. Whatever the molecular basis of the link between cell proliferation and hormone secretion in the parathyroid gland turns out to be, the setpoint hypothesis, in conjunction with variation in the efficacy of PTH in raising plasma calcium (Table 10), accounts for the slowing down of growth in parathyroid adenomas, the persistence of rapid growth in secondary hyperplasia with hypercalcemia, and the small extent of growth in FHH. Integration of the

short-term loop governing hormone secretion and the long-term loop governing proliferation ensures that both in health and in disease, the parathyroid glands will attain the size needed to accomplish their biologic purpose.

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CIeTWR 19 Molecular Basis of Primary Hyperparathyroidism

ARNOLD Centerfor Molecular Medicine and Division of Endocrinology and Metabolism, University of Connecticut School of Medicine, Farmington, Connecticut 06030

ANDREW

INTRODUCTION

feature of cancers is their clonal nature; in other words, cancers arise from a single precursor cell (i.e., "monoclonal") whose progeny of essentially identical daughter cells have a selective growth advantage. Thus, every neoplastic cell in the resulting clinically apparent tumor typically contains an identical detailed pattern of DNA damage to multiple key growth-regulating genes, identifying this clone uniquely. To varying degrees, subpopulations of such a clone may develop due to the overlay of further acquired DNA damage after the initial clone is established, conferring additional selective advantage in a process called clonal evolution. These later changes might be expected to be found in only a subpopulation of the tumor cells. Distinguishing the genetic changes important for initial clonal expansion versus later clonal evolution has been difficult to address, although recent advances in selecting small groups of tumor cells for separate analyses will allow major progress on this issue over the next several years. That said, from the viewpoint of the clinically apparent tumor, the pervasiveness of many specific mutations throughout its entire set of neoplastic cells makes it clear that many of the important underlying genetic events occurred early, before major proliferation or clonal expansion of affected cells to clinical significance. The clonality of tumors also indicates that the summ a t i o n / a c c u m u l a t i o n of factors n e e d e d to transform (or confer the neoplastic phenotype upon) a cell occurs only rarely in a large population of cells in a tissue. Within such a population of cells, the rare emergence of a transformed clone is consistent with the rare occurrence of mutations in certain key rate-limiting

General Principles in Molecular Oncology In recent years we have witnessed an explosion in our knowledge of the molecular basis of neoplasia. This revolution was spearheaded by work on various hematopoietic tumors, but major insights into solid tumor pathogenesis have become commonplace as well. Despite some notable advances, however, knowledge of the molecular mechanisms underlying the pathogenesis of endocrine tumors in general, and of parathyroid tumors i n particular, remains relatively undeveloped in comparison to that for neoplasms such as lymphomas, leukemias, colon, and breast cancers. That said, existing information does indicate that many of the well-established general themes in tumor biology are quite applicable to parathyroid tumorigenesis, in spite of the typically nonmalignant status of these tumors. In addition, one molecular insight into parathyroid tumor development, the discovery of the cyclin D1 (PRAD1) oncogene, has also proved to be of tremendous general significance in h u m a n molecular oncology and in basic cell cycle biology. It is now solidly established that cancer cells contain genetic damage that is central to the abnormal neoplastic phenotypes they characteristically exhibit. In addition to this damage to the actual base sequence in the DNA, certain "epigenetic" factors may also be important; these could include aberrant patterns of DNA methylation, hormonal influences on gene expression, or i m m u n e system stimulation. A critical The Parathyroids, Second Edition

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pathways a n d / o r a requirement for the accumulation within one cell of multiple different damaging DNA alterations. The clonality of tumors does not, however, exclude an important role for field effects or generalized proliferative stimuli directed at the particular tissue as one of several factors contributing to tumorigenesis. The molecular genetic heterogeneity underlying neoplasia should be emphasized. Though certain tumor types might exist in which damage to only one gene is necessary a n d / o r sufficient for transformation, the general rule appears to be that damage to multiple distinct genes, all within the same cell, must accumulate for the ultimate expression of the neoplastic phenotype (1-3). Certain genes may be implicated in tumors of only one or a few cell types (e.g., TSH receptor, RET) while others may be involved in many different types of tumors (e.g., p53, Rb, cyclin D1). For most (and perh a p s all) tumor types, however, it appears that no single genetic change will prove to be both a necessary and sufficient causative agent. More likely, disruption of certain biochemical pathways may be important unifying themes for the emergence of particular tumors, and different combinations of mutated genes may result in similar cellular and clinical consequences. Studies of oncogene additivity or cooperativity, and other functional studies, will help to shed light on these issues. There are two broad categories of normal cellular genes in which clonal DNA damage contributes to neoplasia; these are protooncogenes and tumor-suppressor genes. Protooncogenes often have roles in the normal physiologic control of cellular growth, proliferation, or differentiation, and may function by regulating protein phosphorylation, signal transduction, or gene transcription, for example. Damage to a protooncogene, converting or activating it to an "oncogene," usually causes a deregulation of expression of its normal protein product or the formation of an intrinsically abnormal product. In contrast, tumor suppressor genes normally function to restrain cell proliferation and contribute to neoplasia through their functional inactivation. The types of somatic DNA derangements that can activate protooncogenes include chromosome translocations or inversions, point mutations, proviral insertions, and gene amplification. Inactivation of tumor suppressor genes is often accomplished by internal mutation or deletion, and may be inherited or incurred somatically. The causes of these types of oncogenic "hits" are not well understood. Environmental carcinogens such as ionizing irradiation or chemicals play a direct role in some instances, and errors in normal chromosomal recombinatory mechanisms also appear to occur, perhaps randomly. Certain genetic changes tend to occur more commonly in mitotically active cells, and carcinogens may act either by direct

mutagenesis of DNA or through augmentation of the mitotic rate, which secondarily increases the likelihood of an oncogenic chromosome aberrancy. Still other mechanisms may yield genetic damage without the requirement for high mitotic activity in the cell type. Many excellent reviews are available to the reader interested in exploring these principles further (2-8).

Special Issues in Parathyroid Neoplasia Though the general principles described above are expected to hold true for the specific example of parathyroid neoplasia, a complete molecular description of parathyroid tumorigenesis will ultimately need to explain a number of special features unique to the parathyroid model. Among these mysteries are the increased incidence of parathyroid tumors after exposure to neck irradiation, the heightened frequency with which hyperparathyroidism is found in postmenopausal women, the rarity of parathyroid cancer as relative to the more typical development of benign parathyroid tumors, and the relationship between excess cellular proliferation and the misadjusted set point linking ambient calcium level with parathyroid hormone (PTH) secretion in the tumor cells. Also, evidence exists on several levels that the growth rate of many clinically detectable parathyroid adenomas is quite slow, and may have changed over time (9). Information relevant to these and other special issues is only beginning to be generated, but the continued application of modern methods certainly promises eventually to yield the required molecular/pathophysiologic synthesis.

CLONALITY OF PARATHYROID TUMORS Controversy and uncertainty exist regarding the pathologic etiologies and clinicopathologic categorization of hyperparathyroidism. Distinction between parathyroid adenoma and hyperplasia can be made on the basis of the n u m b e r of abnormal, hypercellular parathyroid glands found in the patient, with a single abnormal gland being defined as an "adenoma." It can, however, be difficult histologically to distinguish a normal from a mildly hypercellular gland, and histologic examination of a particular hypercellular gland offers no features solidly predictive of whether it is a solitary tumor (i.e., adenoma) or one of several (i.e., hyperplasia) in the patient (10,11). Multigland disease can affect an individual's parathyroid glands in a highly nonuniform and asynchronous fashion ("asymmetric hyperplasia"), even to the point of confusing parathyroid hyperplasia with adenoma when only

MOLECULARBASISOF HYPERPARATHYROIDISM / one enlarged gland is found at surgery. Whether "double adenomas" or "multiple adenomas" exist (i.e., together with at least one truly normal parathyroid gland) has therefore been controversial, but the preponderance of clinical evidence suggests this entity is indeed genuine (12-15). Early studies of the clonal status of parathyroid tumors were designed to address some of these uncertainties. Assessment of X-chromosome inactivation patterns using the glucose-6-phosphate dehydrogenase (G6PD) protein polymorphism had indicated that parathyroid adenomas (single-gland disease) were polyclonal as opposed to monoclonal growths (16,17). These data suggested that a parathyroid "adenoma" was really a highly asymmetric form fruste of multigland hyperplasia, and were taken to support the surgical practice of routine bilateral neck exploration a n d / o r subtotal parathyroidectomy for hyperparathyroidism. This conceptualization of the origins of parathyroid tumors certainly did not encourage a search for the types of DNA damage, discussed above, characteristic of monoclonal tumors. The clonal status of parathyroid adenomas was reevaluated several years later, again by X-chromosome inactivation analysis, but using a DNA polymorphismbased method that avoids many of the pitfalls of the protein polymorphism approach. It was determined that most and perhaps all parathyroid adenomas were in fact monoclonal tumors, i.e., that these glands were true neoplastic outgrowths of a single abnormal cell (18). Subsequent studies have confirmed the monoclonality of parathyroid adenomas (19-28). This finding has been taken to support to the surgical practice of resecting only the clearly enlarged gland (and not exploring the other side of the neck) if gross examination and biopsy of an identified ipsilateral gland are completely normal. This approach would, of course, fail if the contralateral side harbored another independent clonal adenoma (12,13,29). Parathyroid carcinomas, not surprisingly, are also monoclonal (25,30,31 ). Even if one puts aside the possibility of multiple adenomas arising de novo in a given patient, a finding of monoclonality in an enlarged parathyroid gland clearly cannot be viewed in isolation as diagnostic for singlegland disease. It has now been established that in various forms of parathyroid hyperplasia, which presumably begin with a stimulus for generalized (polyclonal) parathyroid cell proliferation affecting all the glands, monoclonal tumors can subsequently evolve in some or many of these glands. For example, monoclonality has been documented in parathyroid tumors from patients with nonfamilial (sporadic) primary parathyroid hyperplasia (32) and in the large majority of glands from patients with the

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refractory secondary (or "tertiary") hyperparathyroidism of uremia (32). The clinical or pathophysiologic significance of monoclonality in these settings remains to be determined; it is highly plausible, for example, that such tumors may exhibit a greater degree of pathological autonomy in PTH secretory function as well as higher growth rates than do any truly hyperplastic (polyclonal) neighboring glands in the same patient. Clonal expansion also characterizes the enlarged parathyroids of patients with multiple endocrine neoplasia type 1 (MEN-l) (20,21,33,34), but it is not known whether such outgrowths evolve from a preliminary stage of true polyclonal hyperplasia that might be driven by the inherited haploinsufficiency for the MEN1 gene.

SPECIFIC GENETIC A B N O R M A L I T I E S IN B E N I G N PARATHYROID T U M O R S The determination that parathyroid adenomas were monoclonal neoplasms conveyed the expectation that these tumors, albeit benign, result from some of the same types of genetic damage that characterize clonal malignancies (18). This expectation is now shared by a subset of tumors, also found to be monoclonal, in patients with various forms of multigland "hyperplasia," discussed above. Identification of the particular genes whose tumor-specific activation or inactivation results in these clonal expansions is critical, because building a detailed understanding of the molecular defects that underlie parathyroid neoplasia may ultimately lead to successes in diagnosis, pathologic classification, prevention, a n d / o r treatment. Identifying these genes may also point to particular biochemical pathways of importance in regulating parathyroid cell proliferation, thus speeding the discovery of other genes of similar importance. So far, only two specific genes, cyclin D1 (PRAD1) and MEN1, have been convincingly incriminated in the common sporadic forms of benign parathyroid neoplasia. For cyclin D1, this initial molecular insight has proved to have exceedingly broad implications regarding basic cell cycle biology and human cancer, which speaks well for parathyroid tumors as potentially generalizable models of benign or well-differentiated neoplasia. The principle of molecular heterogeneity predicts that still more parathyroid tumor genes exist, and studies have pointed to specific chromosomal regions that almost certainly contain such genes (22-24,35,36). These target areas for critical (recurrent and clonal) DNA damage, which together confer a selective growth advantage on the parathyroid cell in which they develop (i.e., the clonal precursor cell), are discussed in this section.

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Activation o f the cyclin D1 (PRAD1) O n c o g e n e Initial Cloning of PRAD1

DNA rearrangements, in the form of translocations and related chromosomal events, are among the best characterized types of clonal oncogenic genetic abnormalities (37). Very frequently, these rearrangements involve the tumor-specific juxtaposition of cellular protooncogenes with DNA sequences designed to regulate other genes; this may result in the overexpression or deregulated expression of the protooncogene, thus converting it to an oncogene. In fact, the cloning of DNA immediately adjacent to clonal chromosome breakpoints has proved to be a tremendously effective method for identification of new oncogenes. One example was the discovery of the BCL-2 oncogene in follicular lymphomas with the t(14;18) chromosome translocation. BCL-2 (on chromosome 18) is "activated" and overexpressed as a consequence of its relocation into the part of the immunoglobulin heavy-chain gene, on chromosome 14, that contains sequences responsible for immunoglobulin's strong transcriptional activity in B lymphoid cells (38). It is interesting to note that most oncogenes found to be activated by rearrangement in human cancer have been detected in hematopoietic tumors. Though it is likely that chromosomal translocations occur more frequently in these cell types, the situation may in part also reflect technical advantages in performing cytogenetic analyses on hematopoietic as compared with solid tumors. Improved cytogenetic methods may permit detection of more chromosome translocations in solid tumors, including parathyroid tumors, and thus provide searchlights for the identification of additional oncogenes. The key initial observation in the identification of PRAD1 was a band of abnormal size on a Southern blot of DNA from a parathyroid adenoma, probed with a

;~TH Coding

Break

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P T H genomic DNA fragment (18,39). This band was not present in the same patient's normal leukocyte control DNA, and was thus tumor specific and clonal. Relative intensifies of this band compared with the remaining normal-size band suggested that the underlying DNA alteration affected one of the tumor's two P T H alleles, and that the abnormality was present in every cell in the tumor. It had, therefore, presumably occurred in the original clonal progenitor cell of the mature tumor, likely conferring a selective growth advantage, and hence appeared worthy of additional investigation for its possible pathogenetic importance. Southern analysis of this original adenoma revealed that the clonal alteration responsible for the observed abnormal band was a tumor-specific DNA rearrangement (39). The rearrangement separated the 5' regulatory region and noncoding exon 1 of the PTH gene from its coding exons, with different, non-PTH DNA placed adjacent to each P T H gene section (Figs. 1 and 2). In addition, the tumor possessed one intact P T H gene per cell, which appeared to be the source of PTH production by the adenoma. Though a DNA rearrangement with this structure could have been tumorigenic in a few possible ways, it was hypothesized that in analogous fashion to the immunoglobulin/ BCL-2 model, the P T H gene regulatory region, which strongly drives tissue-specific gene expression in the parathyroid cell, could be activating the expression of a protooncogene placed under its influence by the rearrangement. The DNA sequences of interest, lying adjacent to the PTH breakpoint, were then cloned from a genomic DNA library prepared from this adenoma. From the P T H gene-positive bacteriophage inserts, subclones were made that contained only the breakpoint-adjacent n o n - P T H D N A . This "anonymous" single-copy DNA was mapped using somatic cell genetics and in situ

PTH Coding

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FIG. 1 Schematic diagram illustrating the DNA rearrangement involving the PTH gene and the cyclin D1/PRAD1 gene in a subset of parathyroid adenomas. The chromosomal inversion event is deduced as the simplest cytogenetic mechanism consistent with the molecular details of this DNA rearrangement. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab, Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

MOLWCULA~ BASIS OF HYPERPARATHYROIDISM /

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• Transcription starts with Cyclin 01 's first e~on, using its own promot,dr. • Active Cyclin D1 transcription is driven by ti~,sue-specificenhancer(F) from the 5' PTH gene region. ~ ~_ ~ /" Overexpressed cyclin D1 mRNA ~i!~i!i!i!!ii!iii!ii{!iiiiii ! !i!i!i~i~!!i!iiiiiii!~i] l with normal coding sequence |~!~!:i)~i~.~.~:~i~i!~ii~i~i~i~:~i~i~i!~i~!~!i~i~!~iii~i!i!:~i~:~`~iii~!~.~i~ O v e r e x p r e s s e d cyclin D1 protein, due to gene rearrangement or other activating m e c h a n i s m s ~ - 4 0 % of parathyroid adenomas

G1 ~S +G2 +M Cell cycle deregulation

Other o n c o g e n i c effects

hybridization, was found to be normally located on chromosome 11, band q 1 g, and was initially called D11 $287 (39). Because the normal chromosomal location of the PTH gene is also on chromosome 11, at 11p15, the simplest cytogenetic explanation for the observed tumor-specific DNA rearrangement was a pericentromeric inversion of chromosome 11 (Fig. 1). Although there were several oncogenes that had previously been mapped to 11q13, namely INT-2, HST-1, SEA, and the BCL-1 lymphoma translocation breakpoint, the newly cloned DllS287 region did not appear to be identical to any of these, as evidenced by restriction map comparisons and by direct clone to clone hybridization analysis. DllS287 subclones were then surveyed in attempts to detect an expressed gene or transcription unit in the region. One such probe, located about 1 kb from the breakpoint, detected a distinct 4.5-kb mRNA species on Northern blots (40). This mRNA species was present in normal h u m a n parathyroid tissue, parathyroid adenomas without the PTH/D11S287 rearrangement, normal thyroid, and normal placenta. Thus, the transcript's presence was immediately recognized as not being highly cell type specific. Moreover, the same h u m a n DllS287 probe hybridized to mRNA of the same approximate size, withstanding stringent washing, on RNA blots from other species and tissues, including bovine lymph node, muscle, thyroid, and parathyroid, and mouse heart and liver (41). Most importantly, this mRNA proved to be dramatically (15-fold) overexpressed in the original parathyroid adenoma from which the rearrangement had been cloned (40). Additional independent parathyroid adenomas containing similar PTH rearrangement breakpoints were

FIG. 2 Diagram of the directly observed molecular structure of the PTH/cyclin D1 (PRAD1)DNA rearrangement and its functional consequences. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab,Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

then detected; they also had DllS287 region breakpoints (40,42) that were seen to vary by as much as 15 kb (40), and no other expressed genes were detectable in this 15-kb region. These additional cases also exhibited dramatic overexpression of the DllS287 transcription unit (40). Because of the involvement of DllS287 in clonal DNA rearrangements associated with gross abnormalities in its transcription, analogous to those in lymphoid neoplasia, this highly conserved sequence was considered further as a putative oncogene and was subsequently called PRAD1, for 12arathyroid adenomatosis 1.

PRAD1 Gene Product as a Novel Cyclin The normal cDNA for PRAD1 was cloned from a h u m a n placental cDNA library (41) and screened with the original breakpoint-adjacent genomic fragment from 1 l ql 3 that had hybridized to a distinct transcript. Sequence of the cDNA revealed one long open reading flame, encoding a 295-amino acid protein. The derived amino acid sequence indicated at that time that the PRAD1 protein was not closely related to any known protein or family. However, a weaker but consistent homology between PRAD 1 and the various members of the cyclin classes of proteins, which were known to have important roles in regulation of the cell cycle, was observed (41). The PRAD1 protein's region of maxim u m homology was an approximately 100-amino acid stretch (amino acids 55-160), which matched within the so-called "cyclin box," a 100- to 150-amino acid domain of greatest conservation between all the known cyclins. These other cyclins included A and B forms from yeast, clam, starfish, sea urchin, and Drosophila;

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PRAD1 had similar homology to their cyclin box regions, e.g., 28% identity with Drosophilacyclin A, and 31% identity with clam cyclin A. For comparison, h u m a n versus clam cyclins A are 71% identical in their cyclin boxes (and, as is typical, quite dissimilar throughout their other regions). These sequence comparisons suggested that PRAD1 was a novel cyclin, representing its own family distinct from the other families of cyclins (41). This suggestion was immediately reinforced by functional studies of the PRAD1 gene product, and then confirmed and expanded by countless studies over the past decade. The recognition that PRAD1 encoded a novel cyclin-type protein raised fascinating possibilities for its role in tumorigenesis, based on the role of cyclins in regulating progression through the cell division cycle. The cell cycle comprises stages G 1, S (DNA synthesis), G2, and M (mitosis). Progression through the cell cycle is regulated at two critical checkpoints, the G2/M border and a point in G 1 (called START in Saccharomyces and the "restriction point" in mammalian cells) that once traversed, enables the G1/S transition to proceed (43-47). Control of the G2/M transition is attained through a universal mechanism c o m m o n to all eukaryotic cells. The key feature of M phase is the activation of a s e r i n e / t h r e o n i n e protein kinase, designated cdc2, p34 c~c2,p34, M-phase kinase, and others, depending on the system of original study. Activation of cdc2 kinase induces mitosis through a process of phosphorylation of key proteins, leading to events such as chromosome condensation, cytoskeletal reorganization, nuclear envelope breakdown, and cell shape changes. The mitotic cyclins are another universal c o m p o n e n t of this system. First identified in dividing eggs of marine invertebrates, they are characterized by their accumulation t h r o u g h o u t interphase until the Gz/M transition, and then their rapid destruction during mitosis. Cyclin B, the prototypical mitotic cyclin, can be considered a regulatory subunit that must associate with cdc2, the catalytic subunit, to create an active holoenzyme complex. The disappearance of cyclin B in M phase, effected through the ubiquitin-proteasome degradation pathway, is necessary for kinase inactivation and exit from mitosis. The G1/S transition is also a critical checkpoint, and as the major regulatory point determining the cell's decision to attempt to divide, is an attractive site for attack by an oncoprotein. At the time of discovery of PRAD1, "G 1 cyclins" essential for this transition had been demonstrated only in yeast. These proteins, called CLN1, CLN2, and CLN3, have weak homology to cyclins A and B, and associate with cdc2 kinase specifically at G1/S (48-51). The discovery of PRAD1 thus raised the intriguing possibility that it might encode a h u m a n G 1 cyclin. Consistent with this possibility,

PRAD 1 mRNA and protein levels begin to rise and also peak within G 1, in 70N m a m m a r y epithelial cells (52) and fibroblasts (53) synchronized by serum starvation and subsequent addition of growth factors. The mouse homol0g of PRAD1 also acts in G 1, and was in fact cloned as a macrophage cDNA specifically induced in G 1 in response to colony-stimulating factor-1 (CSF-1) (54). Compelling evidence that the PRAD1 product is truly a functional cyclin was the demonstration that its cDNA could rescue mutant yeast deficient in G 1 cyclins; introduction of PRAD 1 cDNA released these yeast from cell cycle arrest in G 1 (55,56), and was an alternative route to the cloning of PRAD1. Because classic "mitotic cyclins" such as cyclin B can also rescue these G 1 cyclindeficient yeast, these data could not be taken as proof that the PRAD1 cyclin normally functions in G 1 phase. However, such proof has come, in overwhelming fashion, over the past several years and PRAD1 is indeed established as a critical mammalian G 1 cyclin (47). With the subsequent discovery of still other families of h u m a n cyclins, up to about a dozen, the picture has increased in complexity. Given the need for unifying nomenclature, the PRAD1 gene product is now commonly referred to as cyclin D1. In addition to the recognition of other cyclin families, two new cyclins quite closely related to PRAD1/cyclin D1 were isolated by low-stringency hybridization; these members of the Dtype cyclin family are known as cyclin D2 and cyclin D3 (52,57,58). To some extent, the expression of particular D cyclins is cell type specific, but though there may be some capacity for redundancy, most evidence suggests that these cyclins must have functionally distinct roles. Furthermore, multiple new cdc2-1ike kinases have been found, defining the general category of cyclind e p e n d e n t kinases (cdks), many of which have been matched up with specific cyclin partners. For example, the major cdk partner for cyclin D1 is cdk4 or cdk6, d e p e n d i n g on the differentiated cell type. The discovery of cyclin D also led to the identification of a class of proteins found in physical association with cyclin-cdk complexes (46,47,55,59,60), which proved to be inhibitors of cdk activity. These cdk inhibitors include p16, which has high specificity for blocking cyclin D - c d k 4 / 6 kinase activity. Importantly, the p16 gene has been solidly established as a human tumor suppressor gene, participating in a variety of neoplasms such as m e l a n o m a and squamous cell cancers. Unlike cyclin D 1, however, p l 6 does not appear to participate in parathyroid cell neoplasia (35), implying that cyclin D1 overexpression contributes to parathyroid tumorigenesis in ways that are not duplicated or precisely mimicked by p 16 inactivation. The cyclin D l - c d k 4 - p l 6 biochemical pathway also involves the protein product of the retinoblastoma tumor suppressor gene, Rb. The Rb protein (pRb) is an

MOLECULAR BASIS OF HYPERPARATHYROIDISM / important regulator of the G1/S transition of the cell cycle, and is phosphorylated in a cell cycle-dependent fashion (61,62). Early in G 1, the hypophosphorylated form of pRb is thought to bind and sequester a variety of transcription factors, such as those in the E2F family, maintaining the cell in G 1. Release of such factors later in G1, as pRb becomes highly phosphorylated, goes on to drive the cell into S phase. Thus, active (hypophosphorylated) pRb has a growth-restraining effect, which can be reversed or eliminated by inactivation through normal cell cycle-dependent hyperphosphorylation, or through germ-line or somatic mutational events. Much biochemical evidence has shown active pRb to be a major substrate target for cell cycle-dependent phosphorylation by active cyclin D l - c d k 4 / 6 complexes. It is thought, therefore, that overexpression or deregulated expression of cyclin D1 could quite conceivably accelerate the cell's progress through G 1 into S phase, bypassing normal regulatory controls in committing it to divide, and also be well tolerated by the cell during the remainder of the cycle. Such a mechanism would provide an appealing explanation for the benign nature of parathyroid adenomas, because it could yield excessive cellular proliferation without necessarily conferring the phenotypes of invasiveness or metastasis on the tumor cell. The measurable effects of cyclin D1 on proliferation of cells in culture appear to be mediated entirely through its ability to phosphorylate and thereby inactivate pRb; in fact, cyclin D1 becomes dispensible, in terms of its crucial and rate-limiting role at the G1-S checkpoint, in cells with deleted or otherwise genetically inactivated Rb. Rb is a classical example of a h u m a n tumor suppressor gene, involved in numerous tumors such as retinoblastoma, osteosarcoma, and breast and lung cancers. It appears that the cyclin D l - c d k 4 / 6 - p l 6 - R b pathway, viewed as a whole, has become aberrant in virtually every h u m a n cancer, highlighting it along with the p53 gene pathway as apparently essential targets for deregulation if neoplasia is to result. Cyclin D1 stands out as the only cyclin clearly implicated as a h u m a n oncogene (47,63,64). Because genetic abnormalities in many other G1-S regulators, including other cyclins and most cdk inhibitors, apparently do not confer a selective advantage on the cell (i.e., are not seen as clonal changes), it remains to be proved whether the oncogenicity of cyclin D1 is implem e n t e d entirely or at all by the shortening or acceleration of the G 1 phase of the cell cycle. Furthermore, it is plausible that cyclin D1 overexpression contributes to oncogenesis through mechanisms other than, or in addition to, its ability to activate cdk4 and phosphorylate pRb. In other words, tumorigenesis in the intact patient is a highly complex process not fully modeled by cultured cells, and it may be overly simplistic

337

to assume that cyclin D1 acts solely via the D l - c d k 4 p16-Rb paradigm that we now understand. This caution seems especially apt in considering parathyroid neoplasia, given its typically benign nature and slowgrowth properties. It is hoped that newly developed animal models of cyclin D 1-driven parathyroid neoplasia will provide new insight into parathyroid tumorigenesis.

Further Analysis of the Structure of PTH/Cyclin D1 Rearrangements After the cloning and analysis of the full-length complementary DNA (cDNA) representing PRAD1 mRNA, discussed above, it was possible to define the complete i n t r o n / e x o n organization of the normal chromosomal PRAD1/cyclin D1 gene (65). The gene is oriented on l l q 1 3 such that it is transcribed in a centromeric to telomeric direction, and contains five exons and four introns spanning approximately 15 kb. In the parathyroid adenomas with PTH/cyclin D1 rearrangements that have been well characterized to date, the 1 lq13 breakpoints have occurred from 1 to 15 kb upstream of cyclin D1 exon 1, leaving its exon-containing region and immediate p r o m o t e r intact (Fig. 2). Attached in these rearrangements to the cyclin D1 structural gene is the 5' regulatory region, including n o n c o d i n g exon 1, of the P T H gene (Fig. 2). The remarkable up-regulation of cyclin D1 expression in these parathyroid tumors therefore appears to be driven by DNA sequences normally found in this upstream PTH gene vicinity. In fact, these rearrangements provided the strongest initial evidence for localizing the still-undefined tissue-specific enhancer(s) of the P T H g e n e to its 5' regulatory region, a c o m m o n but by no means universal site for such sequences. This general localization of the P T H gene's tissue-specific e n h a n c e r has been confirmed by its success in targeting expression of a cyclin D1 transgene to parathyroid tissue in mice (66). The presence of n o n c o d i n g exon 1 of the P T H gene in the DNA rearranged upstream of cyclin D1 raised the possibility that a P T H exon 1/cyclin D1 fusion mRNA might ensue, and be transcribed from the P T H gene's own promoter. However, this does not appear to be the case. Analysis of the overexpressed cyclin D1 transcript (cDNA) from one such parathyroid t u m o r revealed normal cyclin D1 sequence at its 5' end, with no contribution from the P T H gene (67). The rearranged cyclin Dl's use of its own p r o m o t e r could also predict that its overexpression might be insulated from signals such as 1,25-dihydroxyvitamin D 3 or hypercalcemia, which are inhibitory for PTH gene transcription (see Chapter 2). In addition, it appears that point mutations in cyclin D1 are not necessary for tumorigenesis, because the amino acid coding sequence of this overexpressed cyclin D1 cDNA was entirely normal (67).

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Frequency of Cyclin D1/PRAD1 Involvement in Parathyroid Neoplasia

examined by immunohistochemistry in at least three studies, showing cyclin D 1 overexpression in 20-40% of parathyroid adenomas (68-70). The fraction of parathyroid adenomas for which cyclin D1 overexpression is due to PTH-cyclin D1 rearrangement, or other cyclin DI rearrangements for that matter, remains uncertain. It is important to note that the original Southern blot screens for such rearrangements used probes of PTH or cyclin D1 in

Because cyclin D1 oncogene activation in h u m a n tumors occurs through a variety of mechanisms, of which gene r e a r r a n g e m e n t is but one, the best estimates to date of the frequency of the involvement of cyclin D1 in parathyroid adenomas come from assessm e n t of cyclin D1 expression at the protein level. This pathogenetic c o m m o n d e n o m i n a t o r (Fig. 3) has been

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FIG. 3 Example of cyclin Dl-positive parathyroid adenoma (anticyclin D1 immunoperoxidase staining). (A) Normal parathyroid gland, lacking immunoreactivity; (B) parathyroid adenoma from the same patient, showing strong nuclear immunoreactivity in the majority of cells. Reprinted from Ref. 68, E Hsi, L Zukerberg, W-I Yang, A Arnold. Cyclin D1/PRAD1 expression in parathyroid adenomas: An immunohistochemical study. J Clin Endocrinol Metab, Vol. 81, pp. 1736-1739, 1996. © The Endocrine Society.

MOLECUtAR BASIS OF HYPERPARATHYROIDISM / the vicinity of their coding regions, and with the use of a limited number of restriction endonucleases (18,39,40,42). However, rearrangement breakpoints on 11q13 associated with overexpression of cyclin D1 in B cell lymphomas frequently occur well over 100 kb upstream of the cyclin D1 gene (71), and parathyroid adenomas with rearrangement breakpoints about 60 kb and otter 200 kb upstream of cyclin D1 have been observed (M. E. Williams, Y. Hosokawa, S. Mallya, and A. Arnold, unpublished observations), and would have been missed in the original Southern blot screens, which documented a minimum of about 5% of adenomas bearing PTH-cyclin D1 rearrangements. It is quite conceivable that PTH gene breakpoints could also vary considerably and have eluded detection by the previous approach; indeed, cyclin D1 expression could be deregulated in some parathyroid tumors by rearrangement with actively transcribed genes other than the PTHgene. For all these reasons, more comprehensive approaches will be necessary, and are in progress, to give a better sense of the frequency at which cyclin D1 gene rearrangement is responsible for deregulated expression of the cyclin D1 oncoprotein in parathyroid neoplasia. The first few patients with parathyroid adenomas that were clearly documented to have PTH/cyclin D1 rearrangement and overexpression exhibited clinical features of symptomatic hyperparathyroidism and had unusually large adenomas (6-8 g), without any histologic features suggestive of malignancy (18,39,40,42). However, subsequent study demonstrated cyclin D1 overexpression (Fig. 3) across the entire spectrum of parathyroid adenoma-related phenotypes, including asymptomatic patients with modestly sized adenomas (68). Thus, it appears that deregulation of cyclin D1 is an important contributor to the development of many (20-40%) parathyroid adenomas, which cover the broad spectrum of clinical and pathologic severity characteristic of this disorder. Role of Cyclin D1 in Other Human Tumors

Interest in the role of cyclin D1 in oncogenesis has been intensified by its incrimination in other, nonparathyroid, human tumors (64). In mantle cell (or centrocytic) B cell lymphomas, the upstream cyclin D1 gene region is rearranged with the immunoglobulin heavy-chain gene enhancer on chromosome 14. This t(ll;14) translocation results in the overexpression of cyclin D1, in a fashion analogous to the deregulated expression of oncogenes c-MYC and BCL-2 by their translocation into the immunoglobulin region in other types of B cell lymphomas (72-74). The possibility that a different gene in this region could be the true target of such rearrangements was effectively excluded by

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genetic evidence demonstrating some breakpoints located very close to cyclin D1 (71,75). Thus, cyclin D1 is now established to be the so-called "BCL-I" oncogene, which had been presumed to exist but had eluded workers searching 11 ql 3 in the vicinity of these translocation breakpoints for many years. The difficulty they had was due to the large distance, approximately 120 kb, between the original BCL-1 translocation breakpoint and the first exon of cyclin D1. Detection of cyclin D1 rearrangement or overexpression has become part of the armamentarium of diagnostic pathology labs in distinguishing subtypes of B cell neoplasms. More recently, tumor translocation breakpoints involving cyclin D1 and a different part of the immunoglobulin locus have been described in multiple myeloma (76,77). In addition to being a B cell lymphoma oncogene, cyclin D1 has been implicated in many other types of h u m a n tumors, including breast cancer; squamous cell cancer of the head, neck, and esophagus; bladder cancer; and more. In 12-15% of breast cancers and in up to 40% of the squamous cell tumors, a large stretch of DNA on 11q13 is amplified (present in extra copy number), implying the existence of at least one key gene, a "driver oncogene," whose amplification confers a selective growth advantage on the cell. Though there appears to be some complexity in the patterns of amplification in this region (78,79), cyclin D1 is now considered to be the, or at least one, driver oncogene on the major 11q13 "amplicon." First, no other gene is more consistently present on 11q13 amplicons in breast and squamous cell cancers (80,81). Second, cyclin D1 is overexpressed in these tumors; in contrast, most other genes that may be coamplified along with cyclin D1 typically exhibit poor expression (80). Finally, mammarytargeted overexpression of cyclin D1 in transgenic mice causes mammary carcinoma (82); no other candidate oncogene on the 11q13 amplicon has demonstrated this oncogenic capacity. Further studies have raised fascinating new possibilities regarding the mechanisms through which cyclin D1 exerts its oncogenicity in breast cancer. Two groups determined that overexpressed cyclin D1 can bind to and activate the estrogen receptor in cultured mammary carcinoma cells, without the need for estrogen and without the need for cyclin D1 to bind cdk4 (83,84). Though this mechanism requires further investigation in vivo, it has the potential to explain why virtually all cyclin Dl-overexpressing breast cancers are estrogen receptor positive. Other data have also raised the possibility that cyclin D1 could have non-cdkd e p e n d e n t functions (62,85,86). Such emerging information reinforces the concept that an open mind must be maintained regarding the mechanism(s) by which cyclin D1 contributes to parathyroid neoplasia.

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Inactivation of the MEN1 Tumor Suppressor Gene Multiple Endocrine Neoplasia Type 1 In 1988 the gene responsible for the MEN-1 tumor predisposition syndrome was m a p p e d by linkage analysis to the long arm of chromosome 11, in the general vicinity of the muscle phosphorylase (PYGM) gene (87). The positive finding of linkage initiated an extensive search of this region of 11q13 for the specific "MEN1 gene" (see Chapter 35). It should be noted that though cyclin D1/PRAD1 also m a p p e d to 11q13, the tightest MENlqinked markers were located far upstream (centromeric) of cyclin D1, probably several million base pairs away (72,80,88-92). It was considered that MEN-1 might prove to be due to an inherited mutation in a tumor suppressor gene, akin to the paradigms of familial retinoblastoma and Li-Fraumeni syndromes, which involve inheritance of mutations in the Rb and p53 genes, respectively (61). Therefore, tumors from MEN-1 patients were examined for evidence of somatic genetic events that could have inactivated one allele of a gene in this region and thereby unmask the inherited constitutional MEN1 mutation. In support of this hypothesis, "allelic loss" (or loss of heterozygosity) of polymorphic marker DNAs from chromosome 11 was initially found in two insulinomas from brothers with MEN-1 (87) and was subsequently found in the large majority of MEN-l-associated parathyroid tumors (20,21,33,34,93,94). The n u m b e r of markers from different parts of chromosome 11 exhibiting allelic loss varied widely from tumor to tumor, but the region of overlap of these losses was consistent with the MEN1

region as d e t e r m i n e d by linkage analysis. Furthermore, when the parental origin of the tumor-specific, somatically lost allele was able to be determined it derived from the clinically unaffected parent (21,33); this would be the expected pattern in the event that the retained chromosome contained an inherited mutant tumor suppressor gene (Fig. 4). The positional cloning of MEN1 in 1997 (95) provided further evidence in support of a tumor suppressor function for its gene product. Specifically, many of the observed inherited MEN1 mutations would be expected to eliminate functionality of its gene product, called menin (see Chapter 35). This type of inherited mutation in one allele, together with the commonly found acquired deletion of the remaining (normal) MEN1 allele, would result in absence of menin protein in the resulting parathyroid tumors in this syndrome. The possibility that certain inherited missense mutations might not completely eliminate menin function does not alter the essential theme of MENI's tumor suppressor nature, but might cause a moderated tumorigenic phenotype in some instances. These issues require further investigation. It is important to note that tumor-specific allelic deletions from chromosome 11 are also markers of monoclonality of these MEN-l-associated parathyroid tumors, indicating that despite the usual multigland involvement ("hyperplasia") found in MEN-1 patients, their parathyroid tumors are often (and perhaps always) clonal. It should also be borne in mind that critical acquired DNA damage may need to develop at still other genetic loci in order for clinically significant parathyroid tumors to emerge in MEN-1. Additional

Chromosome 11

normal copy

mutant copy

somatic deletion/mutation of remaining normal allele

Mutant copy of MEN1 tumor suppressor gene on 11q13 is inherited in MEN-1 and present in all parathyroid cells Mutation of one allele of MEN1 gene can occur somatically in other patients present in specific parathyroid cell(s)

mutation of other genes?

benign ~ parathyroid neoplasia

Clonal progenitor cell lacks functional menin gene product: at least 12-17% of sporadic parathyroid adenomas

Other Chromosomes

normal copy

mutant copy

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Somatic mutation of one copy of relevant tumor suppressor qene: no adverse consequences to parathyroid cell

mutation of other genes?

•parathyroid neoplasia

Clonal p r o g e n i t o r cell lacks functional tumor suppressor gene product

FIG. 4 Schematic diagram illustrating the established (for MEN1) and hypothesized roles of inactivation of classic tumor suppressor genes as contributory mechanisms in parathyroid neoplasia. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab, Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

MOLECULARBASIS OF H~Em'ARAT~OmISM study will also hopefully address the interesting question of whether the presence of one mutant plus one normal copy of MEN1 confers an abnormal proliferative phenotype on a parathyroid cell and a preliminary state of generalized true hyperplasia. Finally, it has been hypothesized that some examples of familial primary hyperparathyroidism without other manifestations of MEN may well be variants of MEN-1. Mutational analyses of the MEN1 gene have provided an answer, namely, that most kindreds with familial isolated hyperparathyroidism have no detectable mutations and do not appear to be MEN-1 variants, although such variants do occur (see Chapters 35 and 37). Sporadic Parathyroid Adenomas

The possibility that a chromosome 11-based tumor suppressor gene was involved in sporadic (nonfamilial) parathyroid adenomas was raised by two i n d e p e n d e n t observations. In the well-characterized Rb tumor suppressor gene model, the sporadic (nonfamilial) counterpart of familial retinoblastoma results from the somatic inactivation/loss of both (initially normal) alleles of the Rb gene in the clonal precursor cell. It therefore seemed reasonable to hypothesize that some sporadic parathyroid adenomas might evolve from a cell in which both copies of the MEN1 tumor suppressor gene, m a p p e d to 11q13, became inactivated by somatic mechanisms (Fig. 4). The other key result that focused attention on chromosome 11 was the recognition of chromosome breakpoints, described aboved, involving 11 q13. Study of sizeable numbers of sporadic parathyroid adenomas has determined that allelic loss of chromosome 11 markers occurs in 25-40% of the tumors (21,23,24,94). Examination of polymorphic markers spanning chromosome 11 in one especially informative case revealed that one allele of each marker was lost in this adenoma, indicating that an entire copy of the chromosome was somatically deleted from the tumor genome (19). Careful gene dosage analysis confirmed that this was a true deletion, unaccompanied by reduplication of the remaining chromosome, showing that monosomy 11 can be tolerated by a parathyroid cell and that only a single P T H gene copy per cell is sufficient to allow hyperparathyroid function by the adenoma (19). These latter conclusions were confirmed subsequently by comparative genomic hybridization (24). Given that most (but not all) regions of allelic loss on chromosome 11 included marker loci closely linked to the MEN1 gene, it was expected that inactivating somatic mutations would be identified in the nondeleted MEN1 allele in these tumors, consistent with its hypothesized role as a classic tumor suppressor (Fig. 4). The subsequent cloning of MEN1 has allowed this

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hypothesis to be tested. Interestingly, acquired inactivation of both alleles of the MEN1 gene has been found in 12-16% of parathyroid adenomas (26-28), representing about half of the adenomas with 1 lq deletions. This finding certainly establishes the importance of MEN1 gene inactivation in 12-16% of parathyroid adenomas. However, the large percentage of adenomas with 11q loss but without detectable mutation in the nondeleted MEN1 allele raises the fascinating possibility that inactivation of a different tumor suppressor gene from this chromosome region may contribute to the proliferative phenotype in many of those adenomas. Pursuit of the mechanisms by which MEN1 inactivation can participate in parathyroid neoplasia is in its early stages. Menin, the protein product of the MEN1 gene, was shown to associate with JunD, supporting a role in transcriptional control (96). The normal tissue distribution for expression of MEN1 is essentially ubiquitous and clearly not confined to the tissues susceptible to MEN-l-related tumorigenesis (97). Sporadic adenomas with MEN1 mutations were reported to have a somewhat higher frequency of allelic losses and chromosomal imbalances compared with sporadic adenomas without MEN1 involvement. Although these differences were not statistically significant, they hint at a possible role for MEN1 in maintenance of chromosomal stability (98), a theme that has been raised previously (99,100). The possibility of a relationship or cooperation between cyclin D 1 and menin in the develo p m e n t of sporadic a n d / o r MEN-l-related parathyroid neoplasia will also need to be addressed. Finally, the contribution of MEN1 inactivation to other forms of sporadic parathyroid neoplasia appears to be minor. The 11q13 allelic loss was found in 2-3% of uremia-associated parathyroid tumors (36,101), with MEN1 mutation in a just a subset of these. Such data reinforce the concept that different forms of parathyroid neoplasia are likely to have distinct molecular pathogenetic origins.

O T H E R G E N E T I C A B N O R M A L I T I E S IN SPORADIC PARATHYROID TUMORS For sporadic parathyroid adenomas, there is strong evidence that genes in addition to cyclin D1 and MEN1 participate in their pathogenesis. Highly recurrent clonal allelic losses, which frequently and successfully highlight the genomic locations of tumor suppressor genes, have been observed on chromosomes 1, 6, 11, and 15 in sporadic parathyroid adenomas in molecular allelotyping studies (22-24). O t h e r chromosomes, such as 9 and 13, have also been so implicated, albeit with lower frequency (24,35). Studies of sporadic adenomas using comparative genomic hybridization, a molecular

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cytogenetic method, have generally confirmed these c o m m o n areas of acquired deletion, and have also identified areas of chromosomal gain that may signify the presence of new parathyroid oncogenes (24,98,102). In contrast, traditional cytogenetic analysis has not so far been very helpful in highlighting chromosomal target regions to be intensively searched for new parathyroid oncogenes or tumor suppressor genes. A cytogenetic translocation between chromosomes 1 and 5 has been reported in a single parathyroid aden o m a (103), but it is unclear whether this was an isolated r a n d o m occurrence or will be characteristic of a distinct subset of adenomas. A n u m b e r of specific candidate genes, some of which map to regions of frequent allelic loss, have been examined for clonal defects in parathyroid adenomas. These include genes encoding RAS (42); p53 (104-106); p15, p16, and p18 cdk inhibitors (35,107); the calciumsensing receptor (108); the vitamin D receptor (108a); RET (109-111); RAD51 (112); and RAD54 (113). No tumor-specific mutations that would implicate any of these candidates as an oncogene or tumor suppressor gene in parathyroid adenomatosis have been observed, and these genes do not appear to participate in a primary fashion in typical sporadic adenomas. Although the discussion here relates primarily to c o m m o n sporadic parathyroid tumors, brief mention should be made at this point of inherited syndromes other than MEN-1 that predispose to hyperparathyroidism. Hereditary hyperparathyroidism-jaw tumor syndrome (familial parathyroid adenomatosis with ossifying fibromas of the jaw, or HPT-JT) is clearly distinct genetically from either MEN syndrome (114,115), and the gene responsible for this predisposition has been mapped using linkage analysis to the long arm of chromosome 1 (116). Eventual cloning of the responsible gene will permit an assessment of its role in c o m m o n sporadic hyperparathyroidism. In familial isolated hyperparathyroidism, a subset of kindreds also show linkage to this l q region, and may be variants of the HPT-JT syndrome (117). Other examples of familial isolated hyperparathyroidism are MEN-1 variants (118) and still other causative loci may well exist. Hyperparathyroidism is a c o m p o n e n t of MEN-2A, although not as penetrant or relentless a feature in this syndrome as it is in MEN-1. Heritable mutations in the RET gene are responsible for virtually all cases of MEN2A (see Chapter 36), but such RET mutations have not been observed as acquired clonal defects in sporadic parathyroid adenomas (109-111). Familial hypocalciuric hypercalcemia (FHH) results in large part from germ-line-inactivating mutations, usually heterozygous, in the calcium-sensing receptor (see Chapter 38). Parathyroid cell proliferation is at best only minimally increased in FHH, but homozygous germ-line CaSR deft-

ciency causes neonatal severe hyperparathyroidism with markedly excessive parathyroid cellularity. However, acquired inactivating mutations that would implicate the CaSR as a tumor suppressor in the pathogenesis of sporadic hyperparathyroidism have been sought and not found (108,119). Thus, among the genes currently known to be responsible for inherited predisposition to hyperparathyroidism, only MEN1 has been shown to participate in the cognate sporadic disease. It may be inferred that acquired RET or CaSR mutations do not tend to confer an important selective advantage on normal parathyroid cells under usual circumstances.

M O L E C U L A R P A T H O G E N E S I S OF PARATHYROID CARCINOMA No gene has been d o c u m e n t e d to be a definite participant in the pathogenesis of parathyroid cancer. However, strong evidence indicates that a tumor suppressor gene important in malignant or especially aggressive parathyroid neoplasia is located on chromosome 13. Specifically, a large proportion of these tumors have clonal allelic losses on 13q (30,120,121), which typically cover a large region including the Rb gene. That Rb might be involved was suggested by an immunohistochemical analysis showing no evidence of pRb expression in carcinomas with 13q loss (30), but it is quite conceivable that a different tumor suppressor on 13q, instead of or in addition to Rb, is the pathogenetically relevant target of these deletions. Also, given that a small subset of benign parathyroid adenomas exhibit 13q deletions, an interesting question is whether identical or distinct putative 13q tumor suppressors become inactivated in malignant versus benign hyperparathyroidism. Also noteworthy is the increased incidence of parathyroid carcinoma in the HPT-JT syndrome, linked to l q as mentioned above. Other genetic abnormalities have been sought in parathyroid carcinoma using genomic analyses such as molecular allelotyping and comparative genomic hybridization (25,31). Certain recurrent abnormalities appear to occur preferentially or exclusively in carcinomas as opposed to adenomas, suggesting that genes in these regions contribute specifically to the malignant phenotype. Equally important, a set of chromosomal regions that are frequently lost in benign adenomas are rarely if ever lost in parathyroid cancer (31 ). This observation suggests that parathyroid cancers do not generally evolve from preexisting typical benign adenomas. Even prior to the definitive identification of the involved genes, these data raise the possibility that tumor genome analysis might provide diagnostic or prognostic guidance in assessing parathyroid tumors with "atypical" features, for example.

MOLECULARBASIS OF HYPERPARATHYROIDISM /

E C T O P I C S E C R E T I O N OF P T H Primary hyperparathyroidism is a biochemical diagnosis, and a rare cause of primary hyperparathyroidism is the ectopic secretion of PTH by nonparathyroid tumors. Older literature describing this as a c o m m o n entity was c o n f o u n d e d by poor specificity of assays for PTH fragments. Developing knowledge that led to the ultimate identification of PTHrP as the major cause of hypercalcemia of malignancy later placed the very existence of the true ectopic PTH syndrome in doubt. However, m o d e r n highly specific immunometric assays for intact PTH, combined with molecular biologic approaches, have confirmed the occurrence of this syndrome, and the molecular basis for ectopic PTH production in one such case has been identified. Because tumors may synthesize hormones without releasing them to cause an identifiable clinical syndrome, specific criteria have been recognized for documenting the diagnosis of a true ectopic h o r m o n e syndrome (122). These criteria were unequivocally fulfilled for the diagnosis of one case of the ectopic PTH syndrome (123). This patient had elevated serum PTH (but not PTHrP) levels, four normal parathyroid glands, and an ovarian carcinoma. PTH secretion from the carcinoma was demonstrated and was proved to be the cause of hypercalcemia by a sevenfold increase in PTH concentration in the tumor's venous effluent, an immediate decline in serum PTH level and the develo p m e n t of hypocalcemia after tumor resection, the production of PTH by the ovarian carcinoma cells in culture, and the presence of mRNA encoding PTH (but not PTHrP) in the tumor tissue (123). This ovarian cancer was the only nonparathyroid tumor responsible for hypercalcemia in over 300 consecutive patients with a biochemical diagnosis of hyperparathyroidism (123). In addition to this ovarian carcinoma, reasonable but less definitive support for the diagnosis of ectopic PTH syndrome has been presented through the description of a growing n u m b e r of cases (124-128). The tumor types involved have been metastatic small cell carcinoma, thymoma, neuroectodermal malignancy, squamous cell lung carcinoma, and papillary thyroid carcinoma. These tumors have p r o d u c e d PTH mRNA or protein, with or without PTHrP, and were associated with hypercalcemia and elevated serum PTH levels in the absence of detectable parathyroid gland tumors. Ectopic h o r m o n e production can be considered as an aberration in the tissue specificity of gene expression; it usually involves dysregulation of a normal hormone gene product. Tissue specificity of gene expression is controlled by DNA sequences called "enhancer" and "silencer" elements, often located in the upstream regulatory region of a h o r m o n e gene,

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interacting with the particular mix of DNA binding proteins characteristic of that tissue type. Ectopic h o r m o n e production in a tumor could therefore result from an alteration in that tumor cell type's DNA binding protein environment (activating the intact enhancer of the h o r m o n e gene), or from a change in the e n h a n c e r / s i l e n c e r region adjacent to the h o r m o n e structural gene (thereby conferring responsiveness to the DNA binding proteins typical of the tumor cell type). In the ovarian carcinoma that ectopically produced PTH, described above, a DNA rearrangement was present in the upstream regulatory region of the PTH gene (Fig. 5), replacing the gene's own control elements with DNA sequences that could interact with the ovarian cell's DNA binding proteins and thereby "inappropriately" activate PTH gene transcription (123). In addition, this PTH gene with its rearranged upstream regulatory region was amplified, i.e., present in severalfold extra copy number, in the ovarian cancer cells, which almost certainly heightened the level of PTH production caused by the DNA r e a r r a n g e m e n t (123). The molecular mechanisms underlying other examples of the ectopic PTH syndrome remain to be elucidated.

SUMMARY This is an exciting time for those interested in the molecular basis of the various forms of primary hyperparathyroidism. The cyclin D1/PRAD1 oncogene, discovered through its involvement in the pathogenesis of parathyroid adenomas, is having a broad impact in oncology and cell cycle biology. The MEN1 gene has been identified, also plays an important role in sporadic hyperparathyroidism, and the function of its protein product is being vigorously pursued. New tools will facilitate the identification of still other parathyroid tumor-provoking genes that, given recent evidence, are virtually certain to exist. Genetic causes for MEN-2A and familial hypocalciuric hypercalcemia have been determined, which will continue to yield information of basic and clinical importance in parathyroid and other endocrine diseases. Finally, there is reason to hope that the molecular basis of the relationship between abnormal parathyroid cell proliferation and abnormal hormonal regulatory function, plus other problems unique to parathyroid disease, may soon be elucidated.

ACKNOWLEDGMENT The author wishes to thank Pamela Vachon for her invaluable assistance in the preparation of this chapter.

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CHAPTER19 :

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FIG. 5 Molecular pathology of the ectopic production of PTH by an ovarian cancer. Schematic diagram and restriction map of the rearranged and amplified PTH gene region (bottom) in the PTH-secreting ovarian tumor discussed in the text (123), compared with the normal PTH gene region (top). PTH exons I, II, and III are represented by open bars; normally present introns and flanking regions are denoted by solid bars, and the DNA placed upstream of PTH by the tumor-specific rearrangement are denoted by the cross-hatched bar. Restriction sites are labeled, and fragment sizes are shown in kilobases. For reasons of clarity and space, the map is not drawn to scale. The precise location of the breakpoint of the rearrangement was narrowed to a segment between the Hindlll site at -562 bp (562 base pairs upstream of the start of exon I) and the normally present Bglll site 150 bp further upstream (no longer present in the rearranged gene). Bam, BamHI; Msp, Mspl; Hind, Hindlll; Bgl, Bglll; Eco, EcoRl. Reprinted from Ref. 123, SR Nussbaum, RD Gaz, A Arnold. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990;323:1324-1328. Copyright © 1990 Massachusetts Medical Society. All rights reserved.

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60. Sherr C, Roberts J. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-1163. 61. Weinberg RA. Tumor suppressor genes. Science 1991; 254:1138-1146. 62. Jacks T, Weinberg R. The expanding role of cell cycle regulators. Science 1998;280:1035-1036. 63. Motokura T, Arnold A. Cyclin D and oncogenesis. Curr Opin Genet Dev 1993;3:5-10.

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64. Arnold A. The cyclin D1/PRAD1 oncogene in human neoplasia. J Invest Med 1995;43:543-549. 65. Motokura T, Arnold A. The PRAD 1/cyclin D 1 proto-oncogene: Genomic organization, 5' DNA sequence, and sequence of a tumor-specific rearrangement breakpoint. Genes Chromosomes Cancer 1993;7:89-95. 66. Hosokawa Y, Yoshimoto K, Bronson R, Wang T, Schmidt E, Arnold A. Chronic hyperparathyroidism in transgenic mice with parathyroid-targeted overexpression of cyclin D1/PRAD1. J Bone Miner Res 1997;12(Suppl. 1):Sl10. 67. Rosenberg CL, Motokura T, Kronenberg HM, Arnold A. Coding sequence of the overexpressed transcript of the putative oncogene PRAD1/cyclin D1 in two primary human tumors. Oncogene 1993;8:519-521. 68. Hsi E, Zukerberg L, Yang W-I, Arnold A. Cyclin D1/PRAD1 expression in parathyroid adenomas: An immunohistochemical study. J Clin Endocrinol Metab 1996;81:1736-1739. 69. Vasef M, Brynes R, Sturm M, Bromley C, Robinson R. Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasias: A paraffin immunohistochemical study. Mod Patho11999;12:412-416.

70. Tominaga Y, Tsuzuki T, Uchida K, et al. Expression of PRAD1/cyclin D1, retinoblastoma gene products, and Ki67 in parathyroid hyperplasia caused by chronic renal failure versus primary adenoma. Kidney Int 1999;55:1375-1383. 71. Williams ME, Swerdlow SH, Rosenberg CL, Arnold A. Chromosome 11 translocation breakpoints at the PRAD1 cyclin gene locus in centrocytic lymphoma. Leukemia 1993;7:241-245. 72. Rosenberg CL, Wong E, Petty EM, et al. PRAD1, a candidate BCL1 oncogene: Mapping and expression in centrocytic lymphoma. Proc Natl Acad Sci USA 1991 ;88:9638-9642. 73. Withers DA, Harvey RC, Faust JB, Melnyk O, Carey K, Meeker TC. Characterization of a candidate bcl-1 gene. Mol CeU-Biol 1991;11:4846-4853. 74. Seto M, Yamamoto K, Iida S, et al. Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14) (ql 3;q32) translocation. Oncogene 1992;7:1401-1406. 75. Komatsu H, Iida S, Yamamoto K, et al. A variant chromosome translocation at 1 lq13 identifying PRAD1/cyclin D1 as the BCL1 gene. Blood 1994;84:1226-1231. 76. Gabrea A, Bergsagel P, Chesi M, Shou Y, Kuehl W. Insertion of excised IgH switch sequences causes overexpression of cyclin D1 in a myeloma tumor cell. Mol Cell 1999;3:119-123. 77. Chesi M, Bergsagel PL, Brents I_A, Smith CM, Gerhard DS, Kuehl WM. Dysregulation of cyclin D1 by translocation into an IgH gamma switch region in two multiple myeloma cell lines [see comments]. Blood 1996;88:674-81. 78. Proctor AJ, Coombs LM, Cairns JP, Knowles MA. Amplification at chromosome 11q13 in transitional cell tumours of the bladder. Oncogene 1991 ;6: 789-795. 79. Szepetowski P, Courseaux A, Carle GE Theillet C, Gaudray E Amplification of 1 l q l 3 DNA sequences in human breast cancer: DllS97 identifies a region tightly linked to BCL1 which can be amplified separately. Oncogene 1992;7:751-755. 80. Lammie GA, Fantl V, Smith R, et al. DllS287, a putative oncogene on chromosome llq13, is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-1. Oncogene 1991 ;6:439-444. 81. Schuuring E, Verhoeven E, Mooi WJ, Michalides RJAM. Identification and cloning of two overexpressed genes, U 2 1 B 3 1 / P R A D 1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas. Oncogene 1992;7:355-361. 82. Wang T, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 1994;369:669-671.

83. Zwijsen R, Wientjens E, Klompmaker R, van der Sman J, Bernards R, Michalides R. CDK-independent activation of estrogen receptor by cyclin D1. Cell 1997;88:405-415. 84. Neuman E, Ladha M, Lin N, et al. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4. Mol Cell Biol 1997;17:5338-5347. 85. Hirai H, Sherr C. Interaction of D-type cyclins with a novel myblike transcription factor, DMPI. Mol Cell Biol 1996;16:6457-6467. 86. Zwicker J, Brusselbach S, Jooss K, et al. Functional domains in cyclin DI: pRb-kinase activity is not essential for transformation. Oncogene 1999;18:19-25. 87. Larsson C, Skogseid B, Oberg K, Nakamura Y, Nordenskjold M. Multiple endocrine neoplasia type I gene maps to chromosome 11 and is lost in insulinoma. Nature 1988;332:85-87. 88. Petty EM, Arnold A, Marx SJ, Bale AE. A pulsed-field gel electrophoresis (PFGE) map of twelve loci on chromosome l l q l l q13. Genomics 1993;15:423-425. 89. Janson M, Larsson C, Werelius B, et al. Detailed physical map of human chromosomal region 11q12-13 shows high meiotic recombination rate around the MEN1 locus. Proc Natl Acad Sci USA 1991;88:10609-10613. 90. Larsson C, Weber G, Kvanta E, et al. Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus. H u m Genet 1992;89:187-193. 91. Nakamura Y, Larsson C, Julier C, et al. Localization of the genetic defect in multiple endocrine neoplasia type 1 within a small region of chromosome 11. A m J H u m Genet 1989;44:751-755. 92. Julier C, Nakamura Y, Lathrop M, et al. A detailed genetic map of the long arm of chromosome 11. Genomics 1990;7:335-345. 93. Radford DM, Ashley SW, Wells SA, Jr, Gerhard DS. Loss of heterozygosity of markers on chromosome 11 in tumors from patients with multiple endocrine neoplasia syndrome type 1. Cancer Res 1990;50:6529-6533. 94. Friedman E, De Marco L, Gejman PV, et al. Allelic loss from chromosome 11 in parathyroid tumors. Cancer Res 1992;52:6804-6809. 95. Chandrasekharappa S, Guru S, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404-407. 96. Agarwal S, Guru S, Heppner C, et al. Menin interacts with the API transcription factor JunD and represses JunD-activated transcription. Cell 1999;96:143-152. 97. Bassett J, Rashbass P, Harding B, Forbes S, Pannett A, Thakker R. Studies of the murine homolog of the multiple endocrine neoplasia type 1 (MEN1) gene, menl. J Bone Miner Res 1999;14:3-10. 98. Farnebo F, Kytola S, Teh B, et al. Alternative genetic pathways in parathyroid tumorigenesis. J Clin Fndocrinol Metab 1999;84:3775-3780. 99. Hecht F, Hecht BK. Unstable chromosomes in heritable tumor syndromes: Multiple endocrine neoplasia type 1 (MEN1). Cancer Genet Cytogenet 1991;52:131-134. 100. Scappaticci S, Maraschio P, del Ciotto N, Fossati G, Zonta A, Fraccaro M. Chromosome abnormalities in lymphocytes and fibroblasts of subjects with multiple endocrine neoplasia type 1. Cancer Genet Cytogenet 1991 ;52:85-92. 101. Imanishi Y, Tahara H, Salusky I, et al. MEN1 gene mutations in refractory hyperparathyroidism of uremia. J Bone Miner Res 1999;14(Suppl. 1):$446. 102. Agarwal S, Schrock E, Kester M, et al. Comparative genomic hybridization analysis of human parathyroid tumors. Cancer Genet Cytogenet 1998;106:30-36. 103. Orndal C, Johansson M, Heim S, et al. Parathyroid adenoma with t(1;5)(p22:q32) as the sole clonal chromosome abnormality. Cancer Genet Cytogenet 1990;48:225-228.

MoLwcvlag BASIS OF HYPERPARATHYROIDISM / 104. Cryns VL, Rubio ME Thor AD, Louis DN, Arnold A. p53 abnormalities in human parathyroid carcinoma. J Clin Endocrinol Metab 1994;78:1320-1324. 105. Yoshimoto K, Iwahana H, Fukuda A, Sano T, Saito S, Itakura M. Role of p53 mutations in endocrine tumorigenesis: Mutation detection by polymerase chain reaction-single strand conformation polymorphism. Cancer Res 1992;52:5061-5064. 106. Hakim J, Levine M. Absence of p53 point mutations in parathyroid adenoma and carcinoma. J Clin Endocrinol Metab 1994;78:103-106. 107. Tahara H, Smith A, Gaz R, Zariwala M, Xiong Y, Arnold A. Parathyroid tumor suppressor on lp: Analysis of the p18 cyclindependent kinase inhibitor gene as a candidate. JBone Miner Res 1997;12:1330-1334. 108. Hosokawa Y, Pollak MR, Brown EM, Arnold A. Mutational analysis of the extracellular CaZ+-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995;80:3107-3110. 108a.Brown SB, Brierley TT, Palanisamy N, Salusky IB, Goodman W, Brandi ML, Drueke TB, Sarfati E, Urena P, Chaganti RSK, Pike JW, Arnold A. Vitamin D receptor as a candidate tumor suppressor gene in severe hyperparathyroidism of uremia. J Clin Endocrinol Metab 2000;85:868-872. 109. Pausova Z, Soliman E, Amizuka N, et al. Role of the RET protooncogene in sporadic hyperparathyroidism and in hyperparathyroidism of multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1996;81:2711-2718. 110. Padberg B, Schroder S, Jochum W, et al. Absence of RET protooncogene point mutations in sporadic hyperplastic and neoplastic lesions of the parathyroid gland. Am J Pathol 1995;147:1600-1607. 111. Kimura T, Yoshimoto K, Tanaka C, et al. Obvious mRNA and protein expression but absence of mutations of the RET proto-oncogene in parathyroid tumors. EurJ Endocrino11996; 134:314-319. 112. Carling T, Imanishi Y, Gaz R, Arnold A. RAD51 as a candidate parathyroid tumor suppressor gene on chromosome 15q: Absence of somatic mutations. Clin Endocrino11999;51:403-407. 113. Carling T, Imanishi Y, Gaz R, Arnold A. Analysis of the RAD54 gene on chromosome l p as a potential tumor suppressor gene in parathyroid adenomas. I n t J Cancer 1999;83:80-82. 114. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987;107:54-60. 115. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990;108:1006-1013. 116. Szabo J, Heath B, Hill VM, et al. Hereditary hyperparathyroidism-jaw tumor syndrome: The endocrine tumor gene HRPT2 maps to chromosome lq21-q31. A m J H u m Genet 1995;56:944-950.

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CI-IAPTFI20 Clinical Presentation of Primary Hyperparathyroidism in the United States

SHONNI J. SILVERBERG Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 J O H N E BILEZIKIAN Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

INTRODUCTION

mary hyperparathyroidism does seem to present more along classic lines (1-3) (see Chapters 21 and 22). In addition to describing the clinical features of primary hyperparathyroidism in the United States, this chapter also considers the evolving clinical spectrum of primary hyperparathyroidism, with a retrospective view of what used to be more typical presentations of the disease. Frequent cross-references will facilitate easy access to other chapters in this volume for readers who want to explore some of these points in greater depth.

Primary hyperparathyroidism is a common endocrine disorder, characterized by the excessive and incompletely regulated secretion of parathyroid hormone (PTH) from one or more parathyroid glands. The major actions of PTH, to mobilize calcium from bone, to conserve calcium in the kidney, and indirectly to increase gastrointestinal calcium absorption, lead to one of the major biochemical hallmarks of the disease, hypercalcemia. Another major sign of the disorder is an elevated level of PTH, now readily detected by accurate assays for the hormone. Advances in evaluation of metabolic bone diseases by sensitive circulating and urinary markers of calcium metabolism have permitted a more detailed assessment of patients who do not appear to be suffering from overt clinical consequences of primary hyperparathyroidism. In addition, bone densitometry and analysis of bone by quantitative histomorphometry have provided direct insight into important current features of the disease. The result is a profile of primary hyperparathyroidism that not only is quite different from earlier historical descriptions but also requires consideration of a new set of issues insofar as the clinical m a n a g e m e n t of the disease is concerned. Because primary hyperparathyroidism is the major clinical disorder of the parathyroid glands, it is fitting that this disorder be the focus of an extensive discussion in this volume. This chapter describes major clinical features of primary hyperparathyroidism as it presents in the United States today. The clinical presentation of the disease differs in other parts of the world, where priThe Parathyroids, Second Edition

PREVALENCE A N D INCIDENCE OF PRIMARY HYPERTHYROIDISM It is remarkable that within the lifetimes of some currently practicing endocrinologists, primary hyperparathyroidism has been transformed from an extremely rare endocrine disorder to a relatively common one. In the 1930s and 1940s, primary hyperparathyroidism was appreciated virtually always in the context of a most unusual disorder with characteristic skeletal features known as osteitis fibrosa cystica. In fact, it was said in those days that "the X-ray findings proved to be so characteristic that chemical analysis was needed only for confirmation" (4). Other features of the historical summary of primary hyperparathyroidism by Oliver Cope in 1966 point out how these patients invariably deteriorated with a particularly pernicious bone disease. Studies of the famous sea captain Charles Martell by Bauer and colleagues (5,6) and the work of the Viennese surgeon Mandl (7) gave great impetus to 349

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the correct idea that primary hyperparathyroidism is caused by abnormal function of parathyroid tissue and that removal of the offending a d e n o m a leads to correction of the hypercalcemia. Despite the fact that the disorder used to be rare and that the first series of patients with primary hyperparathyroidism described in 1934 included only 17 patients (8), it was evident even in those days that the incidence of the disease was, in part, a function of how high one's index of suspicion was for it. For example, Raymond Keating, whose work at the Mayo Clinic helped to establish m o d e r n concepts of the disease, was dispatched to the Massachusetts General Hospital in 1942 specifically to learn how the physicians there seemed to recognize patients fairly readily (Aub, Bauer, Albright, and Cope had seen 67 patients by this time); the experience at the Mayo Clinic was much more limited. After this tutorial in Boston, Keating returned to the Mayo Clinic and, with a clear intention to uncover the disease, saw more patients with primary hyperparathyroidism in the next year than he had seen in the preceding 15 years (9). This early experience makes a point that has been relived in the m o d e r n era. In the absence of serendipity or incidental discovery, the disorder is not readily diagnosed without a high index of suspicion. The dramatic change in the incidence of primary hyperparathyroidism occurred in the late 1960s and early 1970s, due primarily to the introduction of the multichannel autoanalyzer. Documentation of this change comes from the work of Heath and colleagues as well as from other groups (10-14). Incidence figures among residents of Rochester, Minnesota, increased nearly fivefold in the first year after the multichannel screening profile became routinely available (1974-1975). Thereafter, allowing for the catch-up detection factor in that first year (the "sweeping" effect), the incidence figures continued to show an impressive fourfold increase in comparison to the premultichannel autoanalyzer era (15). It is difficult to estimate true incidence figures for primary hyperparathyroidism (16), but the experience in Rochester, Minnesota, of 27.7 per 100,000 person years is essentially identical to figures from Sweden (12) and from Birmingham, England (11). On the basis on these figures, an estimate of approximately 100,000 new cases of primary hyperparathyroidism per year in the United States is likely to be accurate. The prevalence of primary hyperparathyroidism (the proportion of the population affected with the disease at a given point in time) is higher than earlier estimates of incidence (the n u m b e r of new cases diagnosed over a specified period of time). Prevalence estimates have been as high as 1 in 100 (17), but 1 per 1000 would appear to be closer to the true prevalence rate in the early autoanalyzer era (18).

A report from Rochester, Minnesota suggests that newly diagnosed cases of primary hyperparathyroidism have been declining continuously since the mid-1970s (19). This experience has not been clearly repeated in other American centers. It is possible that the particular demographics of Rochester, Minnesota, combined with the rather complete discovery of primary hyperparathyroidism in a population that receives virtually all of its care in one system (allowing for ideal epidemiologic surveillence), could account for declining numbers. More research in this area will be necessary to determine the direction of future changes in incidence figures (see later discussion). Primary hyperparathyroidism occurs throughout life, but the incidence peaks in the middle years. Women predominate over men by a 2:1-3:1 margin. The disease is recognized most commonly in women who are in the first postmenopausal decade, between ages 50 and 60 years. It is perhaps because of the effects of estrogens to oppose some of the skeletal actions of PTH that the disease may surface clinically when estrogen levels fall. There do not appear to be any well-established predisposing factors for the development of primary hyperparathyroidism, but a history of irradiation to the neck and u p p e r chest area in childhood is obtained in as many as 15-25% of patients with the disease (20,21). Primary hyperparathyroidism after radioactive iodine therapy for thyroid disease has been reported infrequently (22). Exciting new insights into the molecular bases of some cases of primary hyperparathyroidism are covered in Chapter 19 (23). Newer concepts of parathyroid cell growth properties in primary hyperparathyroidism are presented in Chapter 18 (24).

PATHOLOGY Most patients with primary hyperparathyroidism (80-85%) harbor a single adenoma; the other three glands are normal. In 2-4% of cases, multiple parathyroid adenomas have been described. Histologically the a d e n o m a is described as a confluence of parathyroid cells that may be associated with a rim of normal tissue at the margins. The average a d e n o m a is 0.5 g, although abnormal glands distinctly smaller or larger are seen. Even the smaller adenomas,

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FIG. 4 Changes in bone metabolism following successful parathyroidectomy (PTX) in 20 patients with mild primary hyperparathyroidism. DPD, Urinary deoxypyridinoline; BSP, serum bone sialoprotein; BAP, serum bone specific alkaline phosphatase. For better comparison, values were calculated as percent change versus base line (= 100%). Note the transient increase in BAP and the quick decline in both DPD and BSP (from M.J. Seibel, unpublished data).

Tartrate-Resistant Acid Phosphastae Plasma levels of the tartrate-resistant acid phosphatase (pTRAP) are significantly elevated in patients with PHPT compared to healthy controls (98-100). In untreated patients, pTRAP levels correlate both with sTAP and uOHP, but also with serum PTH concentrations (101). As expected, pTRAP significantly decreases after successful parathyroidectomy (102).

Bone Sialoprotein Although very few studies are presently available, serum bone sialoprotein (sBSP) also seems to be a valid marker of bone turnover in patients with PHPT. In a cross-sectional studies, sBSP levels were significantly elevated over normal (Fig. 3), and a positive correlation was observed with both uPYD and uDPD (88,103). Preliminary observations suggest that serum BSP levels drop rapidly after successful parathyroidectomy (Fig. 4).

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71. Taylor AK, Lueken SA, Libanati C, Baylink DJ. Biochemical markers of bone turnover for the clinical assessment of bone metabolism. Rheum Dis Clin North Am 1994;20:589-607. 72. Yoneda M, Takatsuki K, Oiso Y, et al. Clinical significance of serum bone Gla protein and urinary gamma-Gla as biochemical markers in primary hyperparathyroidism. Endocrinol Jpn. 1986;33:89-94. 73. Charles P, Mosekilde L, Jensen FT. Primary hyperparathyroidism: Evaluated by 47calcium kinetics, calcium balance, and serum bone-Gla-protein. E u r J Clin Invest 1986;16:277-283. 74. Delmas PD, Demiaux B, Malaval L, et al. Serum bone gamma carboxyglutamic acid-containing protein in primary hyperparathyroidism and in malignant hypercalcemia. Comparison with bone histomorphometry. J Clin Invest 1986;77:985-991. 75. Ebeling PR, Peterson JM, Riggs BL. Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases. J Bone Miner Res 1992;7:1243-1250. 76. Hoshino H, Kushida K, Takahashi M, et al. Short-term effect of parathyroidectomy on biochemical markers in primary and secondary hyperparathyroidism. Miner Electrolyte Metab 1997;23:93-99. 77. Minisola S, Romagnoli E, Scarnecchia L, et al. Serum carboxyterminal propeptide of human type I procollagen in patients with primary hyperparathyroidism: Studies in basal conditions and after parathyroid surgery. EurJEndocrinol 1994;130:587-591. 78. Coen G, Mazzaferro S, De Antoni E, et al. Procollagen type 1 Cterminal extension peptide serum levels following parathyroidectomy in hyperparathyroid patients. AmJNephrol 1994;14:106-112. 79. Brahm H, Ljunggren 0, Larsson K, Lindh E, Ljunghall S. Effects of infusion of parathyroid hormone and primary hyperparathyroidism on formation and breakdown of type I collagen. Calcif Tissue Int 1994;55:412-416. 80. Sairanen S, T/ihtela R, Laitinen K, L6yttyniemi E, V/ilim/iki MJ. Effects of short-term treatment with clodronate on parameters of bone metabolism and their diurnal variation. Calcif Tissue Int 1997;60:160-163. 81. Charles P, Mosekilde L, Risteli L, et al. Assessment of bone remodeling using biochemical indicators of type I collagen synthesis and degradation: Relation to calcium kinetics. Bone Miner 1994;24:81-94. 82. Eriksen EF, Charles P, Meisen F, et al. Serum markers of type 1 collagen formation and degradation in metabolic bone disease: Correlation with bone histomorphometry. J Bone Miner Res 1993;8:127-132. 83. LoCascio V, Braga V, Bertoldo F Bettica P, et al. Effect of bisphosphonate therapy and parathyroidectomy on the urinary excretion of galactosylhydroxylyine in primary hyperparathyroidism. Clin Endocrino11994;41:47-51. 84. Hyldstrup L, McNair P, Jensen GF, Nielsen HR, Transbol I. Bone mass as referent for urinary hydroxyproline excretion: Age and sex-related changes in 125 normals and in primary hyperparathyroidism. Calcif Tissue Int 1984;36:639-644. 85. Robins SP, Black D, Paterson CR, Reid DM, Duncan A, Seibel MJ. Evaluation of urinary hydroxypyridinium crosslink measurements as resorption markers in metabolic bone disease. Eur J Clin Invest 1991;21:310-315. 86. Robins SP, Woitge H, Duncan A, Seyedin S, Seibel MJ. Immunoassay of deoxy-pyridinoline: A specific marker of bone resorption. J Bone Miner Res 1994;9:1643-1649. 87. Arbault P, Grimaux M, Pradet V, Preaudat C, Seguin P, Delmas PD. Assessment of urinary pyridinoline excretion with a specific enzyme-linked immunosorbent assay in normal adults and in metabolic bone diseases. Bone 1995;16:461-467. 88. Woitge HW, Oberwittler H, Farahmand I, Lang M, Ziegler R, Seibel MJ. New serum assays for bone resorption. Results of a cross-sectional study. J Bone Miner Res 1999;14:792-801.

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Cytokines in Primary Hyperparathyroidism

INAAM A. NAKCHBANDI Mannheim Faculty of Medicine, University of Heidelberg, 68135 Mannheim, Germany AND REW GREY Department of Medicine, University of Auckland, 92019 Auckland, New Zealand URSZULA MASIUKIEWICZ, MARYANN MITNICK, AND KARL INSOGNA Yale University School of medicine, New Haven, Connecticut 06520

INTRODUCTION

factor-I (IGF-I). In this latter case, IGF-I that is released by osteoblasts in response to PTH acts in an autocrine fashion to induce anabolic effects in osteoblasts. The resorptive response to PTH is t h o u g h t to be mediated by inducing osteoblasts to express proresorptive molecules such as osteoclast differentiation factor, which stimulates osteoclast differentiation a n d / o r activates mature osteoclasts. Thus, in both instances cell-to-cell c o m m u n i c a t i o n is central to the actions of this h o r m o n e . Cytokines have e m e r g e d as i m p o r t a n t mediators of cell-to-cell communication. They are a diverse family of molecules, many of which were initially identified as factors regulating hematopoietic and i m m u n e cell function and differentiation. However, they have, in many instances, been found to have pleiotropic actions that include effects outside the hematopoietic microenv i r o n m e n t (6). For instance, interleukin-6 (IL-6) is released locally by the liver during severe inflammation or infection, and also plays a role in regulating osteoclastogenesis (7,8). However, data indicate that IL-6 can also act systemically to activate the h y p o t h a l a m i c pituitary-adrenal axis (9). Thus, IL-6 can function locally and as a circulating h o r m o n e at distant target tissues. IL-6 is, in turn, regulated by other h o r m o n e s as evidenced by the fact that its expression in bone cells is m o d u l a t e d by estrogen (8). These new findings have added a n o t h e r layer of complexity to the study of cytokines, in that it increasingly appears that certain cytokines are not restricted to local actions that require cell-to-cell c o m m u n i c a t i o n in circumscribed microenvironments, but can act systemically as well to induce

Primary hyperparathyroidism is characterized by biochemical and histomorphometric evidence for increased bone turnover (1-3). Understanding the mechanisms by which parathyroid h o r m o n e (PTH) exerts its skeletal effects is important for devising optimal t r e a t m e n t guidelines for this disorder and for a better appreciation of the anabolic and catabolic effects of this h o r m o n e in bone. Studies have focused attention on cytokines as mediators of the effects of PTH in bone, and these molecules are the topic of this chapter (4,5). PTH has p r o f o u n d effects in bone, inducing both increased bone resorption and bone formation. The ability of exogenously administered PTH to replicate the physiologic effects of native PTH is d e p e n d e n t on both the dose and the m o d e of delivery of the hormone. Intermittent administration is most consistently associated with an anabolic response. By contrast, continuous administration of PTH at a fixed dose leads to a predominantly resorptive response. Chapter 11 discusses the anabolic and catabolic effects of parathyroid h o r m o n e on bone in detail. The principal target cell in bone for PTH appears to be the osteoblast or osteoblast-like cell, which expresses the type 1 P T H / P T H r P receptor. It is currently believed that most of the effects of PTH in bone are mediated by inducing changes in osteoblast function. The anabolic effects of PTH are p r e s u m e d to be both direct, by regulating osteoblast gene and protein expression governing replication and differentiation, and indirect, via an autocrine loop, as is postulated for insulin-like growth The Parathyroids, Second Edition

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changes at target tissues remote from their sites of production. The study of cytokines in disease states has revealed unanticipated complexity, and this is certainly the case for hyperparathyroidism. As will be discussed, PTH has been shown in vitro to regulate the expression of a wide variety of bone-active cytokines. Additional work has also demonstrated that PTH significantly alters the systemic levels of certain cytokines. The relative importance of these various cytokines as well as the contribution of their local versus systemic effects to the overall actions of PTH is an emerging area of investigation. This chapter briefly reviews cytokines for which expression is regulated by PTH and which may, at least in part, mediate the anabolic and catabolic effects of the hormone. Although data are available on the role cytokines play in mediating the effects of PTH in vitro, few in vivo data are available. Further, in hyperparathyroidism, few studies have addressed the role of cytokines in the pathogenesis of this disease (10). This chapter focuses on experimental evidence for a role for cytokines as mediators of PTH actions in bone. These cytokines have been categorized on the basis of whether they are thought to mediate the anabolic or catabolic effects of PTH or to have mixed effects in bone. Finally, a summary of what is known about the changes in tissue expression and circulating levels of cytokines in primary and secondary hyperparathyroidism is presented.

EXPERIMENTAL EVIDENCE THAT CYTOKINES MEDIATE P T H ACTIONS IN B O N E When comparing the effects of intermittent and continuous administration of PTH in animals, it is found that both treatment modalities result in an increase in bone formation, but that there is a p r o m i n e n t resorptive response when PTH is given by continuous infusion (11). It has also been established that intermittent PTH injection leads to an increase in bone mineral density both in animals (3,11) and in humans (12), whereas continuous infusion of PTH in humans leads to a p r o m i n e n t resorptive response and dose-dependent hypercalcemia. The cellular and molecular mechanisms that underlie these differing responses to PTH, based on mode of delivery, are not well understood and only limited data are available on the cytokine profile induced by these two treatment regimens. Continuous infusion of PTH is of limited usefulness as an experimental model of chronic hyperparathyroidism. As noted, the continuous administration of PTH results in an increase in bone remodeling with

activation of both resorption and formation in a manner that partially replicates bone changes in primary hyperparathyroidism (3). However, although basal levels of PTH are elevated in primary hyperparathyroidism, at least in mild disease, a pulsatile secretory c o m p o n e n t still exists. Further, in mild hyperparathyroidism, PTH secretion still responds to changes in ionized calcium such as those induced by dietary intake (13). Therefore PTH infusion does not precisely mimic hyperparathyroidism insofar as levels are constant and u n c h a n g e d with the former but vary somewhat from a tonically elevated base line in the latter.

Cytokines with Anabolic Actions Anabolic cytokines (IGF, vascular endothelial growth factor, and fibroblast growth factor) are t h o u g h t principally to affect bone formation. Insulin-like Growth Factor System

The IGF system includes IGF-I and IGF-II, the IGF receptors, IGF-binding proteins (IGFBPs), and their proteases (14). IGF-I and IGF-II increase replication of cells in the osteoblast lineage and enhance collagen synthesis by mature osteoblasts (15). Further, injections of IGF-I increase bone formation and trabecular connectivity (16,17). IGF-I is produced by neonatal mouse calvaria in response to PTH treatment and is found in primary cultures of rodent osteoblasts (18,19). IGF-I is thought to mediate PTH-related protein (PTHrP)-induced collagen synthesis in osteoblasts because neutralizing antiserum to IGF-I abrogates this anabolic effect of PTHrP (20). There are six conventional and at least four additional provisional IGFBPs, some of which increase the half-life and others that inhibit the actions of IGF-I and IGF-II by inhibiting receptor binding (15). IGFBP-3 expression in osteoblasts is increased by PTH. IGFBP-3 prolongs the half-life of IGF-I, thus amplifying its effects (21). However, PTH also up-regulates IGFBP-4, which inhibits the actions of both IGF-I (22) and IGF-II. Therefore, IGFBP-4 could theoretically inhibit bone formation (15). IGFBP-7/mac25/IGFBPrP1 transcript expression is increased in rat osteoblasts treated with PTH. This protein can also inhibit the actions of IGF-I (23). Whether differences in the mode of PTH delivery in vivo differentially affect these members of the IGFBP family and play a role in the expression the anabolic versus catabolic effects of the h o r m o n e is an area of current investigative interest. Intermittent PTH treatment in animals results in an increase in bone content of IGF-I by 20% (24), an

CYTOKINES IN PRIMARYHYPERPARATHYROIDISM /

increase in IGF-I in bone matrix (25), and an increase in IGF-I mRNA expression in rat bones (26). One report suggested that concomitant with the increase in bone content of IGF-I induced by intermittent PTH treatment, there is a decrease in circulating IGF-I (27). The implications of this finding remain unclear. Continuous PTH administration has been shown to increase IGF-I mRNA expression in rat bones, in a m a n n e r similar to intermittent PTH injection (26). In humans, the serum level of IGF-I reportedly increased during a 20-hour infusion in osteoporotic women (28), but remained constant when PTH was infused in normal healthy volunteers (29). IGF-II levels were not affected by PTH infusion. However, the level of IGFBP3, the binding protein that increases IGF-I half-life and its anabolic effects, was increased by PTH infusion (29). In summary, IGF-I remains an attractive candidate as a mediator for the anabolic effect in bone seen with intermittent PTH administration. Vascular Endothelial Growth Factor

PTH induces vascular endothelial growth factor (VEGF) expression in rat osteoblast-like cells. Because of the importance of new vessel development to bone formation and remodeling, it is possible that PTH induction of this growth factor helps to promote bone formation (30). Fibroblast Growth Factor-2

The expressions of both basic fibroblast growth factor-2 (FGF-2) and the FGF receptors FGFR-1 and FGFR-2 are up-regulated in response to PTH treatment in osteoblastic-like cells (MC-3T3-E1) and in neonatal mouse calvariae (31). FGF-2 can stimulate bone cell replication and it has been suggested that PTHmediated regulation of FGF-2 and FGFR expression in osteoblasts may be part of the mechanism by which this h o r m o n e acts in bone (31). Targeted deletion of the FGF-2 gene leads to diminished bone formation, which is consistent with this notion (32).

Cytokines with Catabolic Actions Catabolic cytokines are thought to play a role in mediating the resorptive actions of PTH and PTHrelated protein. Osteoclast Differentiation Factor

Osteoclast differentiation factor (ODF) is a key mediator of PTH-induced bone resorption. Inhibiting its action blocks the resorptive effects of PTH (33).

413

ODE together with colony-stimulating factor-I, is both necessary and sufficient to induce osteoclastogenesis. ODF is also capable of activating mature osteoclasts (34), and its expression by osteoblasts is increased by both PTH and PTHrP (35). The actions of ODF are controlled in part by a soluble decoy receptor for ODE osteoprotegerin, which antagonizes the actions of ODE PTH inhibits osteoprotegerin expression, consistent with the proresorptive effects of the h o r m o n e (36). Colony-Stimulating Factor-1

Colony-stimulating factor-1 (CSF-1) is the major hematopoietic growth factor released by osteoblasts (37). PTH induces CSF-1 expression in osteoblasts by a transcriptional mechanism (37,38). CSF-1, in turn, enhances osteoclast formation in cocultures of mouse spleen cells and stromal cells (39). PTH-induced osteoclastogenesis appears to, in part, d e p e n d on CSF-1. Thus, in organ culture assays, in which de novo osteoclast formation is required for resorption, neutralizing antiserum to CSF-1 attenuates PTH-induced bone resorption (40). Interestingly, the ability of PTH to augment resorption by mature osteoclasts seems to be inhibited by CSF-1, because PTH-induced bone resorption is enhanced in the presence of neutralizing antisera to this growth factor in an assay system that largely reflects the activity of mature osteoclasts (40). As noted, CSF-1, together with ODE is required for osteoclastogenesis. The ability of PTH to regulate CSF-1 expression as well as that of ODF and osteoprotegerin ligand (OPGL) is consistent with the concept that PTH acts through several signaling pathways in regulating osteoclastogenesis. Stem Cell Factor

Stem cell factor (SCF) is the glycoprotein ligand for c-kit, a receptor tyrosine kinase in the platelet-derived growth factor group that includes Flt3 and c-fms (the receptor for CSF-1) (41). PTH induces SCF expression in h u m a n osteoblasts (42). Further, SCF is a growth factor for mast cells. Blair and co-workers have found that, in patients with secondary hyperparathyroidism due to renal insufficiency, there is a striking accumulation of mast cells in areas of high bone resorption that improves after parathyoridectomy or renal transplantation, consistent with the notion that PTH is mediating this process through local production of SCF (43-45). The receptor for SCF is expressed on osteoclasts, and although not required for osteoclast differentiation, in vitro treatment with SCF augments osteoclastogenesis and osteoclast activity. SCF is unlikely to escape the bone microenvironment (46), and therefore it is unlikely that SCF is released systemically in response to PTH.

414

/

CHAPTER25

Cytokines with Both Catabolic and Anabolic Actions Prostaglandin E 2

Prostaglandins have complex effects in bone with both anabolic and catabolic actions ascribed to these molecules. Prostaglandin Ez (PGE2) is p r o d u c e d in response to PTH by osteoblast-like cells (47), enhances the formation of mouse multinucleated osteoclasts from marrow progenitors, and stimulates osteoclastic bone resorption in rat organ culture (48). PGE 2d e p e n d e n t activation of osteoclasts, which occurs at least in part through stimulating ODF expression, seems to be mediated by the EP2 receptor. On the other hand, PGEz inhibits the formation of h u m a n osteoclasts and causes cytoplasmic contraction of isolated osteoclasts (49). The direct effect to inhibit mature osteoclast function appears to be mediated by the EP4 prostaglandin receptor expressed on osteoclasts (50). In vivo, PGE,~ is a potent stimulator of bone formation (51-54). In ovariectomized rats, PGE 2 increased rat cortical bone mass when administered immediately following ovariectomy (53). It also increased cancellous bone density 4 months following ovariectomy to levels found in sham-operated rats (51). It may be that differences in duration of exposure explain differing responses to PGE 2, which may be relevant to the differing skeletal responses to intermittent versus continuous exposure to PTH. Interleukin-6

Osteoblasts constitutively express interleukin-6 (IL-6) both in vivo and when cultured in vitro, but expression of this cytokine is increased by up to 50-fold by PTH (55). There have been conflicting reports on the effects of IL-6 on bone resorption in vitro that reflect the different types of assays used. In part, these differences relate to the ability of IL-6 to induce early osteoclast progenitor formation (7) and differentiation but not to influence the function of mature osteoclasts (56). It may also partly be due to the fact that the presence of the IL-6 soluble receptor is required for this cytokine to exert a resorptive effect (57). Initial evidence that II,-6 plays a significant role in bone homeostasis in vivo came from the work ofJilka et al., who d e m o n s t r a t e d that bone loss after ovariectomy in mice could be blocked with neutralizing antisera to IL-6. Coupled with earlier work from this group showing regulation of IL-6 gene expression by estrogen, these findings have led to the view that IL-6 plays an i m p o r t a n t role in the bone loss associated with estrogen deficiency (8). The possible role of IL-6 as a mediator of PTH-induced bone resorption in hyperparathyroidism is highlighted below.

Although most of the focus on IL-6 in bone has centered on its ability to stimulate osteoclast formation, some data suggest that it can also have anabolic effects in bone. Thus, IL-6 can increase the n u m b e r of colony-forming units-osteoblasts (CFU-OBs) in murine CFU assays (58). It also stimulates expression of alkaline phosphatase and osteocalcin, both markers of osteoblast differentiation (59,60). Rats that received a single injection of PTH (1-34) at 8 pog/100 g, a dose known to increase bone-forming surfaces, showed a rapid and transient expression of cfos, c-jun, c-myc, and IL-6 mRNA. These transcripts were induced within 1 hour of t r e a t m e n t and returned to base line after 3-6 hours. The induction of these early response genes suggests that PTH may stimulate cell differentiation in trabecular bone. Histone H4 mRNA was down-regulated, suggesting concomitant inhibition of cell proliferation (61). Interestingly, IL-6 transcript expression in bone was induced by intermittent PTH administration, but not when the h o r m o n e was given continuously. This differs from the effects on IL-6 protein levels. By contrast, c-fos transcript expression was increased by both intermittent and continuous PTH treatment (62). Continuous infusion of PTH for 5 days in both mice and rats results in a significant increase in circulating IL-6 levels that correlates strongly with the rise in markers of bone resorption, such as urine collagen crosslinks and serum ICTP (type I collagen carboxy-terminal telopeptide). In mice, at the end of a 5-day infusion, IL-6 levels correlated strongly with the two markers for bone resorption (IL-6 with cross-links; r = 0.95, p < 0.01; IL-6 with ICTP, r = 0.99, p < 0.001). Additional studies by Grey et al. (10) provided evidence of a causal relationship between increasing circulating levels of IL-6 and increasing rates of bone resorption in response to PTH. Thus, neutralizing IL-6 in vivo results in marked attenuation in the resorptive response to PTH infusion in mice. For example, p r e t r e a t m e n t with neutralizing antisera to IL-6 reduced urinary levels of collagen cross-links from a value of 50.4 b~g/mmol creatinine (in mice pretreated with control antibody) to 14.3 b~g/mmol creatinine. Consistent with these data, the increment in markers of bone resorption in response to PTH was markedly diminished in IL-6 knockout mice as compared to wild-type littermate controls. Both of these experiments strongly suggest that IL-6 is required for the full resorptive effect of PTH in vivo.

Data consistent with the conclusion that IL-6 plays an important role in the resorptive effect of PTH in h u m a n s have recently emerged. In both pre- and postmenopausal women, continuous infusion of PTH for 36 hours results in significant increases in both circulating IL-6 and urinary N-telopeptide (another marker

CYI'OKINES IN PRIMARY HYPERPARATHYROIDISM /

for bone resorption) concentrations, which are then followed by a rise in serum calcium. As is the case in rodents, increases in circulating levels of IL-6 occurred prior to the rise in markers of bone resorption, which, in turn, preceded the increase in serum calcium, suggesting a causal relationship between the PTHinduced rise in IL-6 and the subsequent increase in bone resorption (63).

Interleukin-11 IL-11 is another i m m u n e modulatory cytokine the expression of which in osteoblasts is stimulated by PTH treatment in vitro. IL-11 induces osteoclast formation in vitro (64,65). In vivo, however, diminished expression of IL-11 in the marrow of the senescence-accelerated mouse (SAMP6) is associated with diminished bone formation as well as decreased osteoblastogenesis and osteoclastogenesis, resulting in a decrease in bone mass (66). Overexpression of IL-11, on the other hand, results in increased bone formation (67). Continuous infusion of PTH results in a surprising decrease in circulating levels of IL-11. This effect is most likely due to a direct effect of PTH-induced IL-6 to inhibit IL-11 production. Thus PTH-induced IL-11 production by osteoblast-like cells is significantly increased by pretreatment with neutralizing antisera to IL-6, an effect not seen with control antisera (68).

Leukemia Inhibitory Factor Expression of leukemia inhibitory factor (LIF), also called differentiation-inducing factor, is a u g m e n t e d by PTH treatment in osteoblasts. In vitro evidence suggests a role for LIF in osteoblast proliferation and differentiated osteoblast function (55,59). In vivo, targeted disruption of the LIF receptor results in decreased bone volume in the primary spongiosa of developing bone (69), whereas overexpression of LIF results in sclerotic bone marrow, excessive woven bone, and ectopic bone formation (70). LIF has also been reported to induce osteoclast formation and bone resorption (56).

TGF-[3 is expressed by h u m a n osteoblasts (74) and PTH stimulates its expression (75,76). Further, TGF-[3 decreases bone resorption by inducing apoptosis in osteoclasts (72). Intermittent PTH injection in rodents results in a significant increase in bone TGF-[31 content (25). This could reflect direct up-regulation of TGF-[3 gene expression in response to PTH (75). As noted, TGF-[3 is anabolic in bone and it is possible that this cytokine participates in the anabolic response seen with intermittent PTH administration. BMPs have complex anabolic effects in bone. BMP2 is known to stimulate osteoblastic maturation and differentiation, has anabolic effects in bone, and is able to induce ectopic bone formation (77). PTH is not thought to regulate BMP expression.

CHANGES IN CYTOKINES IN STATES OF ALTERED P A R A T H Y R O I D F U N C T I O N Secondary Hyperparathyroidism Secondary hyperparathyroidism has been reported to occur in 10-13% in postmenopausal women with osteoporosis. In a retrospective study of 187 patients with selective femoral osteopenia, we found 17 patients to have subtle calcium malabsorption and secondary hyperparathyroidism with a mean PTH of 53 n l E q / m l in a sensitive midmolecule assay (normal,

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FIG. 1 Chemical composition of 30 stones from patients with idiopathic hypercalciuria and 14 stones from patients with primary hyperparathyroidism. All patients are males. M.A.P., Magnesium ammonium phosphate. Note that no patient with primary hyperparathyroidism had a pure calcium oxalate stone. (Reprinted from Ref. 16, with permission.)

Maurice-Estepa et aL (19) found carbonate apatite was the most frequent crystalline phase of phosphate among 1148 patients who passed phosphate-containing stones. The carbonate content was less than 10% in stones from patients with primary hyperparathyroidism and other, noninfection stone-forming states, and was greater than 15% in stones that formed in the presence of urinary tract infection with urea-splitting bacteria. Brushite was also found in stones from patients with primary hyperparathyroidism. Thus, the presence of either carbonate apatite or brushite in stones is consistent with the presence of primary hyperparathyroidism. However, in a series of patients with primary hyperparathyroidism and renal stones reported by Parks et al. (15), the majority of patients (20 of 31 ) formed calcium oxalate stones. For the others, two had pure calcium phosphate, two had a mixture of calcium phosphate and oxalate, five had a mixture of calcium oxalate and uric acid, one had calcium oxalate and cystine, and one had a struvite (ammonium-magnesium phosphate) stone. Taken together, these series indicate that stones from patients with primary hyperparathyroidism vary from pure calcium phosphate to pure calcium oxalate or are a mixture of the two.

PREVALENCE OF STONES

Common Primary Hyperparathyroidism The frequency of stone disease in primary hyperparathyroidism varies among series (Table 2). The incidence has been noted to decrease since the condition was first described because of the increasing numbers

of asymptomatic patients discovered by routine biochemical screening. In 1948, Albright and Reifenstein (5) reported that 80% of their patients with primary hyperparathyroidism had renal calculi. A series of TABLE 2

Prevalence of Renal Stones in Patients with Primary Hyperparathyroidism

Series Keating (1961 ) Hodgkinson (1963) Cope (1966) Pyrah (1966) Lloyd (1968) Purnell (1971 ) Pratley (1973) Mallette (1974) Broadus (1979) Mundy (1980) Siminovitch (1981 ) Ranni-Sivula (1985) Deaconson (1987) Lafferty (1989) Silverberg (1990) Haddock (1998) Total

No. of patients

Percentage with stones a

380 50 343 68 138 171 60 57 50 111 448 289 258 100 62 128

64 68 57 40 59 51 78 39 42 7 41 11 28 18 18 56

2713

42

aThe percentages may reflect a small contribution of patients with nephrocalcinosis but without nephrolithiasis, in that some series did not clearly distinguish those findings. Adapted and reprinted from Ref. 20 with permission.

440

/

CHAPTER27

171 patients with primary hyperparathyroidism evaluated at the Mayo Clinic (21) consisted of twice as many women as men. No clinical findings or biochemical tests distinguished those with stone disease from those without. Of this series, 59% developed urologic complications, including nephrolithiasis in 46%, nephrocalcinosis in 5%, and impairment of renal function in 8%. Calcification of the papillary tips was not included in this evaluation, so the incidence of nephrocalcinosis may have been underestimated. Nephrolithiasis accompanied all cases of nephrocalcinosis. Active stone disease as documented by new stone passage, appearance on X-ray, or passage of gravel within a year prior to parathyroidectomy was present in only 10.5% of patients. The prevalence of nephrolithiasis in 16 series of patients published since 1961 (Table 2) varied from 7 to 78%, with an average incidence of 42%. From these and other series, it appears that nephrolithiasis is currently the most frequent complication of primary hyperparathyroidism, The incidence of primary hyperparathyroidism in patients presenting with nephrolithiasis has, on the other hand, remained quite stable. The occurrence of primary hyperparathyroidism among all stone formers in series published since 1960 ranges from 3 to 13%, with an average of 7% (20). Thus, the frequency of primary hyperparathyroidism as a cause of stone disease in patients presenting with nephrolithiasis is rather low.

Mild Primary Hyperparathyroidism In recent years, routine screening of serum calcium levels with automated biochemical techniques has led to the increased diagnosis of asymptomatic primary hyperparathyroidism. For middle-aged adults, the annual incidence is 100-200 cases per 100,000 population, which accounts for approximately 60,000 new cases diagnosed each year in the United States (21). Primary hyperparathyroidism is being recognized more frequently, and the clinical presentation is changing. At the Mayo Clinic, the frequency of stone disease in patients with primary hyperparathyroidism has decreased from 51 to 4% following the advent of routine measurement of serum calcium in the late 1960s (22).

Multiple Endocrine Neoplasia Type 1 Multiple endocrine neoplasia type 1 (MEN-l) is an autosomal dominant disorder characterized by benign tumors of the parathyroid, pancreatic islet, and anterior pituitary cells. Among patients with this disorder, 95% present with hyperparathyroidism, and fewer than onethird have either gastrinoma or prolactinoma. The clinical manifestations of primary hyperparathyroidism in MEN-1 are very similar to those in sporadic cases of pri-

mary hyperparathyroidism, with a few important exceptions. Patients with MEN-1 tend to appear at a younger age (20-40 years), and the sex ratio does not favor females (23). In addition, parathyroid hyperfunction in MEN-1 is generally multiglandular, with hyperplasia of all four glands. In series of patients with primary hyperparathyroidism reported from academic medical centers, 50% had asymptomatic hypercalcemia, up to 50% nephrolithiasis, and 250 m g / d a y for women and >300 m g / d a y for men) is frequently encountered in patients with hyperparathyroidism and has been implicated in the pathogenesis of renal stone formation (31). Excess calcium in the urine may come from increased intestinal absorption, increased bone resorption, or from both. As a result, ultrafilterable calcium increases and may be sufficient to exceed distal tubular reabsorptive capacity. Increased intestinal calcium absorption has been documented in primary hyperparathyroidism using various methods, including metabolic balance, double isotope, and fecal excretion of radiolabeled Ca (32). 1,25(OH)zD is the major stimulator of intestinal calcium absorption, and PTH stimulates its synthesis in the renal proximal tubule (33). In addition, low serum phosphate concentrations, caused by PTH inhibition of

TABLE 4 Metabolic Causes of Stone Formation in Primary Hyperparathyroidism Hypercalciuria Reduced inhibitory activity Increased urinary promoter activity Reduced urinary pyrophosphate Low urine citrate excretion Reduced urinary magnesium Hyperuricosuria Mild renal tubular acidosis

442

/

CHAPTER27

proximal tubular phosphate reabsorption, may also stimulate 1,25(OH)2D synthesis (33). In support of a regulatory role of 1,25(OH)zD in the increased intestinal calcium absorption, Kaplan et al. (32) found that the mean plasma concentration of 1,25(OH)zD was greater in 18 patients with primary hyperparathyroidism than in normal subjects and was highly positively correlated with fractional calcium absorption. Increased intestinal calcium absorption and elevated serum calcitriol levels were found in some but not all hyperparathyroid stone formers. In a study by Peacock (10), patients with primary hyperparathyroidism and stone disease uniformly displayed an increase in fractional intestinal calcium absorption. Broadus et al. (34) also attributed stone formation to increased intestinal calcium absorption and calcitriol excess. Among 50 unselected patients with primary hyperparathyroidism, 22 had a history of one or more kidney stones. An abnormal response to a calcium load test (>0.20 mg calcium per 100 ml glomerular filtration) was associated with a greater serum 1,25(OH)zD level, a higher incidence of stones, and a higher 24-hour urinary calcium excretion compared to patients showing a normal response (....,,. ........!~ ............ ........ A g o n i s t

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Agonist

C-Terminus

CHAPTER 4, FIGURE 2 Superposition, using the heavy backbone atoms of residues 22-32, of agonist and antagonist containing (top) the midregion lactam, c[Lys13-Asp17]PTH(1-34)NH2 and c[Lys13-Asp17]PTH(7-34)NH2 , respectively, (middle) C-terminal region lactams, c[Lys26 -Asp 30 ]PTH(1-34)NH 2 and c[Lys26 -Asp 30 ]PTH(7-34)NH 2, respectively, and (bottom) bicyclic mid- and C-terminal region lactams, c[Lys 13-Asp 17 ,Lys26 -Asp 30 ]PTH(1-34)NH 2, and c[Lys13-AsplF,Lys26-Asp3°]PTH(7-34)NH2 , respectively. The structures were taken from the molecular dynamic trajectories to illustrate the consequences of the flexible hinge centered around Arg-19. Reprinted with permission from Ref. 13. Copyright 1997 American Chemical Society.

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CHAPTER 4, FIGURE 4 Helical wheel representations of hPTHrP(22-31), RS-66271(22-31), and hPTH(22-31), illustrating the amphiphilic nature of the helices. Hydrophobic (red) and hydrophilic (blue) amino acids are shown. Reprinted with permission from Ref. 83. Copyright 1997 Amercan Chemical Society.

Radiolabeled Photoreactive Ligand

Receptor

Ligand-Receptor Complex

Binding

UV

Ligand-Receptor Conjugate

Ligand-Receptor Conjugated Fragment

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~,. Radiolabel ¢--- Photoreactivemoiety CHAPTER 4, FIGURE 8 Schematic approach to photoaffinity scanning of PTH receptors. Photocross-linking is followed by fragmentation of the resultant radiolabeled hormone-receptor photoconjugate. Comparison of the fragmentation pattern elucidated by SDS-PAGE analysis with the theoretical restriction digestion map of the receptor identifies the putative contact site. Mass spectroscopic and microsequence analysis will identify the cross-linked residue in the receptor.

Y

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IC3 CHAPTER 4, FIGURE 9 Depiction of the PTH1-Rc from a molecular dynamic simulation. The transmembrane oL-helices are depicted as cylinders. The regions of the receptor that have been experimentally determined are depicted as ribbons. The regions of the receptor that have been shown to cross-link with PTH analogs, PTH1Rc[173-181] (26,27) and M 425 (28), are depicted in gray (241).

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PTH Ligand C H A P T E R 4, FIGURE 10 Structural features and topological orientation of PTH1-Rc[168-198] located at the C-terminal region of the extracellular N terminus followed by the ectopic portion of the first TM domain (241, 242). (A) Schematic representation of the experimentally determined conformation. The structure consists of three (x-helices, two of which have been determined to lie on the surface of the membrane; the third, at the top of TM1, is membrane embedded. (B) The orientation of this peptide is shown with respect to the surface of the dodecylphosphocholine micelles used in the NMR study. The hydrophobicity of the molecule is indicated (blue, polar; red, hydrophobic). The decane molecules of the water/decane simulation cell used in the structure refinement are shown in green as CPK space-filling spheres.

CHAPTER 4, FIGURE 11 Model for the binding of hPTH(134) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains hPTH1 -Rc[173-189] and hPTH1 -Rc[409-437]) (A, side view; B, top view). The amphipathic o~-helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high-resolution, low-energy structure of hPTH(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of hPTH(1-34) are denoted in yellow. The C-terminal amphipatic oL-helix of hPTH(1-34) is aligned in antiparallel arrangement with the amphipatic oL-helix of the extracellular N-terminus hPTH1-Rc[173-189], contiguous with TM1 and encompassing the 17-amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue M 414 and M 425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc[S4°9-W437]) are shown (28).

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CHAPTER 6, FIGURE 1 PTHrP as a polyhormone. (A) PTHrP cDNA encodes a prepropeptide and mature forms of 139, 141, and 173 amino acids. The three isoforms are identical for the first 139 amino acids, which is the portion depicted here. Proposed biologically active domains within the protein are shown. SP, Signal peptide; P, propeptide, (B) The PTHrP bipartite nuclear localization sequence. This segment encompasses amino acids 88-106 of the mature form (indicated in the single-letter amino acid code) and comprises two basic clusters separated by 10 intervening "spacer" amino acids, resembling the Xenopus laevis nucleoplasmin nuclear localization sequence (72). Furthermore, this region conforms to the structural requirements for a nucleolar localization sequence, as described in key regulatory proteins of human retroviruses (HTLV-1 Rex, and HIV-1 Tat and Rev), consisting of an "arginine hinge" (KRK, in blue) and an adjacent Q inserted between two putative nuclear localization sequences (22). (C) Subcellular distribution of PTHrP in transfected COS-7 cells. Plasmid constructs encoding PTHrP forms having either an intact coding region, deletions within the coding region or fused in frame to the Escherichia coil lacZ gene were expressed in COS-7 cells and the subcellular localization of the recombinant protein was determined by indirect immunofluorescence. SP, Secretory pattern; N, nucleolar, C, cytoplasmic. Size of lettering on the right-hand side is indicative of the levels of PTHrP immunoreactivity in the various subcellular compartments (24).

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CHAPTER 6, FIGURE 2 Nuclear/nucleolar localization of endogenous PTHrP in vitro and in situ. (A) Human keratinocyte HaCaT cell line. [Reproduced from Gillespie M. Role of phosphorylation of parathyroid hormone-related protein (PTHrP). International Bone Forum 1997; 30 with permission.] (B) Cultured osteoblast-like cells harvested form calvariae of newborn mice. Insert: immunogold labeling of an osteoblast nucleolus in a section from a fetal mouse tibia (N, nucleus; Nu, nucleolus). [Reproduced and modified from (24), with permission.] (C) Neurons (arrow) and glial cells (arrowhead) in the ventral horn of a normal mouse.

CHAPTER 6, FIGURE 4 (A) Nuclear/nucleolar uptake of PTHrP by UMR-106.01 osteoblasts. Cells incubated with exogenous PTHrP(1-108) conjugated to a chromophore show intranuclear distribution of labeled PTHrP after 50 minutes, which is blocked by an excess of unlabeled ligand (pathway A). [Reproduced from (45), with permission.] (B) In COS-7 cells transiently expressing ubiquitin and PTHrP, ubiquitinated forms of PTHrP were present in total cell lysates treated with MG 132, a proteasome inhibitor, but not with vehicle DMSO, calpain inhibitor II, or cysteine proteinase inhibitor E-64 (pathway B). [Reproduced from (56) with permission.] (C) COS-1 cells transfected with a PTHrP cDNA in which the unique initiator ATG is altered to an ATC. Non-AUG-initiated PTHrP forms localized exclusively to the nucleolar compartment (pathway C). Regardless of the path utilized (A, B or C in Fig. 3), once PTHrP relocates to the cytosolic compartment, it would bind to importin 13and be directed to the nucleus/nucleolus on the strength of its fully functional NLS

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CHAPTER 6, FIGURE 3 Potential pathways (A, B, and C) utilized by PTHrP to gain access to the cytosol. In pathway A, secreted PTHrP undergoes internalization at the cell surface in a "receptor"-dependent manner. Endocytosis could be mediated by the type 1 PTH receptor (PTHR1) or a binding protein that is distinct and recognizes either the N-terminal domain or other regions of PTHrP (R). In pathway B, PTHrP, after entering the ER lumen, "dislocates" back to the cytosol via the Sec61p translocon, a key component of the mammalian cotranslational protein translocation system, which functions as a two-way channel shuttling proteins both into the ER and back to the cytosol. Ubiquination of preproPTHrP may serve as the signal for retrograde transport of the peptide. In pathway C, innitiation of translation in PTHrP mRNA downstream from the initiator methionine generates a protein with a shorter signal peptide. Such a protein would fail to be targeted for secretion and remain in the cytosol for subsequent nuclear import. Experimental evidence supporting each of these pathways is illustrated in Fig. 4.

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CHAPTER 6, FIGURE 5 Regulation of PTHrP nuclear import. (A) Phosphorylation site for CDK2-CDC2 in PTHrP. The NLS (NOS) in PTHrP spans amino acids 88-106 of the mature protein (yellow-colored box) and is preceded by a consensus sequence (blue-colored box) for phosphorylation by CDK2-CDC2. The asterisk denotes the threonine (T) residue that undergoes phosphorylation. Below, the analogous region in the SV40 large T antigen is depicted for comparison. (B)Phosphorylation of PTHrP at T 85 by cyclin-dependent kinases negatively regulates its nuclear translocation. The G-specific cyclin-CDKs do not phosphorylate PTHrP, which then localizes to the nucleolus. Phosphorylation at T 85 by CDK2-CDC2 in the other phases of the cell cycle leads to the exclusion of the protein from the nucleus. Although phosphorylation of PTHrP is shown here to inhibit its interaction with importin 13 (113),this has not been vigorously demonstrated.

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CHAPTER 6, FIGURE 6 Abnormal endochondral bone development caused by deficiency of type 1 PTH receptor and PTHrP. (A) Contrasting effects of type 1 PTH receptor and PTHrP targeted disruption. Unlike in wild-type phalanges (a), blood invasion and bone replacement are delayed in the receptor-null bones (b), as illustrated by the persistence of proliferating chondrocytes. Conversely, in PTHrP-null phalanges (c), these processes are advanced and chondrocytes hypertrophy prematurely, leading to precocious ossification. (B) Partial rescue of the delay in vascular invasion and endochondral ossification in the type 1 PTH receptor-null mice by further ablation of the PTHrP gene. The impairment in blood invasion and bone formation is partially rescued in receptor/ligand double-negative mutant bones (b), as demonstrated by the more timely appearance of the ossification zone, compared to the receptor-negative single mutant (a). This argues in favor of PTHrP actions that are independent of the type 1 PTH receptor. [Reproduced from (48), with permission.]

CHAPTER 16, FIGURE 3 Overexpression of PTHrP end the PTHrP receptor disrupts heart development. (A) Whole mounts at E9.5 of double-trensgenic (left) end wild-type (right) embryos. The double trensgenic exhibits 8 greatly enlarged heart with pericardia1 effusion end vascular pooling (arrows). (B) Histologic sections of double-transgenic (left) end wild-type (right) embryos at E9.5. The trebeculee within the ventriculer cavity (v) of the wild-type embryo ere prominent (large arrows), whereas in the double trensgenic, trebeculee ere severely reduced or absent (asterisks). Prominent gaps ere also evident between the cerdiomyocytes in the double-trensgenic hearts (smell arrowheads); 8, atria; bar = 1001~m. (C) Left panel shows the localization of SMP8 lacZtrensgene in 9.5-day embryo. Staining is apparent in heart, hindgut, end somites. Right panel is an unstained control (from Qien J., et al., Endocrinology 1999; 140:1826-1833, with permisison)(49).

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CHAPTER 22, FIGURE 1 Visible and palpable parathyroid tumor. At surgery a 9.2-g parathyroid adenoma was found and removed successfully.

CHAPTER 22, FIGURE 2 Swollen and deformed knee due to osteitis fibrosa cystica. The X-rays of this knee are shown in Fig. 3.

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CHAPTER 26, FIGURE 1 (A) Low-power photomicrograph illustrating the extended osteoid perimeter (stained red) on mineralized trabecular surfaces (stained green). Original magnification, x6. (B) Extended eroded perimeter (ES) appearing as irregular scalloped surface covered by osteoclasts (OC). Original magnification, x25. (C) Extended osteoid perimeter (OS) seen at higher magnification. Peritrabecular fibrosis (arrows). Original magnification, x25. Goldner's trichrome. (D) Extended double tetracycline labels (DL) seen by fluorescence microscopy covering trabecular surfaces. Original magnification, x25. Unstained section.

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CHAPTER 44, FIGURE 5 Schematic representation of the role of PTHrP and the PTH/PTHrP receptor during mammary gland and tooth development. Upper panel: Branching morphogenesis depends on the interaction between mammary epithelium and the mesenchyme that condenses underneath (dense mesenchyme). PTHrP is produced by the mammary epithelium, and acts through the PTH/PTHrP receptor, which is expressed in the dense mesenchyme (72). Lower panel: During tooth development, PTHrP is expressed in the bone surrounding the tooth and in the dental mesenchyme. Stimulation of bone resorption following PTH/PTHrP receptor activation by PTHrP is requriedd for normal tooth eruption (71).

CHAPTER 44, FIGURE 9 Section of the upper tibia end from a fetus affected by Blomstrand lethal chondrodysplasia (BLC) (A) and an age-matched control (B). Note the severely reduced size of the growth plate, the irregular boundary between the growth plate and the primary spongiosa, and the increased cortical bone thickness (from Ref. 163, with permission, and Anne-Lise Delzoide, personnal collection).

CHAPTER 50, FIGURE 1 A young boy with APS1. This boy with APS1 has severe mucocutaneous candidiasis. There is striking hyperkeratosis from infection of the skin of the hands and face in addition to involvement of the oropharynx and fingernails.

C a'TEk 29 Medical Management of Primary Hyperparathyroidism J O H N L. STOCK Department of Medicine, University of Massachusetts Medical School, Worcest~ Massachusetts 01605; and Eli Lilly and Company, Indianapolis, Indiana 46285

ROBERT MARCUS Department of Medicine, Stanford University School of Medicine, and Aging Study Unit, VA Medical Cent~ Palo Alto, California 94304

INTRODUCTION

of any etiology (12-14). Patients with PHPT may be volume depleted due to decreased oral intake and increased renal losses of free water. Immediate rehydration and treatment of nausea and vomiting followed by saline diuresis usually result in a prompt decrease in the serum calcium concentration. The acute treatment of hospitalized patients with intravenous saline and furosemide has been well studied (12), but the value of salt and water loading and chronic oral furosemide in the outpatient setting is not well documented. Potential complications of this therapy include congestive heart failure if salt loading is too vigorous, prerenal azotemia with worsening of hypercalcemia if excessive diuretics are used, and other electrolyte abnormalities. Thiazides and related diuretics, including metolazone and indapamide, actually decrease calcium excretion at distal tubular sites and should be discontinued in patients with PHPT. Lithium carbonate may also decrease urinary calcium excretion (15), and, by direct effects of the drug on the parathyroid glands, magnify hypercalcemia. Short-term administration of lithium carbonate decreases the sensitivity of parathyroid cells to inhibition by calcium; chronic administration of this drug may predispose to development of parathyroid adenomas (15). At the least, lithium toxicity, if present, should be corrected in any patient with PHPT, but the discontinuation of this psychotropic drug is often problematic and depends on the underlying psychiatric diagnosis and other available options for therapy. Immobilization is known to result in hypercalciuria and hypercalcemia as a consequence of increased bone resorption and decreased bone formation (16), and

Surgery is the treatment of choice for patients with symptomatic or complicated primary hyperparathyroidism (PHPT) (1-3). Although the role of medical management for asymptomatic PHPT is still controversial (3,4), there are subsets of symptomatic patients who may benefit from medical rather than surgical intervention. These include patients who refuse surgery, do not have access to an experienced parathyroid surgeon, are too ill for surgery, have had unsuccessful previous neck explorations (5,6), or have inoperable parathyroid carcinoma (7-9). Occasionally, a trial of medical therapy to normalize the serum calcium concentration can assist the patient and physician in deciding whether symptoms are related to hypercalcemia (10). Patients may require stabilization of severe hypercalcemia prior to surgery (5). In its most extreme form, this has been called parathyroid crisis or acute PHPT (see Chapter 34) (11). This chapter describes the nonsurgical modalities available for the treatment of PHPT. After an introduction to the general principles of medical care for patients with PHPT, specific agents that decrease parathyroid h o r m o n e (PTH) action a n d / o r decrease PTH secretion are discussed.

GENERAL PRINCIPLES The general principles of medical treatment of symptomatic hypercalcemia in PHPT are the same as those for the treatment of symptomatic hypercalcemia The Parathyroids, Second Edition

459

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

460

/

CHAPTER29

patients with PHPT should be instructed to avoid sustained bed rest and to increase their general levels of activity. Although the hypercalcemia of PHPT is predominantly related to increased bone resorption, a c o m p o n e n t of it may respond to changes in diet. Some patients with PHPT show hyperabsorption in a calcium tolerance test, hypercalciuria, and elevated serum 1,25-dihydroxyvitamin D [1,25 (OH) 2D] concentrations (17). However, in a study of 18 unselected patients with PHPT, a high-calcium diet (1000 mg) suppressed PTH and 1,25(OH)2D concentrations in some subjects (18). Thus, the benefits of a low-calcium diet might include a lower urinary calcium excretion in certain patients, but should be balanced by a concern about aggravating the hypersecretion of PTH, with its possible skeletal consequences. The benefit of a higher calcium diet might be to suppress parathyroid function and thus theoretically delay progression of disease in a subset of patients, but could be offset by an increase in urinary calcium excretion in some of these subjects. Because no long-term studies currently allow one to tailor dietary r e c o m m e n dations, we suggest a m o d e r a t e dietary calcium intake for most patients with PHPT. SPECIFIC PHARMACOLOGIC

serum calcium or creatinine values. The mechanisms of the effects of oral phosphate therapy were investigated in a series of 10 patients with PHPT and elevated serum levels of 1,25 (OH) 2D who received 1500 mg of elemental phosphorus daily for 1 year (22). Phosphate treatm e n t led to decreases in serum 1,25 (OH) 2D levels, calciuric responses to oral calcium loading, and urinary calcium excretion, suggesting a decrease in calcium hyperabsorption in these subjects (Fig. 1).

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Albright et al. (19) described the use of oral phosphate for the t r e a t m e n t of PHPT in 1932. In elegant metabolic studies of three patients, they attributed the decrease in serum calcium concentrations and urinary calcium excretion with phosphate therapy to the increase in the Ca × P product in the serum. They also predicted the theoretical dangers of such therapy: "parathyroid poisoning," or the deposition of calcium deposits in the kidney and other organs with resultant uremia, as well as the risk of producing phosphate kidney stones. In 1966 Goldsmith and Ingbar (20) described the short-term use of oral phosphate in four patients with PHPT, with a resultant decline in the serum calcium concentration in all and actual normalization of the serum calcium concentration in one subject. Purnell gave 14 subjects with persistent or recurrent PHPT 1.0-2.5 g of elemental phosphorus daily (orally) for up to 51 months (21). In seven patients with serum calcium concentrations >11.0 m g / d l , calcemia normalized in one, decreased in four, and was u n c h a n g e d in two after 1 m o n t h of therapy. Although these effects were generally sustained with longer term treatment, an increase in serum creatinine concentrations was noted in three patients. In those patients affected by nephrolithiasis, there appeared to be a palliative effect on disease progression. In patients with milder disease and a serum calcium concentration 25 subjects) of patients undergoing noninvasive localizing studies (ultrasound, thallium technetium scanning, computed tomography, and magnetic resonance imaging) before successful surgery. Table 1 summarizes the true-positive and falsepositive rates of the four rnodalities as reported. The sensitivities varied between 60 and 70%, with a falsepositive rate of all studies of approximately 15%. Although some investigators reported a higher sensitivity with a specific modality, it often occurred at the expense of an increased false-positive rate. A few institutions reported superior sensitivity using a modality with which they had extensive experience, but the purpose of the study was to evaluate the results that could be expected in institutions with broad experience in parathyroid localization but without dedication to a single modality. The NIH experience was not included because very few patients are seen at this institution before initial surgery. The sensitivity of 60-70%, with a false-positive rate of 15%, for all noninvasive localizing studies contrasted with the widely reported surgical success rates of 90-95 % in patients undergoing initial operations (1). In addition, studies that looked at operating room times or surgical success rate did not establish a significant difference between patients without and patients with preoperative localization (45-49). For these reasons, the following statement summarized my conclusion: "There has never been a well-controlled study which showed a decrease in operating time or improvement in surgical success, attributable to preoperative localization in patients undergoing initial surgery." This controversy concerning preoperative localization on nonoperated patients has been put to rest by the introduction of a truly sensitive technique, MIBI scintigraphy with SPECT. Several studies have shown that the dual-isotope technique improves the sensitivity when compared with the single-isotope delayed-scanning technique (17) and that SPECT increases the sensitivity as well as provides the more precise anatomic localiza-

TABLE 1 Sensitivity and False-Positive Rate of Noninvasive Localizing Studies a Study

Sensitivity (%)

False-positive rate (%)

Ultrasound Thallium/technetium scintigraphy

65 55

12.5 13.5

74

18

Computerized tomography Magnetic resonance imaging

63

aAdapted from Ref. 44; JL Doppman, DL Miller. Localization of parathyroid tumors in patients with asymptomatic hyperparathyroidism and no previous surgery. J Bone Miner Res 1991 ;6:$153-$158.

PREOPERATrVE LOCALIZATION OF TISSUE

tion (19). Using the procedure as described above, detection rates of adenomas have been over 95% in the hands of many experienced investigators (7-12). The sensitivity for detecting all hyperplastic glands drops to 60-70%, but in many patients more than one gland is imaged, thereby establishing a diagnosis of hyperplasia. As in all localization studies, the sensitivity is dependent on the size of the adenoma, but we (18), as well as others, have demonstrated that sestamibi detects smaller lesions than thallium/technetium scanning did. Finally, we have a test that outperforms an experienced parathyroid surgeon, and although there remains a difference of opinion (50), most patients now undergo sestamibi scanning prior to their initial surgery. The widespread application of preoperative localization with sestamibi scintigraphy has been fueled by the interest in shortening hospitalization for patients undergoing parathyroidectomy. Although there are proponents for the conservative or nonoperative treatment of such patients (51), most authorities remain convinced that surgery should be performed in all patients with documented hyperparathyroidism (52), principally because of the long-term effects on bone density. The accuracy of sestamibi scanning has led to the routine performance of targeted surgical explorations based on sestamibi localization, often under local anesthesia on an outpatient basis (53-55). The surgeon removes the previously localized adenoma and initiates closure without an effort to identify the other three glands. A rapid PTH determination is performed 10 minutes after surgical excision of the adenoma and demonstrates a greater than 50% decline of PTH levels in patients who are cured. The application of fast PTH assay in the operating room (56-58) has enabled the surgeon to abandoned the classic routine of identifying the remaining three parathyroid glands, surgical detection of which often requires more time than removal of the adenoma. Although large series have not been reported, a 50% decrease in PTH levels at 10 minutes postexcision has proved to correlate well with surgical cure (56). In the 3-5% of patients with negative sestamibi studies, a classic four-gland exploration will be required.

REFERENCES 1. Savata RM, Jr, Beahrs OH, Scholz DA. Success rate of cervical exploration for hyperparathyroidism. Arch Surg 1975;110:625-628. 2. Carty SE, Norton J. Management of patients with persistent or recurrent primary hyperparathyroidism. World J Surg 1991;15:716-723. 3. Libutti SK, Bartlett DL, Jaskowiak NT, Skarulis M, Marx SJ, Spiegel AM, Fraker, DL, Doppman JL, Shawker TJ, Alexander HR. The role of thyroid resection during reoperation for persistent or recurrent hyperparathyroidism. Surgery 1997;122:1183-1186. 4. O'Doherty MJ, Kettle AG, Wells P, Collins, REC, Coakley AJ. Parathyroid imaging with technetium-99m-sestamibi: Preoperative localization and tissue uptake studies. J Nucl Med 1992;33:313-318.

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5. Wei JR Burke GJ, Mansburger AR. Prospective evaluation of the efficacy of technetium 99m sestamibi and iodine 123 radionuclide imaging of abnormal parathyroid glands. Surgery 1992;112:1111-1117. 6. Taillefer RM, Boucher Yvan, Potvin C, Lambert R. Detection and localization of parathyroid adenomas in patients with hyperparathyroidism using a single radionuclide imaging procedure with technetium-99m-sestamibi (double-phase study). J Nucl Med 1992;33:1801-1807. 7. Johnston LB, Carroll MJ, Britton KE, Lowe DG, Shand W, Besser GM, Grossman AB. The accuracy of parathyroid gland localization in primary hyperparathyroidism using sestamibi radionuclide imaging. J Clin Endocrinol Metab 1996;81:346-352. 8. Lee VS, Wilkinson RH, Jr, Leight GS, Jr, Coogan AC, Coleman RE. Hyperparathyroidism double-phase techentium 99m sestamibi imaging. Radiology 1995;197:627-633. 9. Hindie E, Melliere D, Simon D, Perlemuter L, Galle E Primary hyperparathyroidism: Is technetium-99m-sestamibi/iodine-123 subtraction scanning the best procedure to locate enlarged glands before surgery? J Clin Endocrinol Metab 1995;80: 302-307. 10. Hindie E, Melliere D, Jeanguillaume C, Perlemuter L, Chehade F, Galle E Parathyroid imaging using simultaneous doublewindow recording of technetium-99m-sestamibi and iodine-123. J Nucl Med 1998;39:1100-1105. 11. Neumann DR, Esselstyn CB, Jr, Go RT, Wong CO, Rice TW, Obuchowski NA. Comparison of double-phase 99m Tc-sestamibi with 123-I 99m Tc-sestamibi subtraction SPECT in hyperparathyroidism. AmJ Roentgenol 1997;169:1671-1674. 12. Caixas A, Berna L, Hernandez A, Tebar FJ, Madariaga P, Vegazo O, Bittini AL, Moreno B, Faure E, Abos D, Piera J, Rodriquez JA, Farrerons J, Puig-Domingo M. Efficacy of preoperative diagnostic imaging localization of technetium 99m-sestamibi scintigraphy in hyperparathyroidism. Surgery 1997;121:535-41. 13. Neumann DR, Esselstyn CB, Jr, MacIntyre wJ, Chen EQ, Go RT, Kohse LM, Licata AA. Primary hyperparathyroidism: Preoperative parathyroid imaging with regional body FDG PET. Radiology 1994;192:509-512. 14. Sisson JC, Thompson NW, Ackerman RJ, Wahl RL. Use of 2-[F-18]-fluoro-2deoxy-D-glucose PET to locate parathyroid adenomas in primary hyperparathyroidism (letter to the Editor). Radiology 1994;192:280-280. 15. Miller DL, Doppman JL, Krudy AG, et al. Localization of parathyroid adenomas in patients who have undergone surgery. Part I. Non-invasive imaging methods. Radiology 1987; 162:133-137. 16. Coakley J, Kettle AG, Wells CP, O'Doherty MJ, Collings REC. Technetium-99m-sestamibi--a new agent for parathyroid imaging. Nucl Med Commun 1989; 10:791-794. 17. Chen CC, Holder LE, Scovill WA, Tehan AM, Gann DS. Comparison of parathyroid imaging with technetium-99mpertechnetate/sestamibi subtraction, double-phase technetium99m-sestamibi and technetium-99m-sestamibi SPECT. J Nucl Med 1997;38:834-839. 18. Chen CC, Skarulis MC, Fraker DL, Alexander HR, Marx SJ, Spiegel AM. Technetium-99m-sestamibi imaging before reoperation for primary hyperparathyroidism. JNucl Med 1995;36:2186-2191. 19. Billotey C, Sarfati E, Aurengo A, Duet M, Mundler O, Toubert M-E, Rain J-D, Najean Y. Advantages of SPECT in technetium99m-sestamibi parathyroid scintigraphy. J Nucl Med 1996;37: 1773-1778. 20. Doppman JL, Skarulis MC, Chen CC, Chang R, Pass HI, Fraker DL, Alexander HR, Niederle B, Marx SJ, Norton JA, Wells SA, Spiegel AM. Parathyroid adenomas in the aortopulmonary window. Radiology 1996;201:456-462. 21. Doppman JL, Shawker TH, Krudy AG, et al. Aspiration of enlarged parathyroid glands for parathyroid hormone assay. Radiology 1983;148:31-35.

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22. MacFarlane DL, Fraker DL, Shawker TH, Doppman JL, Chang RA, Skarulis MC, Marx SJ, Spiegel AM, Alexander HR. Use of preoperative fine-needle aspiration in patients undergoing reoperation for primary hyperparathyroidism. Surgery 1994;116:959-965. 23. Jeanguillaume C, Urena PK, Hindie E, Prieur P, Petrover M, Menoyo-Cologne V, Janin A, Chiappini-Briffa D, Melliere D, Boulahdour H, Galle E Secondary hyperparathyroidism: Detection with 1-123 Tc-99m-sestamibi subtraction scintigraphy versus US. Radiology 1998;207:207-213. 24. Doppman JL, Shawker TH, Krudy AG, et al. Parathymic parathyroid, CT, US, and angiographic findings. Radiology 1985;157:419-423. 25. Fraker DL, Doppman JL, Shawker TH, Marx SJ, Spiegel AM, Norton JA. Undescended parathyroid adenoma: An important etiology for failed operations for primary hyperparathyroidism. WorldJ Surg 1990;14:342-348. 26. Billingsley KG, Fraker DL, Doppman JL, Norton JA, Shawker TH, Skarulis MC, Marx SJ, Spiegel AM, Alexander HR. Localization and operative management of undescended parathyroid adenomas in patients with persistent primary hyperparathyroidism. Surgery 1994;116:982-989. 27. Doppman JL, Shawker TH, Fraker DL, Alexander HR, Skarulis MC, Lack EE, Spiegel AM. Parathyroid adenoma within the vagus nerve (case report). AmJRoentgenol 1994;163:943-945. 28. Peck WW, Higgins CB, Fisher MR, et al. Hyperparathyroidism; comparison of MR imaging with radionuclide scanning. Radiology 1987;163:415-420. 29. Kneeland JB, Krubsack AJ, Lawson TL, et al. Enlarged parathyroid glands; high resolution local coil MR imaging. Radiology 1987;162:143-146. 30. Auffermann W, Gooding GAW, Okerlund MD, et al. Diagnosis of recurrent hyperparathyroidism; comparison with MR imaging with other imaging techniques. AmJ Roentgenol 1988; 150:1027-1033. 31. Seelos KC, DeMarco R, Clark OH, et al. Persistent and recurrent hyperparathyroidism; assessment with gadopentate dimeglumine-enhanced MR imaging. Radiology 1990;177:373. 32. Lee VS, Spritzer CE, Coleman RE, Wilkinson RH, Jr, Coogan AC, Leight GS, Jr. The Complementary roles of fast spin-echo MR imaging and double-phase 99mTc-sestamibi scintigraphy for localization of hyperfunctioning parathyroid glands. Am J Roentgeno11996;167:1555-1562. 33. Karstrup S, Transbol I, Holm HH, Glenthoj A, Hegedus L. Ultrasound-guided chemical parathyroidectomy in patients with primary hyperparathyroidism: A prospective study. Br J Radiol 1989;62:1037-1042. 34. Karstrup S, Holm HH, Granthoj A, et al. Non-surgical treatment of primary hyperparathyroidism with sonographically guided percutaneous injection of ethanol: Results in a selected series of patients. Am J Roentgenol 1990; 154:1087-1090. 35. Charboneau JW, Hay ID, van Heerden JA. Persistent primary hyperparathyroidism: Successful ultrasound-guided percutaneous ethanol ablation of an occult adenoma. Mayo Clin Proc 1988;63:913-917. 36. Solbiati L, Giangrande AL, DePra L, et al. Percutaneous ethanol injection of parathyroid tumors under US guidance: Treatment for secondary hyperparathyroidism. Radiology 1985; 155:607-610. 37. Miller DL, Doppman JL, Krudy AG, et al. Localization of parathyroid adenomas in patients who have undergone surgery. Part II. Invasive procedures. Radiology 1987;162:138-141. 38. Doppman JL, Skarulis MC, Chang R, Alexander HR, Bartlett D, Libutti SK, Spiegel A. Hypocalcemic stimulation and nonselective venous sampling for localizing parathyroid adenomas: Work in progress. Radiology 1998;208:145-151. 39. Doppman JL. The treatment of hyperparathyroidism by transcatheter techniques. Cardiovasc Interventional Radiol 1980;3:268-281.

40. Miller DL, Doppman JL, Chang R, et al. Angiographic ablation of parathyroid adenomas; Lessons from a 10-year experience. Radiology 1987;165:601-607. 41. Doherty GM, Doppman JL, Miller DL, et al. Results of the multidisciplinary strategy in managing mediastinum parathyroid adenoma as the cause of persistent primary hyperparathyroidism. Ann Surg 1992;215:101-1061 42. Sugg SL, Fraker DL, Alexander R, Doppman JL, Miller DL, Chang RC, Skarulis MC, Marx SJ, Spiegel AM, NortonJA. Prospective evaluation of selective venous sampling for parathyroid hormone concentration in patients undergoing reoperations for primary hyperparathyroidism. Surgery 1993:114 (6):1004-1010. 43. Jaskowiak N, Norton JA, Alexander HR, Doppman JL, Shawker T, Skarulis M, Marx S, Spiegel A, Fraker DL. A prospective trial evaluating a standard approach to reoperation for missed parathyroid adenoma. Ann Surg 1996;224:308-322. 44. Doppman JL, Miller DL. Localization of parathyroid tumors in patients with asymptomatic hyperparathyroidism and no previous surgery. JBone Miner Res 1991; (6) :S153-S158. 45. Wilson SD, Hoffmann RG, Cerletty JM, et al. Parathyroidectomy for primary hyperparathyroidism; the influence of preoperative localizing studies on cure rate and operating time. Presented at the 1991 Annual Meeting Society for Endocrine Surgeons, San Jose, California, March 1991. 46. Serpel JW, Campbell PR, Young AE. Preoperative localization of parathyroid tumors does not reduce operating time. B r J Surg 1991;78:589-590. 47. Thompson N. Localization studies of patients with primary hyperparathyroidism. Br MedJ 1988;75:97-98. 48. Bruining HA, Birkenhager JC, Ong GL, Lamberts SWJ. Causes of failure in operations for hyperparathyroidism. Surgery 1987;101:562-565. 49. Levin KE, Clark OH. The reasons for failure in parathyroid operations. Arch Surg989;124:911-915. 50. Shen W, Sabanci U, Morita ET, Siperstein AE, Duh Q-Y, Clark OH. Sestamibi scanning is inadequate for directing unilateral neck exploration for first-time parathyroidectomy. Arch Surg 1997;132:969-976. 51. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JP. A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. NEnglJ Med 1999;341:1249-1255. 52. Utiger RD. Treatment of primary hyperparathyroidism (editorial). N EnglJ Med 1999;341:1301-1302. 53. Hindie E, Melliere D, Perlemuter L, Jeanguillaume C, Galle E Primary hyperparathyroidism: Higher success rate of first surgery after preoperative Tc-99m sestamibi-I123 subtraction scanning. Radiology 1997;204:221-228. 54. Borley NR, Collins REC, O'Doherty M, Coakley A. Technetium99m sestamibi parathyroid localization is accurate enough for scandirected unilateral neck exploration. BrJ Surg 1996;83:989-991. 55. Sfakianakis GH, Irvin III GL, FossJ, Mallin W, Georgiou M, Deriso GT, Molinari AS, Ezuddin S, Ganz W, Serafini A, Jabir A, Chandarlapaty SKC. Efficient parathyroidectomy guided by SPECTMIBI and hormonal mea~surements. J Nucl Med 1996;37:798-804. 56. Irvin III GL, Sfakianakis G, Yeung L, et al. Ambulatory parathyroidectomy for primary hyperparathyroidism. Arch Surg 1996; 131:1074-1078. 57. Carty SE, Worsey J, Virgi MA, Brown MA, Watson CG. Concise parathyroidectomy: The impact of preoperative SPECT 99m-Tc sestamibi scanning and intraoperative quick parathormone assay. Surgery 1997;122:1107-16. 58. Proye CAG, Goropoulos A, Franz C, Carnaille B, Vix M, Quivreux J'L, Couplet-Lebon G, Racadot A. Usefulness and limits of quick intra-operative measurements of intact (I-84) parathyroid hormone in the surgical measurement of hyperparathyroidism. Sequential measurements in patients with multiglandular disease. Surgery 1991;110:1035-1042.

CHAPTER

31

The Surgical Management of Hyperparathyroidism

SAMUEL A. WELLS, JR. AND GERARD M. DOHERTY Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110

H I S T O R Y OF PARATHYROID S U R G E R Y

The patient was a streetcar conductor, whose condition transiently improved following removal of a parathyroid tumor; however, the hypercalcemia recurred and no other parathyroid tissue was found at repeat operation or at autopsy performed after the patient succumbed from complications of hyperparathyroidism. The first operation performed for hyperparathyroidism in the United States involved a sea captain, Charles Martel, who was treated in 1926 at the Massachusetts General Hospital. However, the disease was not corrected until the seventh operation 7 years later (6). In the meantime, the first successful operation for hyperparathyroidism was performed at Barnes Hospital in 1929, when I.Y. Olch removed an enlarged parathyroid gland from a patient with bone disease and kidney stones (7). The term hyperparathyroidism was first used in the report of this case. Since the first operations for parathyroid surgery over 75 years ago we have learned a great deal about parathyroid gland physiology and now understand at the molecular level many bone and mineral diseases associated with parathyroid gland disorders. The surgical management of patients with hyperparathyroidism has also changed over time and has particularly evolved recently with the introduction of sophisticated preoperative imaging techniques and the use of "minimally invasive surgery." The current practice for the operative management of patients with hyperparathyroidism is the topic of this chapter.

The parathyroid glands were discovered in 1880 by Ivar Sandstrom, a Swedish student, working as an assistant in a histology laboratory at the University of Uppsala in Sweden (1). He identified the parathyroid glands in several species of animals, including humans, and his manuscript includes both gross and microscropic descriptions of the structures. Sandstrom's observation was one of the last great anatomic discoveries. However, his report was accepted for publication only in the periodical published by his medical school, and went largely unnoticed until 10 years later, when the Frenchman, Gley, rediscovered the parathyroid glands and showed in experimental animals that their removal led to tetany (2). Von Recklinghausen described osteitis fibrosa cystica generalisata, the bone disease associated with hyperparathyroidism (3). Other pathologists had also reported the association of bone disease and enlarged parathyroid glands, but it was generally accepted that the parathyroid enlargement was secondary to the skeletal pathology. The theory was not challenged until 1915, when Schlagenhaufer reasoned that it would be unlikely for secondary hyperparathyroidism to affect only one gland and predicted that in some cases the parathyroid tumor was the primary abnormality and the bone disease was secondary (4). The first parathyroidectomy for hyperparathyroidism was performed in 1925 by Felix Mandl of Vienna (5).

The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CLINICAL FEATURES OF PRIMARY HYPERTHYROIDISM Clinically, there are three types of hyperparathyroidism. Primary hyperparathyroidism (PHPT) defines patients whose hyperparathyroidism results from de novo enlargement and overactivity of one or more parathyroid glands. In secondary hyperparathyroidism (SHPT) the parathyroids are generally enlarged in reaction to the chronic hypocalcemia caused by disease in another organ system, most commonly the kidney or the gastrointestinal tract. Tertiary hyperparathyroidism (THPT) refers to the condition of autonomous parathyroid hyperfunction that occasionally develops in patients with SHPT. Although each type of hyperparathyroidism is managed operatively, most of our discussion will concern patients with primary hyperparathyroidism. The clinical presentation and methods of laboratory and radiologic diagnosis of PHPT have been discussed in previous chapters, but it is relevant to address certain facets of the clinical disease that are of particular concern to the surgeon. The classic signs and symptoms of PHPT relate to kidney stones and bone disease. Patients who present with bone disease have a more severe form of hyperparathyroidism than do those who present with renal stones, because the serum calcium concentration is higher, the onset of PHPT is more abrupt, and the resected parathyroid tumors are larger. Furthermore, patients with radiologic evidence of bone disease, or those with an elevated alkaline phosphatase preoperatively, are at increased risk of prolonged hypocalcemia following parathyroidectomy (i.e., "hungry bone" syndrome) and may require calcium and vitamin D therapy for several months before the serum level of calcium returns to normal. Patients with renal stones are often cured by parathyroidectomy, even though some may continue to have hypercalciuria or later excrete stones that were in situ at the time of parathyroidectomy.

PATIENT HISTORY The majority of patients with PHPT are now either asymptomatic at the time of diagnosis or they have mild and nonspecific symptoms such as muscle weakness, fatigue, lethargy, constipation, urinary frequency, or loss of memory. Indeed, if one carefully questions patients with PHPT, most will be found to have one or more of these symptoms. Often patients present to a physician with complaints of weakness, tiredness, and loss of energy, but these symptoms are common in older patients and the diagnosis of PHPT is not sus-

pected. This is unfortunate because the signs or symptoms of PHPT, though having minimal, if any, effect on younger patients, may significantly alter the life quality of older patients. Furthermore, the onset of the signs and symptoms of PHPT are subtle and insidious. Many older patients think their symptoms are due to increased age, only to find that their sense of well being and their energy and strength improve markedly after their parathyroid tumor is removed. The past history and family history are very important parts of the evaluation in patients with PHPT. Patients should be questioned about a history of external beam radiotherapy to the neck. Years ago it was common for infants and young children who presented with upper respiratory symptoms, incorrectly thought to be due to an enlarged thymus gland, to be treated with low-dose radiotherapy. Approximately 30% of these children developed thyroid nodules subsequently, and about 30% of these were malignant. The incidence of PHPT in these children with X-ray exposure is also increased and the cause is almost always found to be due to a single enlarged gland. At the time of neck exploration for PHPT it is important to evaluate the thyroid gland for the presence of pathology and perform a thyroidectomy if a malignancy is found. Any patient with PHPT should be studied for the presence of tumors of the pancreatic islet system or the pituitary gland. If either of these is present the diagnosis of multiple endocrine neoplasia (MEN) type 1 must be excluded. The diagnosis of MEN-1 in a patient with PHPT should lead to a thorough evaluation of the extended family. The occurrence of medullary thyroid carcinoma (MTC) or pheochromocytoma in a patient with PHPT suggests the presence of MEN-2a and similarly calls for screening of the patient's family. A history of PHPT alone in a family suggests the presence of familial hypocalciuric hyperparathyroidism (FHH) or familial primary hyperparathyroidism. Each of these familial endocrinopathies is inherited in an autosomal dominant pattern, characterized by complete penetrance. In MEN-1 and MEN-2A there is variable expressivity of the component diseases. Virtually all patients with MEN-1 have PHPT at the time of diagnosis, 50% have islet cell tumors (most often a gastrinoma or an insulinoma), and less than 20% develop a pituitary tumor (most often a prolactinoma). In patients with MEN-2A, MTC is uniformly present, however, only 50% of patients develop pheochromocytomas and 25% develop PHPT. In patients who are less than 40 years of age at the time of presentation with PHPT, the examining physician should suspect MEN-l, MEN-2A or FHH. It is important to establish the diagnosis of familial hyperparathyroidism prior to operating for PHPT, for

SURGICAL MANAGEMENTOF HYPERPARATHYROIDISM /

two reasons. Patients with these familial endocrinopathies have generalized involvement of the parathyroid glands and the operation should be planned accordingly. Furthermore, and perhaps most importantly, patients with F H H are not candidates for parathyroidectomy. They develop neither renal stones nor bone disease although they have hypercalcemia. More importantly, their hyperparathyroidism is virtually impossible to cure surgically, unless one performs a total parathyroidectomy. It is also important to note that the offspring of parents who both have F H H have a 25% chance of inheriting a mutated allele from each parent. In such cases the child will develop neonatal severe hyperparathyroidism (NSHP), which is a medical emergency with an excessive morbidity and mortality. The children have extraordinarily high levels of serum calcium and are often dehydrated. Because it is difficult to reduce the serum calcium permanently by medical means, these children are candidates for parathyroidectomy. Occasionally, during an operation on a patient thought to have sporadic PHPT, the surgeon finds four enlarged parathyroid glands. The patient should have an appropriate operation and in the postoperative period the presence of either MEN-l, MEN-2A, or F H H should be excluded. Although the PHPT is not lifethreatening in these patients, the pancreatic islet cells tumors in patients with MEN-1 and the MTC and pheochromocytomas in patients with MEN-2A may prove lethal if untreated. If the diagnosis of MEN-1 is made first and patients are found to have a pancreatic gastrinoma, the hyperparathyroidism should be corrected first, because normalization of the serum calcium level will reduce gastric acid secretion. However, if an insulinoma is detected, it should be treated first, because there is no g o o d medical therapy for insulin o m a and the associated hypoglycemia may be lifethreatening. Virtually all patients with MEN-2A have MTC at the time of diagnosis and if a total thyroidectomy is p e r f o r m e d all four parathyroid glands should be identified and an appropriate operation performed. Failure to detect the presence of a p h e o c h r o m o c y t o m a in a patient with MEN-2A who is undergoing operation for PHPT or MTC may be catastrophic because severe hypertension may develop, either during induction of anesthesia, during the operative procedure, or in the postoperative period. The diagnosis of MEN-l, MEN-2A, and F H H has been greatly simplified because the genetic mutation for each has been identified. The m e n i n gene on chromosome 11 is mutated in patients with MEN-l, the RET protooncogene on c h r o m o s o m e 10 is mutated in MEN-2a, and the calcium-sensing receptor gene on chromosome 3 is mutated in F H H (8-11). Each of

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these mutations occurs in the germ line so they can be detected by direct DNA analysis of genomic DNA from a patient's white blood cells.

THE P H Y S I C A L E X A M I N A T I O N On physical examination of patients with PHPT one can rarely feel an enlarged parathyroid gland. A palpable neck mass in this setting most often represents a thyroid nodule or an enlarged lymph node. On the other hand, if a neck nodule is palpable and the serum calcium concentration is above 14 m g / d l one should suspect a parathyroid carcinoma (12). These patients often present with hoarseness and systemic manifestations of severe hypercalcemia, including nausea, vomiting, and dehydration. On physical examination the neck mass is firm and immobile and at operation the parathyroid tumor is usually fixed to the trachea or surrounding tissues. Occasionally, patients with a parathyroid carcinoma will require urgent surgery to control hypercalcemia, particularly when it is not possible to control the serum calcium concentration medically.

Laboratory Studies Most often surgeons see patients in referral from colleagues in internal medicine, endocrinology, or pediatrics, who have already established the diagnosis of PHPT biochemically. Thus the laboratory work need not be repeated unless there is some question about the results of a specific test.

Imaging Studies Most patients with PHPT will not require specific radiologic studies prior to surgery. Often bone densitometry studies are p e r f o r m e d to determine the degree of calcium loss from the skeleton. In patients who are being followed medically the clinician may choose to perform serial bone densitometry studies to monitor bone mass. Renal imaging studies may be helpful to determine whether a patient has occult renal stones, or to measure the stone b u r d e n in a known stone former. The use of imaging procedures to localize enlarged parathyroid glands was formerly applicable only to patients with a failed operation, who required repeat neck exploration. More recently, it has been shown that 99mTc-labeled sestamibipertechnetate (MIBI) scintigraphy combined with single-photon emission c o m p u t e d tomography successfully detects single parathyroid adenomas in approximately 95% of patients with PHPT (13). Unfortunately, the technique is not nearly as useful (60-70%) in patients with multiple-gland disease.

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However, the introduction of this technology has remarkably changed the surgical management of patients with PHPT and patients commonly have a sestamibi scan once the diagnosis of PHPT is established. Consequently, patients frequently present for surgical consultation bringing radiographic tests showing the location of an enlarged parathyroid gland. Unfortunately, there has been no prospective study of the indications for preoperative localization studies and their place in the management of patients with primary hyperparathyroidism. The combination of preoperative localizing studies and minimally invasive surgery has led to a dramatic change in the management of patients with PHPT, because the procedure can be performed on an outpatient basis at substantial cost savings. The discussion will be contined in the section on operative management of patients with PHE

Indications for Operative Intervention The decision, whether to operate on patients with PHPT, is influenced by several considerations and is somewhat controversial. The accepted indications for operation include the presence of either bone disease, kidney stones, or muscle weakness or tiredness of a degree that it limits the patient's function. A serum calcium above 12 m g / d l in a patient who has minimal or no symptoms is also an indication for neck exploration. Prolonged follow-up of patients with PHPT can become expensive, especially if bone densitometry studies and laboratory tests are performed repeatedly over time. It is the opinion of most surgeons that once the clinical diagnosis of PHPT is established in a medically fit patient, they should undergo a neck exploration, because the cure rate is high and the operation is usually simple. The indications for operative intervention have become liberalized since the introduction of new techniques for minimally invasive surgery. In a study by Silverberg and associates of 121 patients with PHPT (101 of whom were asymptomatic), 61 had parathyroidectomy and 60 were followed expectantly (14). Whether patients were symptomatic or asymptomatic, the parathyroidectomy resulted in correction of the hypercalcemia and an increase in bone mineral density of the lumbar spine and the femoral neck, but not the radius. Of the 52 asymptomatic patients not having a neck exploration, the serum calcium levels and the bone densitometry values did not change; however, 14 (23%) of them developed other indications for parathyroidectomy. Unfortunately, in patients with asymptomatic PHPT there have been no prospective randomized clinical trials comparing immediate parathyroidectomy to nonoperative management. This is an important issue that has significant clinical, scientific, and economic considerations.

CONSIDERATIONS IN THE SURGICAL TRF~TMENT OF PATIENTS WITH HYPERPARATHYROIDISM

Embryology and Anatomy of the Parathyroid Glands It is extremely important for the operating surgeon to have a clear understanding of the embryology and anatomy of the parathyroid glands. Although it is not commonly done today, many skilled parathyroid surgeons in the past spent hours in the autopsy suite, or the anatomy laboratory, searching for parathyroid glands and learning normal parathyroid anatomy, before they began to operate on patients with parathyroid or thyroid disease. This remains an important exercise because the normal parathyroid glands are small and may be difficult to recognize, even by the experienced eye.

Embryology Phylogenetically, the parathyroid glands first appear in Amphibia. In humans the upper parathyroids derive from the fourth pharyngeal pouch and the lower parathyroids originate from the third. During embryogenesis both sets of glands are intimately associated with the derivatives of their respective pouches, the lower glands with the thymus and the upper glands with the lateral thyroid complex. As the embryo matures the parathyroid glands descend to assume their normal positions (Fig. 1). The upper glands come to rest on the posterior surface of the midportion of the thyroid lobes, close to the point where the inferior thyroid artery enters the thyroid parenchyma. The lower parathyroid glands descend to reside close to the anterior-lateral surface of the lower thyroid pole. In some cases the migratory patterns are imperfect, which has important clinical implications. Either an upper or lower parathyroid gland may fail to migrate and remain embedded in the pharyngeal musculature. Conversely, a lower parathyroid gland may have an arrested descent and remain high in the neck as an "undescended parathymus." In this situation the thymus gland also fails to descend and remains as a thick cord of tissue medial and parallel to the carotid artery. The undescended parathyroid gland may reside adjacent to or within any portion of the undescended thymic tissue. Conversely, during migration a parathyroid gland may fail to separate from the thymus gland and descend into the anterior or deep mediastinum. On very rare occasions a portion of the thymus, containing an enlarged parathyroid gland, may descend to fill the space bounded by the aortic arch and the pulmonary artery, the so-called "middle mediastinal parathyroid adenoma."

SURGICAL MANAGEMENT OF HYPF~AgATm~omISM

FI6. 1 The migratory patterns of the parathyroid glands during embryogenesis. The upper parathyroid glands a r e derived from the fourth pharyngeal pouches and the lower parathyroid glands are derived from the third pharyngeal pouches. (Reprinted with permission from Langman J. Medical embryology, 3rd ed., Baltimore:Williams & Wilkins, 1975:266.)

Anatomy Usually, there are four parathyroid glands, but there may be more. It has been questioned whether there are ever less than four parathyroid glands. The phenomenon does appear to occur rarely. In Alveryd's report of 354 adults studied at autopsy, 90.6% had four glands, 3.7% had five glands, 5.1% had three glands, and 0.6% had only two glands (15). In only one of Alveryd's 18 patients with less than four identified parathyroid glands was the combined weight of the glands sufficient to suggest that none had been overlooked. Moreover, Norris serial sectioned 109 embryos from the base of the skull to the thoracic diaphragm and found at least four parathyroid glands in every specimen (16). Akerstr6m and associates performed autopsy studies on 503 cadavers and found four parathyroid glands in all but 18 specimens. There was a striking constancy to the location of the parathyroid glands, as shown in Fig. 2 (17). From these studies one can surmise two important facts. It is extremely unusual for a patient to have less than four parathyroid glands and most often the parathyroid glands are where they should be. It is often tempting for the inexperienced surgeon to assume, after an arduous neck exploration during which only

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two or three parathyroid glands are found, that the patient naturally has less than four. One must always assume that each patient has at least four parathyroid glands. During the process of neoplastic transformation the enlarged parathyroid glands may migrate to an ectopic site (Fig. 3). Bulky upper parathyroid glands tend to descend into the posterior neck or the upper posterior mediastinum. Enlarged lower glands usually descend into the anterior mediastinum, adjacent to the thymus gland. This inferior migration of enlarged parathyroid glands is due to an increasing weight and the intermittent, but persistent, negative pressure generated in the thoracic cavity. Even when parathyroid glands are in a "normal" position they may be difficult to find because of pathologic changes within the thyroid gland. A normal or enlarged parathyroid gland may be entrapped between thyroid nodules and hidden from view. If, during a neck exploration for PHPT in a patient with a multinodular goiter, three normal parathyroid glands are found but the fourth cannot be found, it may be necessary to remove the thyroid lobe ipsilateral to the missing gland. Often in this situation the pathologist will incorrectly interpret the parathyroid gland as being intrathyroidal. The normal parathyroid glands are flat and weigh less than 30-45 mg each. They are yellowish tan in color, although enlarged parathyroid glands may be deep reddish brown. The glands are usually ovoid in shape but they may be lobulated or stellate. When the glands enlarge, their shape will be influenced by the surrounding structures, but they often assume an elongated shape with little adherence to adjacent tissues.

Preoperative Preparation It is important that the surgeon explain the operative procedure to the patient. The surgeon should review the possible causes of hyperparathyroidism and discuss the specific surgical treatments for the various disease states, e.g., operations for single-gland disease and multiple-gland disease. Most importantly, the possible complications of parathyroid surgery should be explained, including damage to the nerves that control phonation, postoperative hypocalcemia, and failure to find a parathyroid tumor due to its ectopic location. Patients should also be told of complications that are common to all surgical procedures, such as wound infection and postoperative bleeding. The patient may wish to know more about expected outcomes because many will have already read a great deal about the disease and will be familiar with the various treatments available.

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FIG. 2 Locations of the superior (A) and inferior (B) parathyroid glands. The most common locations are indicated by the darker shading. [Reprinted with permission from Akerstr6m G, Malmaeus J, and Bergstro R: Surgical anatomy of human parathyroid glands. Surgery 1984;95: 14-21 (17).]

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SURGICAL MANAGEMENT OF HYPERPARATHYROIDISM //

Operative Technique The primary goal of surgery in patients with PHPT is to cure the hypercalcemia without intraoperative or postoperative complications. In the past seven years the surgical m a n a g e m e n t of patients with hyperparathyroidism has changed markedly, primarily due to three innovations: (1) sestamibi scanning has proved to be highly accurate in localizing enlarged parathyroid glands; (2) techniques of minimally invasive surgery have been designed, and in concert with sestambi localization and neck explorations these are frequently performed in an outpatient setting with either light general, regional, or local anesthesia; and (3) the development of rapid immunoassays for parathyroid hormone, which can be performed intraoperatively to document that hyperfunctioning parathyroid tissue has been completely resected. These techniques have been introduced only recently and there has been insufficient time and experience to clearly judge their worth in clinical practice. However, it appears clear that the new technologies will have an important place in the management of patients with PHPT. Regardless of the operative technique for patients with PHPT there are certain important factors that need to be considered. There is an extremely low mortality following parathyroidectomy and the morbidity of the operation relates to postoperative bleeding, wound infection, hoarseness, and hypocalcemia. Bleeding usually occurs within the first 24 hours postoperatively and is associated with a sudden increase in venous pressure due to the patient's straining or coughing. Postoperative wound infection is rare and usually occurs in patients who suffer from a chronic skin irritation. Hoarseness results from injury to either the recurrent laryngeal nerve or the external branch of the superior laryngeal nerve. It is important that these nerves be identified during the operation and that they be kept from harm's way.

General Technique of the Standard Operation In patients who either do not have a preoperative localization procedure, or in whom the findings are controversial, a standard open neck exploration is performed through a cervical incision u n d e r general anesthesia. The goal of operation is to identify each parathyroid gland and to remove those that are enlarged. The remaining normal-sized parathyroid glands should be confirmed as such by biopsy. Some surgeons have reported that biopsy of all four parathyroid glands is associated with an increased incidence of postoperative hypoparathyroidism, but we have not found this to be the case. It must be emphasized that

493

great care needs to be taken in performing the parathyroid biopsy because only a very small amount of tissue is taken from the portion of the parathyroid gland opposite to the arterial supply. Even though the terminologies of adenoma and hyperplasia are common descriptors of parathyroid pathology, we do not find the terms useful and prefer to characterize hyperparathyroidism as being either due to single-gland disease or multiple-gland disease. Nearly all patients who have a single enlarged parathyroid gland and three normal glands are cured following resection of the large gland. Patients who have two or three enlarged glands have a less good outcome. This disease state is commonly referred to as double or triple parathyroid adenomas, or early parathyroid hyperplasia. Little has been written about the long-term evaluation of patients with this condition following parathyroidectomy, however. Our group evaluated 375 consecutive patients with PHPT and found 85 (15%) who had enlargement (greater than 50 mg) of two or three parathyroid glands (18). At operation the enlarged parathyroid glands were removed and the remaining normal-sized glands were biopsied. Of these patients, 76 were followed from 12 to 140 months after surgery and 8 (10.5 %) had either persistent (2 patients at 1 and 4 months) or recurrent (6 patients from 45 to 133 months) hyperparathyroidism. When compared to age- and gender-matched patients with PHPT due to single-gland disease, patients with two-or three-gland disease had lower serum calcium concentrations and a lower combined weight of the resected parathyroid tissue. This indicated a mild form of PHPT with a higher incidence of persistent or recurrent hypercalcemia compared to patients with single-gland disease, but a much lower incidence of postoperative hypercalcemia compared to patients with four-gland disease. Patients with PHPT due to generalized parathyroid enlargement represent the most difficult m a n a g e m e n t problems. Surgery is not often curative for this disease, because regardless of the procedure performed there is a high rate of persistent or recurrent hyperparathyroidism. The original operation for this disease was radical, subtotal, 3g-gland parathyroidectomy. The strategy was to remove three of the four enlarged parathyroid glands and one-half or less of the fourth. An attempt is made to remove a substantial portion of the parathyroid mass so that the patient remains normocalcemic. The problem is that the forces that led to the hypertrophy and excess secretion of parathyroid hormone are still at play. There have been almost no long-term reports of patients with four-gland disease who have been managed by this operation, however, the few publications that have addressed this issue provide evidence that the failure rate exceeds 70%. An alternate

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approach to patients with four-gland disease is total parathyroidectomy with autotransplantation of fragments of the most normal-appearing parathyroid tissue to a heterotopic site, such as the brachioradialis muscle of the n o n d o m i n a n t forearm. This operation is associated with a transient period of hypocalcemia, while the graft is vascularized. There is also an appreciable likelihood that these patients will develop graft-dependent hyperparathyroidism, but this complication can be managed by removing a portion of the graft under local anesthesia. Placement of the autotransplant in the arm allows one to monitor graft function by comparing PTH concentrations in venous blood obtained from the two arms. A markedly elevated level of PTH in blood draining the graft excludes a fifth hyperfunctioning parathyroid gland in the neck or mediastinum. In patients who develop hypercalcemia after 3~gland parathyroidectomy a repeat neck exploration under general anesthesia is required. Most surgeons have a methodical way of exploring the neck, and in the process identify all four parathyroid glands. In most circumstances the operation can be performed in less than 1 hour. If one or more parathyroid glands cannot be identified, biopsies should be taken of the identified normal glands and their location marked with a black silk suture so that they can be readily identified at a subsequent operation after localization procedures have been performed. Above all, normal parathyroid glands should not be removed.

Minimally Invasive Parathyroid Surgery A variety of less invasive operative strategies have been developed recently to take full advantage of advances in parathyroid localization and testing. The

goal of these strategies has been to maintain the outstanding success rate of the conventional neck exploration in curing hyperparathyroidism, while decreasing the invasiveness, and potentially the cost, of the procedure. Sestamibi scanning and rapid PTH measurement have had substantial effects, whereas the application of videoscopic surgical techniques remains investigational. 99mTechnetium-labeled sestamibi was originally developed for cardiac imaging. It was found incidentally to image parathyroid tissue on delayed scans and has since been used extensively for noninvasive parathyroid imaging (Fig. 4) (19,20). This technique has advantages in lateral, oblique, and three-dimensional imaging compared to the formerly used technetiumthallium scanning because of its single-nuclide nature and short half-life, high-energy profile. Sestamibi scans can identify the site of abnormal tissue in 75-85% of patients, but is less useful in patients with small adenomas or multiple-gland disease. The success of sestamibi in identifying the site of abnormal parathyroid tissue has been an impetus for the reassessment of directed, unilateral strategies of parathyroidectomy. Techniques for the rapid, accurate measurement of serum PTH have been developed that allow the intraoperative assessment of the effect of parathyroidectomy (21,22). A two-site immunoassay for intact PTH with standard curves adjusted to the short incubation period is used. The time to PTH determination is typically 20-30 minutes from the blood draw, which makes it practical to perform the assay during the operation. Experience has demonstrated that if the PTH level decreases by at least 50% within 10 minutes after removal of the enlarged parathyroid gland, the patient will be normocalcemic postoperatively (Fig. 5). Thus a directed, unilateral approach to parathyroidectomy, without visualization of the other normal

FIG. 4 99mTechnetium-labeled sestamibi scan of a patient primary hyperparathyroidism due to a right lower parathyroid adenoma. The activity is distributed in the normal thyroid gland, salivary glands, and heart in the initial scan performed 10 minutes after radionuclide injection (white arrows in left panel). Two hours after injection, the radioactive tracer has washed out of the thyroid gland, but remains in a right-sided parathyroid gland that overlies the lower pole thyroid activity from the initial scan.

SURGICAL MANAGEMENT

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tion, except that the focus is limited to identifying the enlarged parathyroid gland identified on the preoperative scan. Ten minutes after resecting the abnormal gland, a second blood sample is taken to determine the PTH level; if this PTH level is 50% less than the base line value, then the operation is terminated (29). If, however, the PTH level does not decrease, the exploration is extended to a conventional exploration and the other parathyroid glands are identified.

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Sestamibi-Guided Parathyroidectomy Single gland disease

Multiple gland disease

FIG. 5 Intraoperative parathyroid hormone levels from patients who underwent conventional bilateral neck exploration for primary hyperparathyroidism. Of these 72 patients, 55 had single-gland disease and 17 had multiple-gland disease as defined by the gross parathyroid morphology and gland weights. A decrease to 50% of the base line intraoperative PTH level 10 minutes following parathyroid gland resection predicted successful parathyroidectomy in 93% (51/55) of patients; if the PTH remained >50% of base line, 76% (13/17) of patients had multiple-gland disease. (Data from Gordon LI, et aL Surgery 1999;126:1030-1035.)

glands, appears to be justified by the low failure rate reported from early experience with this procedure (23,24).

Concise Parathyroidectomy This is the most frequently used approach (25-28). The surgeon obtains a sestamibi scan in advance of the operative day in an attempt to identify the site of the abnormal parathyroid gland. If the sestamibi scan does not identify a gland, then the patient undergoes the conventional full-neck exploration. If the sestamibi scan identifies an enlarged parathyroid gland, the patient is taken to the operating room and a limited neck exploration and parathyroidectomy are performed. The operation can be performed u n d e r general anesthesia, cervical block, or local anesthesia with sedation. A transverse incision is made at the level of the thyroid isthmus, on the side of the midline ipsilateral to the abnormal parathyroid gland. Often this incision is less than 2.5 cm. Blood is drawn at the outset of the operation (often from the anterior or internal jugular vein) to determine the base line PTH level. The strap muscles are opened, either by separating them in the midline for a relatively anteriorly placed lower parathyroid gland, or by splitting the sternohyoid and sternothyroid muscles. Exploration proceeds as for the conventional opera-

In this approach, the sestamibi concentration in the parathyroid tissue is used to aid the surgeon in identification of the enlarged gland (30,31). The positioning and anesthetic requirements are the same as for concise parathyroidectomy. A sestamibi scan is obtained on the day of the procedure, within 2 hours of the operative start time. The operation is begun by using a handheld gamma probe to localize the site where the per-second counts are highest in the neck. A limited incision is fashioned over that site and dissection is carried directly to the parathyroid gland using the probe for directional guidance. The parathyroid gland is removed, and the operation terminated. Some surgeons use intraoperative PTH measurements to confirm resection of all hyperfunctioning parathyroid tissue, but many do not (32).

Videoscopic Cervical Exploration Some surgeons use a videoscopic approach to resect an abnormal parathyroid gland that has been identified on preoperative sestamibi scan (33,34). This is done using a variety of different work-space maintenance techniques (carbon dioxide gas pressure, or gasless) and anatomic approaches (lateral approach anterior to the sternocleidomastoid muscle or central approach from the suprasternal notch). The technique is still in its developmental stage, and the optimal approach remains to be determined. Investigators using this technique use a sestamibi scan in order to identify the abnormal gland preoperatively, and in general identify and resect only that gland. Given the use of a limited incision to identify and remove parathyroid glands in the directed, open approaches, m u c h of the advantage of a videoscopic approach has been nullified.

The Failed Operation In approximately 95% of patients with PHPT an operation will be successful. When the operation is unsuccessful it is usually due to one or more of several factors: inexperience of the operating surgeon, enlargement of more than one parathyroid gland,

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ectopic location of an enlarged parathyroid gland, or an erroneous diagnosis. The importance of patience and experience cannot be overemphasized, and as with most operative procedures the surgeon with the most experience with a given technique gets the best results. When the neck exploration has been unsuccessful one should allow the patient to convalesce from the procedure and then reconsider the diagnosis of PHPT by biochemical tests. In patients who were found to have more than one enlarged parathyroid gland it is important to exclude familial hyperparathyroidism, especially FHH. Other diagnoses should be considered in hypercalcemic patients in whom four normal-sized parathyroid glands were found. Once the biochemical diagnosis of PHPT is confirmed, attempts should be made to identify by localization techniques the site of the missed enlarged parathyroid gland. The noninvasive localization procedures should be performed first and only if they are unsuccessful should invasive localization procedures be performed. The specific radiographic localization procedures will not be mentioned here because they are discussed in Chapter 30. However, the importance of these procedures cannot be overemphasized because the specific identification of a lesion will markedly shorten the time of the repeat operative procedure and make the patient less susceptible to the complications attendant to repeat neck explorations when no hyperfunctioning parathyroid tissue has been identified. In almost every series of patients undergoing repeat neck exploration for PHPT the missed enlarged parathyroid gland is most often found in the neck, or it is retrievable through a cervical incision (Table 1) (35). The technique for repeat neck exploration is similar to that used for a standard neck exploration. The previous scar should be excised and if the parathyroid gland has been localized by imaging techniques, only that side of the neck need be explored if the patient has sporadic PHPT. Even if the enlarged parathyroid gland is in the deep anterior mediastinum close to the thymus gland it can be removed with the thymus through a cer-

TABLE 1

Location of Missed Parathyroid Glands Found during Reoperation a

Location

Persistent

Recurrent

Total

Cervical Mediastinal

208 20

35 1

243 21 264

aReprinted with permission from Brennan MF, Norton JA. Reoperation for persistent and recurrent hyperparathyroidism. Ann Surg 1985;201:40-44 (35).

vical excision (36). Rarely, it is necessary to perform a thoracotomy or a mediastinotomy to remove the enlarged parathyroid gland.

Renal Osteodystrophy and Secondary Hyperparathyroidism Most patients with advanced renal disease who are maintained on chronic dialysis have evidence of parathyroid-induced bone disease, with elevated serum levels of PTH. The management of patients with this disorder is discussed in Chapter 39. Due to improvements in the medical management of patients with chronic renal failure, the frequency of parathyroidectomy has decreased. Nevertheless, there are indications for parathyroidectomy in patients with secondary hyperparathyroidism due to chronic renal failure, or in patients with autonomous hyperplastic parathyroid glands (tertiary hyperparathyroidism), including hypercalcemia either in patients who are candidates for renal transplantation or in patients who have a wellfunctioning kidney, pruritis, bone pain, and extensive soft tissue calcification. Patients with secondary hyperparathyroidism almost always have generalized parathyroid gland enlargement and should be treated with either radical subtotal parathyroidectomy or total parathyroidectomy and parathyroid autotransplantation. Rothmund and associates performed a prospective randomized trial comparing these two procedures and found that total parathyroidectomy and autotransplantation afforded better results in that there was a lower incidence of postoperative hypoparathyroidism and a higher incidence of bone healing (37).

Postoperative Management After a standard neck exploration for PHPT the patient should have minimal ambulation on the day of the operation but can eat a normal diet on the day after surgery. Patients are usually discharged on the day after surgery but they may require prolonged hospitalization or careful evaluation as an outpatient if the serum calcium remains low and they have symptoms of hypocalcemia. Patients who have minimally invasive operations are usually discharged the same day of surgery, although they still require careful postoperative evaluation.

REFERENCES 1. Sandstrom I. O m e n ny k6rtel hos menniskan och fitakillga d/iggdjur. Uppsala Liika F6rh 1879;15:441. 2. Gley E. Sur les fonctions du corps thyroide. C R Soc Biol 1891;43:841.

SURGICAL MANAGEMENT OF HYPERPARATHYROIDISM 3. Von Recklinghausen FD. Die fibr6se oder deformierte Ostitis, die Osteomalacie und die osteoplastiche Carcinose in ihren gegenseitigen Beziehungen. Festschr Rud Virchow (Berlin) 1891 ;1:89. 4. Schlagenhaufer E Zwei Ffille von Parathyreoidea Tumoren. Wien Klin Wochenschr 1915;28:1362. 5. Mandl E Therapeutischer Versuch bei einem Falle von Ostitis fibrosa generalisata mittels Extirspation eines Epithelk6rperchentumors. Zentrabl Chir 1926a;5:260. 6. Cope O. The story of hyperparathyroidism at the Massachusetts General Hospital. N EnglJ Med 1966;21:1174-1182. 7. Barr DE Bulger MA. The clinical syndrome of hyperparathyroidism. Ann J Med Sci 1930;179:449. 8. Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404-407. 9. Donis-Keller H, Dou S, Chi D, et al. Mutation in the RET protooncogene are associated with MEN-2A and FMTC. Hum Mol Genet 1993;2:851-856. 10. Mulligan LM, Kwok jBj, Healey CS, et al. Germ-line mutations of the RETprotooncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-460. 11. Pollak MR, Brown EM, Chou Y-HW, et al. Mutations in the CA2+sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-1303. 12. Wang CA, Gaz RD. Natural history of parathyroid carcinoma: Diagnosis, treatment, and results. A m J Surg 1985;149:522-527. 13. Neumann DR, Esselstyn CB, Jr, Go RT, Wong CO, Rice TW, Obuchowski NA. Comparison of double-phase 99mTc-sestamibi with 123-I 99mTc-sestamibi subtraction SPECT in hyperparathyroidism. A m J Roentgeno11997;169:1671-1674. 14. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JE A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255. 15. Alveryd A. Parathyroid glands in thyroid surgery. Acta Chir Scand 1968;389:1-120. 16. Norris EH. The parathyroid glands and the lateral thyroid in man: Their morphogenesis, histogenesis, topographic anatomy and prenatal growth. Contrib Embryo11937;26:47. 17. Akerstr6m G, Malmaeus J, Bergstro R. Surgical anatomy of human parathyroid glands. Surgery 1984;95:14-21. 18. Wells SA, Leight GS, Hensley M, et al. Hyperparathyroidism associated with the enlargement of two or three parathyroid glands. Ann Surg 1985;202:533-538. 19. Wei, JP, Burke GJ. Cost utility of routine imaging with Tc-99msestamibi in primary hyperthyroidism before initial surgery. 1997;1097-1101. 20. Bhatnagar A, Vezza PR, Bryan JA, Atkins FB, Ziessman HA. Technetium-99m-sestamibi parathyroid scintigraphy: Effect of P-glycoprotein, histology and tumor size on detectability, J Nucl Med 1998;39:617-620. 21. Irvin, GL, Dembrow VD, Prudhomme DL. Clinical usefulness of an intraoperative "quick parathyroid hormone" assay. Surgery 1993; 114:1019-1023.

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22. Nussbaum SR, Thompson AR, Hutchenson BA, Gaz RD, Wang C. Intraoperative measurement of parathyroid hormone in the surgical management of hyperparathyroidism. Surgery 1988;104:1121-1127. 23. Duh Q-Y, Uden P, Clark OH. Unilateral neck exploration for primary hyperparathyroidism: Analysis of a controversy using a mathematical model. WorldJ Surg 1992;16:654-662. 24. Shen W, Sabanci U, Morita ET, Siperstein AE, Duh Q-Y, Clark OH. Sestamibi scanning is inadequate for directing unilateral neck exploration for first-time parathyroidectomy. Arch Surg 1977;132:969-974. 25. Carry S, Worsey M, Virji, M, Brown M, Watson C. Concise parathyroidectomy: The impact of preoperative SPECTOomTc sestamibi scanning and intraoperative quick parathormone assay. Surgery 1997;122:1107-1116. 26. Boggs JE, Irvin GL, Molinari AS, Deriso GT. Intraoperative parathyroid hormone monitoring as an adjunct to parathyroidectomy. Surgery 1996;120:954-958. 27. Irvin GL, Sfakianakis G, Yeung L, Deriso, GT, Fishman LM, Molinari, AS, Foss JN. Ambulatory parathyroidectomy for primary hyperparathyroidism. Arch Surg 1996;131:1074-1078. 28. Chen H, Sokoll, LJ, Udelsman R. Outpatient minimally invasive parathyroidectomy: A combination of sestamibi-SPECT localization, cervical block anesthesia, and intraoperative parathyroid hormone assay. Surgery 1999;126:1016-1022. 29. Gordon LI, Snyder WH, Wians F, Nwariaku F, Kim LT. The validity of quick intraoperative parathyroid hormone assay: An evaluation in seventy-two patients based on gross morphologic criteria. Surgery 1999;126:1030-1035. 30. Norman J, Chheda H. Minimally invasive parathyroidectomy facilitated by intraoperative nuclear mapping. Surgery 1997; 122:998-1004. 31. Norman J, Chheda H, Farrell C. Minimally invasive parathyroidectomy for primary hyperparathyroidiam: Decreasing operative time and potential complications while improving cosmetic results. Ann Surg 1998;64:391-396. 32. Murphy C, Norman J. The 20% rule: A simple, instantaneous radioactivity measurement defines cure and allows elimination of frozen sections and hormone assays during parathyroidectomy. Surgery 1999;1126:1023-1029. 33. Miccoli P, Bendinelli C, Vignali, E, Mazzeo S, Cecchini G, Pinchera A, Marcocci C. Endoscopic parathyroidectomy: Report of an initial experience. Surgery 1998;124:1077-1080. 34. Henry JF, Defechereux T, Gramatica L, de Boissezon C. Minimally invasive videoscopic parathyroidectomy by lateral approach. Langenbecks Arch Surg 1999;384:298-301. 35. Brennan ME Norton JA. Reoperation for persistent and recurrent hyperparathyroidism. Ann Surg 1985;201:40-44. 36. Wells SA, Cooper JD. Closed mediastinal exploration in patients with persistent hyperparathyroidism. Ann Surg 1991 ;214:555-561. 37. Rothmund M, Wagner PK, Schark C. Subtotal parathyroidectomy versus total parathyroidectomy and autotransplanation in secondary hyperparathyroidism: A randomized trial. World J Surg 1991;15:745-750.

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CI4APTF32 Ectopic Locations of Parathyroid Glands

NORMAN

W. T H O M P S O N

Arbor, Michigan 48105

A N D P A U L G. G A U G E R

Division of Endocrine Surgery, Department of Surgery, University of Michigan, Ann

INTRODUCTION

The following discussion considers ectopic glands based on their embryologic origin.

A thorough knowledge of the location of ectopic parathyroid glands is of fundamental importance to the surgeon undertaking a neck or mediastinal exploration for either primary or secondary hyperparathyroidism. Although relatively infrequent, ectopic parathyroid glands are the most common cause of failure when the neck is explored by an experienced parathyroid surgeon. Because of the possible ectopic location of a hyperplastic parathyroid gland in any neck exploration, it is essential that as each gland is identified it is characterized as a superior (branchial pouch IV origin) or inferior (branchial pouch III origin) gland based on its anatomic location and relationships to other anatomic structures. Thus, when three normal glands have been found and the missing gland has not been identified after a careful search in the usual locations, a systematic exploration can be made in the known ectopic locations for that gland based on its embryologic origin. With a disciplined exploration, nearly all cervical and upper mediastinal glands can then be identified. Ideally, only deep mediastinal glands in either the anterior or the middle mediastinum (1-2%) should remain undetected after such a search (1). Rarely, the cause of hyperparathyroidism may be due to an ectopic supernumerary (fifth gland) determined by finding four normal glands. In this unusual situation, the surgeon's search cannot focus on the embryologic domain of one specific gland, but must extend to the entire neck and all known accessible ectopic sites before termination of the exploration. The Parathyroids, Second Edition

S U P E R I O R PARATHYROID G I A N D S The incidence of true superior parathyroid gland ectopia is rare. Normal superior parathyroid glands are more likely to be found in a relatively discrete and predictable area than are those glands designated as inferior. Nearly 95% of normal superior glands can be found within a 2-cm radius of the junction of the recurrent laryngeal nerve and the inferior thyroid artery. Only 2-4% of normal superior glands will be found caudal to the inferior thyroid artery within the tracheoesophageal groove and about the same percentage is found more cephalad along the superior pole of the thyroid gland (2,3). Less than 0.1% is even further cephalad in approximation to the pharyngeal wall (4). Nevertheless, abnormal parathyroid glands can descend to an ectopic location (pseudoectopia) in more than 40% of all patients with hyperparathyroidism due to superior gland disease (1). By far, the most common location is along the esophagus, often in the tracheoesophageal groove (Fig. 1). Pseudoectopia occurs when the enlarged gland descends u n d e r or posterior to the inferior thyroid artery. In this plane, unless retained by investing fascia or vessels, an enlarged gland can acquire an ectopic localization as it descends anterior to the prevertebral fascia, into the posterior mediastinum. The extent of descent is probably limited by the end artery supplying the tumor, which is always a 499

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

500

/

C-6

CHAPTER32

~ ..

,, ...::..

.. ~;.:,, ...

FIG. 1 Locations of enlarged superior parathyroid glands. Approximately 40% of superior gland adenomas are found in ectopic locations as a result of developmental mechanical forces (pseudoectopia).

branch from the inferior thyroid artery (3,5). These "acquired ectopic" superior parathyroid glands can always be removed through a cervical incision, even when they are fairly deep in the posterior mediastinum (Fig. 2). It is noteworthy that more than half of these pseudoectopic adenomas will not be visualized even after the initial mobilization of the thyroid lobe and its medial retraction. The caudally descended superior gland is always covered by a layer of fascia extending from the thyroid medially as the thyroid sheath, and laterally to the carotid artery where it becomes the carotid sheath. Caudally the pretracheal fascia extends laterally before evolving into the carotid sheath. This fascia lies anterior to an ectopic superior parathyroid gland as well as the recurrent laryngeal nerve as it ascends the neck toward the larynx. Because abnormal superior glands are so frequently found in a paraesophageal location in the lower neck or upper posterior mediastinum, these locations must be routinely evaluated whenever a parathyroid gland has not been detected in the usual anatomic area. Using a routine but disciplined approach to exploration for the superior gland, an enlarged gland in this ectopic location should never be overlooked (1,3,4,6,7). In our opinion, the key maneuver at exploration is initially to incise the fascia lateral to the upper thyroid pole, vertically proceeding caudally to the inferior thyroid artery. This allows exposure of the prevertebral fascia posteriorly, improved medial rotation of the entire thyroid lobe with appropriate retraction, and direct visualization caudally into the posterior mediastinum. An index finger can be inserted behind the inferior thyroid artery into

the tracheoesophageal groove and or behind the esophagus (Fig. 3). Rarely, a truly ectopic parathyroid gland may be found in the midline posteriorly, adherent to the esophageal wall rather than freely movable on a vascular pedicle. Such parathyroid tumors are covered by a thin layer of fascia fixing their position and establishing their embryonic heritage, rather than an acquired location. Occasionally, a large gland will be found behind the lower pharynx or upper esophagus, but completely free except for its vascular pedicle. Enlarged parathyroid glands along the upper pole of the thyroid gland are infrequent (2%) and are considered as embryologically ectopic because of their fixed position beneath the sheath of fascia covering the thyroid gland (Fig. 4). Even when very large, they may be overlooked because they can be molded around the underlying thyroid tissue, compressed within its sheath, and may be initially considered a portion of the upper pole. However, when the sheath has been incised, these abnormal glands may literally "spring out" of their confined space. These glands should not be considered intrathyroidal even when found compressed into a groove between thyroid lobulations or nodules, if present. In our experience with more than 3500 parathyroid explorations and observations in more than 5000 thyroidectomy specimens, we have found only one truly ectopic intrathyroidal superior gland beneath the true thyroid capsule, on the left side, within the Tubercle of Luckerkandl. As a result, we consider there to be little if any basis in ever performing a thyroid lobectomy for a missing superior parathyroid gland. We a b a n d o n e d this step in our explorations many years ago. During the past decade we came to appreciate that an abnormal superior parathyroid gland could be ectopically located in the region of the pharynx at the level of the pyriform sinus (4). This is the site of the fourth branchial pouch and the embryonic origin of the superior parathyroid gland, as well as a component of thymic tissue (5,8). This location is more than 2 cm cephalad to the upper thyroid pole and is medial to the carotid sheath. We have observed that several of these adenomas were actually covered by pharyngeal muscle, although external to the submucosa of the pharyngeal wall (Fig. 5). We consider these parapharyngeal parathyroid glands to be undescended superior glands (4). They are much rarer than undescended inferior glands, which may be found at the same level of the neck but in a more lateral location within the carotid sheath (9,10). Another extremely rare ectopic site where a superior parathyroid gland may be found is in the neck, but lateral to the carotid sheath. Only a few such cases have been described (11). We have e n c o u n t e r e d only one such case in 3500 patients, which presented as a large right superior gland approximately 4 cm above the

EcToeIc LOCATIONS OF PARATHYROID GLANDS /

501

FIG. 2 (A, B) Magnetic resonance imaging demonstrates a huge (6 x 5 cm) right superior parathyroid adenoma in the posterior mediastinum in a patient with severe hypercalcemia (serum calcium, 16 mg/dl). (C) Operative photo showing the adenoma from the same patient as in A and B. This is a right lateral view with the thyroid lobe retracted medially, causing tension on the inferior thyroid artery and its branch to the adenoma. The cephalic end of the adenoma is emerging from under the inferior thyroid artery. A finger was inserted laterally and posteriorly and with gentle pressure caused the adenoma to be easily delivered out of the posterior mediastinum.

502

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CIJaPTWI~32

FIG. 3 Drawing showing the key maneuver in detecting an occult parathyroid gland in the tracheoesophageal groove. Note that the pretracheal fascia has been incised vertically and the index finger inserted along the prevertebral fascia. This is done whenever a normal superior gland has not been identified in the area around the recurrent laryngeal nerve and inferior thyroid artery junction.

level of the clavicle. This was easily palpable and was initially considered to be a scalene lymph node involved by lymphoma. The patient, a 22-year-old woman, also had marked hypercalcemia and biochemical evidence of primary hyperparathyroidism. The surgical plan was to excise the node for definitive biopsy and to perform a parathyroid exploration. At surgery, three normal parathyroid glands were identified but the right superior gland was not detected after a preliminary exploration. With further dissection, a large arterial branch from the right inferior thyroid artery was noticed, crossing laterally and posterior to the carotid sheath and its contents. With retraction of the entire carotid sheath anteriorly, this artery was traced into the scalene fat pad where it terminated in a 3-cm parathyroid adenoma.

INFERIOR PARATHYROID GLANDS Ectopic inferior parathyroid glands are more comm o n than those arising from superior parathyroid glands if only embryologic factors are considered in their definition (5). Ectopic locations are not caused by mechanical forces influencing the movement of an enlarged gland. Ectopic sites of inferior parathyroid glands are determined during embryogenesis. The

majority (80%) of inferior parathyroid glands migrate with the thymus from branchial pouch III and, after separation, settle in an area within 2 cm of the inferior pole of the thyroid gland (1,3). Approximately 15% of inferior parathyroid glands will remain within the thyrothymic tract or upper pole of the thymic tissue but still within 2 to 4 cm of the inferior thyroid pole. Fewer than 5% of inferior parathyroid glands are located on the anterior or anterior lateral surface of the thyroid gland, most often beneath its sheath. These are all classic locations for normal or abnormal parathyroid glands and are sites that would routinely be explored during parathyroidectomy. Approximately 15% of inferior parathyroid gland adenomas will be found within thymic tissue that descends from the lower thyroid pole as thyrothymic tract (fascia, fat, vessels) or atrophic thymus gland (an encapsulated structure composed primarily of fat in older adults) (1,2,3,5,7). In children and young adults, thymic tissue can usually be identified within a capsule as grayish pink in color and may extend up to the inferior thyroid artery level. Although intrathymic parathyroid glands are considered to be ectopic, the great majority should be readily accessible through a standard cervical incision. Several maneuvers are needed for those more caudal than 4 to 6 cm below the thyroid gland. First, is the identification of the lateral edge of the thymus or tract after reflecting the sternothyroideus muscle laterally at the base of the neck. The fascia lateral to the thymus is then incised to allow finger dissection along and around the thymic horn or cervical thymus. At this point, the thyrothymic tract may be divided, maintaining a clamp on the caudal end to be used for cephalic traction as an index finger is used to gently free the thymus from surrounding tissue as its passes into the mediastinum anterior to the innominate vessels. A right-angle clamp is used to grasp the emerging thymic tissue at intervals as the traction pulls more and more thymic tissue into view (Fig. 6). Care must be taken to observe this tissue for a possible parathyroid adenoma before reapplying a potentially tumor-crushing clamp. Several points need to be emphasized. This dissection must be done gently or the thymus will tear off before much tissue can be mobilized. Nearly all intrathymic adenomas above the level of the aortic arch can be retrieved through a cervical incision. Bleeding is not a problem if this is done gently, even though occasionally the thymic vein may be torn off without its being ligated or clipped. Frequently, this specific vein will be seen posteriorly as the thymus is retracted anteriorly and can be divided between clips. Although we have performed cervical thymectomy in several h u n d r e d patients with primary hyperparathyroidism, in all patients with multiple endocrine neoplasia type 1 (MEN-l) and in all patients with secondary hyperparathyroidism (potential supernumerary glands),

ECTOPIC LOCATIONSOF PARATHYROIDGlaNr)s MEN-1 Hyperparathyroidism

................

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Right inferior gland totally i posterior tO ~I~!!i !i !! right inferior pole ~ ~

503

18 yr. WM Operati0.n 4/7/99

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undescended right superior gland

/

Largest gland est. 4-500 mg

iil !! i~i ~i~i~i~'i~~i~~i~i~~!i! ~~~~i I

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Subtotal parathyroidectomy 34/5 th glands Cervical thymectomy

D . . I 80 mg viable remnant !eft inferior gland with large clip

................... ~.:.~.:~ ....... ............. ...............

FIG. 4

Drawing showing location of right superior gland. This gland was beneath the

sheath of the thyroid but associated with some extrathyroidal thymic tissue that is occa-

sionally seen in children and teenagers. Thymic tissue is rarely found near adult superior parathyroid glands.

we have never encountered any significant hemorrhage. In young patients, in whom the thymus and its capsule may not be atrophic, it may be possible to tease out even deep mediastinal ectopic parathyroid adenomas, thus avoiding a sternal split (Fig. 7). Inferior parathyroid glands may be found entirely within the thyroid capsule in 1-4% of patients with

hyperparathyroidism (1,2,12). Nearly all intrathyroidal glands are within the lower one-third of the thyroid lobe, although some may extend into the middle third of the gland. These totally intrathyroidal abnormal glands can be palpated in only about 50% of cases but can be readily detected by intraoperative ultrasonography. There are several means by which enlarged

504

/

CI4APTW~.32

A Reoperative 1° HPT Sternocleido

Operation March 12, 1992

2.5 cm Right superior gland adenoma partially under pharyngeal muscles

Previous Operation Memphis, Tenn. Left superior excised - normal Left inferior Bx - normal Right inferior Bx - normal Ca++ 12.0 mg/dl Thallium Tc scan + Cephalic to upper lobe right thyroid

Strap muscles

Prevertebral fascia

Lateral approach medial to carotid sheath... between SCM and strap muscles

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Inferior thyroid A.

adenoma in wall of pharynx

B Reoperative Primary HPT Carotid bifurcation

z..d

Right superior adenoma adherent to pharyngeal wall - 3 cm above thyroid level of pyriform sinus

64 yr. WF

0peration Oct. 17, 1996 Undescended right superior gland adenoma 460 mg

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FIG. 5 (A) Drawing showing a patient's large undescended right superior parathyroid adenoma in the wall of the pharynx at the level of the pyriform sinus. This was localized by a thallium scintiscan following a failed exploration. (B) Drawing showing a patient's right superior parathyroid adenoma adherent to the pharynx but not covered by any of its muscle fibers. The reoperation was through a "lateral" approach between the strap muscles and sternocleidomastoid muscles. Localization was achieved by SVS of PTH. (C) Drawing showing undescended left superior parathyroid adenoma at reoperation through a lateral approach. During the first operation, a right superior gland weighing 150 mg was excised and a left superior gland was, in retrospect, mistakenly identified. Localization was by SVS and cervical ultrasound.

E C T O P I C L O C A T I O N S OF P A R A T H Y R O I D G L A N D S

C

/

505

Reoperative Hyperparathyroidism (1°) Operation Jan. 24, 2000 i

:

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Excision

Left superior para adenoma

Left superior adenoma from behind pharynx

Right superior excised at first operation wt. 150 mg

Lateral approach

Localization US and selv. sampling 2000 in Left high jug.

Left inferior parathyroid

Right inferior parathyroid .............~i~~iii~~il ..........

~::::~!iiii~.:::i~i~i~i~i~i:i|il

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Left inferior gland

FIG. 5 (continued)

Noted left superior at first operation NOT seen during procedure #2 :. mistaken identity

intrathyroidal glands can be detected besides preoperatively. The simplest noninvasive means of localization is by cervical ultrasound, which can detect nearly 100% of intrathyroidal parathyroids. Characteristically, these glands are hypoechoic and homogeneous. Specificity is not as high because some thyroid nodules may also have these features. Sestamibi scanning is considerably less sensitive in detection and even less specific because both benign and malignant thyroid neoplasms may concentrate and retain sestamibi. Localization studies are frequently n e e d e d because intrathyroidal parathyroid adenomas are frequently overlooked at initial exploration and require a second operative procedure for their excision. However, with a systematic plan of exploration for the inferior parathyroid gland, the ectopic intrathyroidal parathyroid gland should be detected unless it occurs as a s u p e r n u m e r a r y fifth gland

::

Undescended left superior parathyroid adenoma

or as a second adenoma. W h e n a n o r m a l or abnormal inferior gland has not been detected after a reasonable search in the usual locations, attention should be directed to the lower pole of the thyroid. This is done after an u n d e s c e n d e d or intrathymic gland parathyroid a d e n o m a has been ruled out. At one time, we routinely p e r f o r m e d a vertical thyroidotomy in the lower third of the gland and then enucleated the a d e n o m a when detected. In several cases, however, the capsule of an a d e n o m a was inadvertently incised in p e r f o r m i n g this maneuver. Although to our knowledge this has not resulted in subsequent parathyromatosis, we now prefer to resect the lower third of the thyroid gland. We do not routinely do this p r o c e d u r e when two other normal parathyroid glands are found as well as an adenoma. However, one of our patients did require a reoperation for a second a d e n o m a that was nonpalpable within the

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CHAeTER32 Reoperative 1° HPT 66 yr. WF Operation July 10, 1996

Age 61 Feb. 13, 1991 1st operation Petosky, MI 3 Glands excised 1. 2.3 cm LS. "Adenoma"

Right superi( gland not identi

2. Normal left inferior 3. Normal antimediastinal Right inferior(?) (surgeon's operative note)

2nd operation University of Michigan July 10, 1996

Operation Thyroid isthmus

1. Digital ant. mediastinal exploration 2. Cervical thymectomy 3. Transplantation 50 mg left 2 cm

marked with 3 clips

2 cm "adenoma" intrathymic Left Innominate A.

Thymus

Transplant site 60 mg

...... ~

~

~{}

.

Medial head _ left S.C.M. 3 clips 3 cm

FIG. 6 Drawing based on a surgeon's operative note during the first exploration, during which a left superior adenoma was excised. Reoperative drawing shows the procedure through previous incision. A right angle on the thymus allowed the gland to be teased from the anterior mediastinum. This contained another large adenoma that had been located behind the upper sternum. Regional localization was by SVS of PTH.

lower pole of the thyroid gland. In our experience, this is unlikely to ever happen again. Another important location for an ectopic inferior parathyroid gland is the area immediately around the carotid artery bifurcation within the carotid sheath. These glands are referred to as undescended inferior parathyroid glands and are nearly always associated with some thymic tissue, which may trail caudally to the base of the neck (Fig. 8) (1,3,5,7,9,10). In older adults, this tissue is usually so atrophic that it appears to be only a cord of fatty tissue. Although most undescended inferior ectopic glands are near the carotid bifurcation, rarely a gland may be found at an even higher level along the internal carotid artery behind the submandibular gland or in a more caudal location within the carotid sheath (Fig. 9). We have found hyperplastic glands as low as the base of the neck, where they are usually "molded" around the carotid artery by the carotid sheath and

therefore are not easily palpated or visualized until the sheath has been open (Fig. 10). For practical purposes, an undescended inferior gland cannot be excluded until the carotid sheath has been opened from the level of the clavicle to the internal carotid artery. We have observed that when an ectopic undescended inferior gland is present, there is invariably a trail of either thymic or fatty tissue from the cervical thymus (if present) or mediastinal thymus crossing laterally into the carotid sheath and then cephalically. We refer to this as the "yellow brick road" and when found and followed will lead to a parathyroid gland at its termination (Fig. 8). Only 1-2% of parathyroid adenomas are located in the deep mediastinum, a space defined as any site caudal to the aortic arch (1,2,5,6,13,14). M t h o u g h these adenomas are most commonly within the thymic tissue or adherent to its surface within the anterior mediastinum, an increasing n u m b e r of glands are being

ECTOPIC LOCATIONSOF PARATHYROIDGLANDS /

B

507

36 yr. WF Deep mediastinal parathyroid a d e n o m a excised through cervical incision Cervicomediastimal

thymectomy

!iiii~i~¸~il!!

~,i~ ¸....... Thyrothymic tract Thymus

i~!~

Intrathymic left inferior a d e n o m a

~'

3 x 1.5 cm

Pericardial level .

FIG. 7

(A) Operative photograph during primary operation in a young woman with an intrathymic 2-cm a d e removed from the deep mediastinum by cervical thymectomy. Note the bilateral intact thyrothymic tracts. The adenoma was 12 cm caudal to the manubrium. (B) Surgeon's drawing. noma

reported within the middle mediastinum (15,16). There is good evidence to support the theory that all ectopic glands in the anterior mediastinum represent either inferior parathyroid glands or supernumerary glands derived from branchial pouch III tissue (2,5). Deep anterior mediastinal ectopic inferior glands most commonly receive their blood supply from the internal mammary artery (anterior thoracic) rather than the inferior thyroid artery (Fig. 11) (14). Options for treatment include a traditional median sternotomy and thymectomy, embolization after identification of the adenoma by selective angiography (internal mammary artery), and thorascopic excision after precise anatomic localization with imaging studies (Fig. 12). The embryonic origin of middle mediastinal parathyroid glands has not been clearly established, although there is some evidence to suspect the superior parathyroid gland because of a close relationship of the evolving great vessels and heart and superior parathyroid glands (16). However, ectopic middle mediastinal adenomas are usually not associated with a missing superior gland and are most often classified as supernumerary glands. The two most common locations for middle mediastinal

adenomas are in the aortopulmonary window and behind either main pulmonary artery and anterior to either main stem bronchus or the tracheal carina (6,13,16). The rarest middle mediastinal location is within the pericardium. These adenomas are most commonly localized by sestamibi scanning but have been detected by computerized tomography (CT), magnetic resonance imaging (MRI), and selective arteriography. Although some middle mediastinal adenomas may receive their blood supply from the internal mammary artery, more often it is from a bronchial artery (14). Embolization is not a good option for these tumors because the vasonervosum of the left vagus nerve and left recurrent laryngeal nerve also may be supplied by the same artery. Unless clearly localized in the aortopulmonary window, a left thoracotomy offers the best surgical approach (6,15). Aortopulmonary window adenomas can be excised thoracoscopically or through a sternal split, although this may be difficult. We have excised middle mediastinal tumors through all three of these .approaches and favor a left thoracotomy especially when the patient has had a previous sternal split, which is often the case (Fig. 13).

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Primary Hyperparathyroidism Undescended Right Inferior Parathyroid Adenoma Operation March 20, 1999

Ca bifur Left superior gland

F su g q

lormal left inferior gland

"C

Thyro tn

Following the "yellow brick road" Atrophic thymic fat

B

Reoperative Parathyroidectomy

Right inferior parathyroid 1.4 grams within carotid sheath "Undescended" inferior gland

Distinct thymic t r a c ~ ~ ~ ; i in carotid sheath ....~,~.~,~:.: Vagus nerv

"'

~::i~

Thyrocervic

' ~)

::;i~~':

| 1 t

Recurrent " I~ ~ .~i~ill ~:i:~ ...... laryngeal nerve ...................... , ~ :i .... ~ ...... Rim of thymic tissue ~ ~ .:: ..... along pleura ::::

.............i:::~....

40 yr. BM 14 yr Hx renal dialysis PTH > 1,450 Renal osteodystrophy Operation Aug. 26, 1992

1st operation: Roland Gandy, MD Toledo Ohio July 6, 1992 Excism of left inferior Bx right superior

FIG. 8 (A) Drawing depicting the primary exploration in a patient with an undescended right inferior gland adenoma at the carotid bifurcation. Note the trail of thymic fat within the carotid sheath, which was first seen rising from the thyrothymic tract during the exploration. (B) Drawing showing the undescended right superior gland adenoma during reoperation. Thymic tissue with fat within the carotid sheath led to its detection. Localization was determined to be regional using SVS, which showed an increased PTH level that was elevated in the mid- and high jugular vein samples.

ECTOPIC LOCATIONS OF PARATHYROIDGLANDS /

Reoperation Persistant 1° HPT Parathyroid "adenoma" with surrounding fat...? posterior to jugular veins atrophic thymus ? undescended right inferior gland

65 yr. WF Operation July 8, 1997

360 mgm

Cervical reexploration Lateral approach to level above carotid bifurcation

Venus brar source of 1" I

300 mg adenoma found at level just below angle of jaw...posterior to high jugular vein after opening carotid sheath above level of bifurcation

Approached througt carotid sheath Initial expl. ne

Localization: SVS highest level right superior thyroid V 1300/pg VS peripheral 100 FIG. 9 Drawing showing a very high undescended right inferior gland adenoma at reoperation. A sestamibi scintiscan was negative but the adenoma was posterior to the submandibular gland, which concentrated sestamibi. SVS of PTH regionalized the missing gland to the high neck.

Reoperative Primary Hyperparathyroidism 43 yr. WF !st operation: Feb. 7, 1995 excision left inferior gland within upper thymus left superior- not identified excision right superior gland Right inferior not identified

i~~:III:IIIII~II!I I~I~!:II~II~ i~:......

Thymic ton(, within carotid she~ }Iml

Right inferior parathyroid adenom~

I/ml

115 pg/ml Left mid mominate 125 pg/m! Superic ca~ PTH 150 pg/ml

2nd operation: Dec. 20, 1995 Cervical approach excision of thymus and tongue within carotid sheath right with parathyroid adenoma 250 mg Abnormal descent Right inferior gland N__Qsternal split

~'ised)

FIG. 10 Drawing showing a partially descended right inferior parathyroid gland adenoma at reoperation within grossly apparent thymic tissue that could be seen within the carotid sheath. The adenoma was neither palpable nor visualized until the carotid sheath was opened.

509

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CHAPTER32

FI6. 11 Selective internal mammary arteriogram using a subtraction technique. The arrow points to the adenoma in the anterior mediastinum.

FI6. 12 Chest X-ray of a patient on the day following hyperosmotic dye injection and selective arterial occlusion of the internal mammary arterial branch to a deep mediastinal adenoma. Dye retention as noted here implies necrosis of the adenoma and a good result. This patient's serum calcium returned to normal and remained there during a 3-year follow-up.

ECTOPIC LOCATIONSOF PARATHYROIDGLANDS /

511

Middle Mediastinal Parathyroid Adenoma Left Lateral Thoracotomy

2 previous operations in Leiden, Holland January, 1996 Ligamentum arteriosum ~ f / / a g u s nerve AortopulmonarYwindow"~ ~ Esophagus

~.~

~:ii :i:~2~!~

left nerve

..

,~

,,...

nerve ~ :.~ : i ) ~ ~

I

. ,~;........ Left lung ''II)>;II ..... ;"

Left pulmonary artery Left Lateral View of Mediastinum and Aortopulmonary Windows Divided ligamentum arteriosurn ,\, ~ Vagusnerve ~! .!i>~~ /Left main stem bronchus Anthracotic ,,=,, n o d e s ~ ~

i~ "

Hyperplastic parathyroid pulmonary ~:,~i artery

SUPERNUMERARY PARATHYROID GLANDS In anatomic studies, 5-6% of individuals are found to have a fifth (supernumerary) gland (2). However, in studies specifically searching for small supernumerary glands, the incidence is greater than 10% (3). This has particular clinical relevance in patients with hyperparathyroidism due to multiple gland disease, particularly those with MEN-1 and secondary hyperplasia, in whom growth factors and other stimuli may cause these rudimentary glands, which otherwise might be insignificant, to enlarge and cause persistent or recurrent hyperparathyroidism after a subtotal or total parathyroidectomy. However, occasionally a supernumerary gland may be the only abnormal gland causing primary hyperparathyroidism. Most supernumerary glands are in an ectopic location. The most common site for a supernumerary gland is the cephalic portion of the thymus. (1-3). Because of this fact, cervical thymectomy should be routinely performed in all patients with sec-

FIG. 13 Drawing showing a left lateral thoracotomy approach to a middle mediastinal parathyroid adenoma located posterior to the left pulmonary artery and anterior to left main stem bronchus. This patient had previously undergone both cervical and anterior mediastinal explorations.

ondary hyperparathyroidism and in those with MEN-1 (Fig. 14). A secondary benefit in MEN-1 is the reduction of the possibility of the development of a malignant thymic carcinoid. In our experience, supernumerary glands, as the sole cause of primary hyperparathyroidism, have been most common in the cervical thymus or deep mediastinum. Intrathyroidal supernumerary glands are also a relatively common occurrence. Other rare sites, such as the nasopharynx, have been reported. We have found hyperplastic parathyroid glands within the vagus nerve in two patients (Fig. 15). Of interest is that both were supernumerary, as were the 12 cases of intravagal parathyroid glands reported in the literature (17). It would appear that the only time a surgeon should be concerned about this possible site is after an initial exploration reveals only four normal parathyroid glands. Fortunately, this is a very infrequent occurrence in patients with biochemically proven primary hyperparathyroidism.

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MEN-1 Hyperparathyroidism 55 yr. WF 1 HPT + ZES Bilobed ~ft superior gland Right superior glan(

Operation Feb. 23, 1995 ST parathyroidectomy leaving 60 mg right inferior gland partial thymectomy

Ectopic thryoid tissue Intrathymic right inferior glan~

Left inferior gland

~ernumerary 15th) gland t +

-v ~-

inferior gland

:::....

]i~------- Metal clip on edge of remnant

FIG. 14 Drawing showing supernumerary gland location in the left lobe of the thymus in a patient with the MEN-1 syndrome and hyperparathyroidism. Both inferior glands were also within the cervical thymus. Cervical thymectomy is routinely done in MEN-1 patients because of the 15-20% incidence of supernumerary glands, most of which are within the thymus.

lntra Vagal Parathyroid Adenoma 5th gland (4 normal glands location) Reoperation

43 yr. WM

~i;~~"'::;;:)~:i::,~+(....

:J!~i:!~;

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elsewhere

~i~,~i 4 normal glands Bx right thyroid Iobectomy and excision 2 normal glands

~/:i.+ 1

Operation Oct. 9,1997 .

.

.

.

i :

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~'~: :i,

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1.4 x 1.0 cm u~/:,.~:J One small nerve branch ran within adenoma

,:fll

/l~i,¢: ~

1. Sel. venous sampling 2. Cervical V.S.

FIG. 15 Drawing showing location of an ectopic left intravagal parathyroid adenoma at reoperation in a patient found to have four normal glands at initial exploration. Great care must be taken in dissecting intravagal adenomas from the nerve or ipsilateral (recurrent laryngeal nerve) dysfunction will result.

ECTOPIC LOCATIONS OF PARATHYROID GI~aXl)S

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REFERENCES 1. Thompson NW, Eckhauser FE, Harness JK. The anatomy of primary hyperparathyroidism. Surgery 1982;92:814-822. 2. Gilmour JR. The gross anatomy of the parathyroid glands. J Pathol 1938;46:133-143. 3. Akerstrom G, Malmaeus J, Bergstrom R. Surgical anatomy of human parathyroid glands. Surgery 1984;95:14-21. 4. Simeone DM, Sandelin K, Thompson NW. Undescended superior parathyroid gland: A potential cause of failed cervical exploration for hyperparathyroidism. Surgery 1995:949-956. 5. Gilmour JR. The embryology of the parathyroid glands, the thymus and certain associated rudiments. J Pathol 1937;45:507-519. 6. Bondeson AG, Thompson NW. Mediastinal parathyroid adenomas and carcinomas. In: Shields TW, ed. Mediastinal surgery. Philadelphia:Lea & Febiger, 1991:289-316. 7. Wang CA. Parathyroid re-exploration: A clinical and pathological study of 112 cases. Ann Surg 1977;186:140. 8. Joseph ME Nodol JB, Pilch BZ, Goodman ML. Ectopic parathyroid tissue in the hypopharyngeal mucosa (pyriform sinus). Head Neck Surg 1982;5:70-74. 9. Edis AJ, Purnell DC, van HeerdenJA. The undescended "parathymus"; An occasional cause of failed neck exploration for hyperparathyroidism. Ann Surg 1979;190:64-68.

10. Billingsley, KG, Fraker DL, Doppman JL, et al. Localization and operative management of undescended parathyroid adenomas in patients with persistent primary hyperparathyroidism. Surgery 1994; 116:982-990. 11. Udekwu AO, Kaplan EL, Wu T, Arganini M. Ectopic adenoma of the lateral triangle of the neck: Report of two cases. Surgery 1987;101:114-118. 12. Wheeler MH, Williams ED, Wade JSH. The hyperfunctioning intrathyroidal parathyroid gland: A potential pitfall in parathyroid surgery. WorldJSurg 1987;11:110-114. 13. Shields TW. Mediastinal parathyroids. In: Shields TW, ed. Mediastinal surgery. Philadelphia:Lea & Febiger, 1991:19-22. 14. Doppman JL, et al. The blood supply of mediastinal parathyroid adenomas. Ann Surg 1977;185:488-494. 15. McHenry C, et al. Resection of parathyroid tumors in the aortopulmonary window without prior neck exploration. Surgery 1988;104:1090-1094. 16. Curly I, Wheeler M, Grant C, Thompson NW. The challenge of the middle mediastinal parathyroid. WorldJSurg 1988;12:818-823. 17. Pawlik TM, Richards M, Giordano TJ, Burney RE, Thompson NW. Intravagal parathyroid adenoma. A report of two cases. Arch Surg 2000 accepted.

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CI-IAPTVR 3 3

Parathyroid Carcinoma

ELIZABETH SHANE Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032

INTRODUCTION

National Cancer Data Base reported 286 cases of parathyroid carcinoma, the largest series to date (26). In most series, this entity accounts for less than 1% of patients with primary hyperparathyroidism (1-3,11, 17,27-31). The disease may be somewhat more common in Japan than in Western countries, accounting for 5% of patients with primary hyperparathyroidism (18,32-34), and in an Italian study 5.2% of patients operated on for primary hyperparathyroidism were eventually found to have parathyroid carcinoma (15).

Parathyroid carcinoma is an u n c o m m o n cause of parathyroid hormone-dependent hypercalcemia. The collective published experience with this rare neoplasm has provided a distinctive clinical profile that differs in a n u m b e r of respects from that of benign primary hyperparathyroidism (1-3). The distinguishing features of parathyroid carcinoma assume even greater prominence when viewed within the current context of primary hyperparathyroidism, which commonly presents today as a mild asymptomatic disease (4-8). In this chapter, the clinical features, natural history, and prognosis of parathyroid cancer are reviewed. Surgical approaches to parathyroid cancer are outlined as well as medical therapies of the hypercalcemia that accompanies recurrent or metastatic disease. Because the ultimate prognosis depends to a major extent on successful resection of the tumor at the time of the initial operation, major emphasis is placed on those features of parathyroid carcinoma that help to differentiate it from primary hyperparathyroidism due to benign adenomatous or hyperplastic disease.

ETIOLOGY The etiology of parathyroid cancer is unknown. No clear pattern of predisposing factors has emerged in the cases described thus far. However, there are a number of clinical situations that may predispose to the development of parathyroid carcinoma. Several cases of parathyroid carcinoma have been reported in patients with a history of neck irradiation (35-37). There have also been a n u m b e r of reports of carcinoma occurring within an adenoma or a hyperplastic parathyroid gland (38-45), and there is a report of parathyroid carcinoma occurring in a patient with prolonged secondary hyperparathyroidism secondary to celiac disease (10). Despite these associations, Shantz and Castleman (28), in an extensive review of 70 cases, found no evidence for malignant transformation of previously pathologic tissue.

INCIDENCE Approximately 290 cases of parathyroid carcinoma were reported in the English literature between 1930 and 1992. Since 1992, more than 100 additional cases have been reported (9-25). Moreover, in 1999 the

The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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End-Stage Renal Disease Parathyroid carcinoma has been described in several patients with end-stage renal disease. Miki et al. (16) reviewed 12 such cases, originally published between 1982 and 1996, of patients with parathyroid carcinoma on maintenance hemodialysis. All demonstrated hyperplasia of other parathyroid glands (16,37,38,46-50) and one had a history of prior neck irradiation (37). The diagnosis was made an average of 6 years after the start of hemodialysis. In all cases, parathyroid carcinoma was diagnosed during or after parathyroidectomy on the basis of local invasion (n = 5), tumor pathology (n = 4), or distant metastases (n = 2). The average age of the patients was 49 years. Only 50% of these patients presented with signs of hypercalcemia; the mean serum calcium level was 10.8 m g / d l with a range of 8.5 to 12.6 m g / d l , considerably lower than serum calcium levels generally observed in patients with parathyroid carcin o m a (see below). Parathyroid h o r m o n e levels were more than twice the u p p e r limit of normal in all patients, not an unusual finding in patients on maintenance hemodialysis. Although the tumor recurred in one-third of the patients, only one died of hypercalcemia due to recurrent disease. The authors of the review concluded that no preoperative features distinguished hemodialysis patients with parathyroid carcin o m a from those with parathyroid hyperplasia and that the clinical course may be more benign because of the tendency for renal insufficiency to lower serum calcium levels.

Familial Hyperparathyroidism Carcinoma has been reported in association with familial hyperparathyroidism (9,51-56), particularly in the autosomal d o m i n a n t form with isolated hyperparathyroidism that is not part of the multiple endocrine neoplasia type 1 (57). In one such family, there was no evidence of antecedent hyperplasia in unaffected glands, and chromosomal abnormalities commonly observed in other solid tumors were identified (a reciprocal translocation between chromosomes 3 and 4, trisomy 7, and a pericentric inversion in chromosome 9) (55). Analyses of tumor DNA from one family m e m b e r with parathyroid carcinoma showed no evidence of ras gene mutations, PTH gene arrangement, or allelic loss from chromosome 11q13, the locus of the gene for multiple endocrine neoplasia type 1. In addition, a greatly increased risk of parathyroid carcinoma is associated with the hereditary hyperparathyroidism-jaw tumor syndrome (52,56,57), recently localized to chromosome lq21-31 (58).

PATHOGENESIS Over the past decade, evidence has accumulated for the involvement of mutations of both protooncogenes and tumor suppressor genes in the development of parathyroid tumors. In this regard, a chromosomal inversion activating one copy of the PRAD1 (parathyroid a d e n o m a l ) gene by placing it next to the regulatory region of the PTH gene has been reported in 5% of parathyroid adenomas (59,60). Cell cycle regulators have also been implicated in the pathogenesis of parathyroid carcinoma. The retinoblastoma (Rb) gene is a tumor-suppressor gene on chromosome 1 lq13. A variety of h u m a n neoplasms are related to inactivation of the Rb gene. In 1994, Cryns et al. studied nine parathyroid cancers for evidence of loss of Rb gene DNA and for altered expression of Rb protein. The cancers were compared to 21 parathyroid adenomas (61). All 11 parathyroid cancers lacked an Rb allele and most had complete absence of nuclear staining for the Rb protein. In contrast, only one parathyroid adenoma lacked the allele and none had abnormal staining for the Rb protein. Subramaniam et al. used a mouse monoclonal antibody to detect Rb gene expression immunohistochemically in three parathyroid carcinomas and 11 benign adenomas. Evidence of Rb gene inactivation was observed in two of the three cancers and only one of 11 adenomas (62). Pearce et al. observed allelic deletions of the 13q12-14 region, involving both the Rb gene and the hereditary breast cancer susceptibility gene in 3 of 19 parathyroid adenomas, all of which had aggressive clinical or histopathologic features, and in 1 of 1 parathyroid cancers. Yoshimoto and colleagues demonstrated allelic deletions on chromosome 13q in parathyroid adenomas from two members of a family with isolated primary hyperparathyroidism, one who also had parathyroid cancer and one with only an adenoma (9). Allelic losses of Rb or D13S71 at 13q14 in a parathyroid cancer were also reported by Dotzenrath (63). In addition, Cryns and colleagues have found evidence for involvement of another cell cycle regulator, the p53 tumor suppressor gene (31). There was both p53 allelic loss and abnormal p53 protein expression in some cases of parathyroid cancer, suggesting a role for p53 in the pathogenesis of some of these tumors. Both wild-type Rb and p53 proteins halt progression through the cell cycle. Abnormal function at this point related to mutations in either gene could therefore predispose to the clonal development of parathyroid carcinoma.

PARATHYROIDCARCINOMA / CLINICAL FEATURES The clinical features of parathyroid carcinoma (1-3,11,12,14,15,17,26-31,34) are due primarily to the effects of excessive secretion of parathyroid hormone by the functioning tumor, rather than to infiltration of vital organs by tumor mass. Thus, signs and symptoms of hypercalcemia often dominate the clinical picture with contributions from typical hyperparathyroid bone disease and features of renal involvement, such as nephrolithiasis or nephrocalcinosis. The challenge to the clinician rests on differentiating between hyperparathyroidism due to parathyroid carcinoma and that due to its much more common benign counterpart. It is of great importance that parathyroid carcinoma be considered in the differential diagnosis of parathyroid hormone-dependent hypercalcemia, because the morbidity and mortality associated with this diagnosis are substantial and optimal outcomes are associated with complete resection of the tumor at the time of the initial operation (1-3,11,12,14,15,17,26-31,34,64). All too often the diagnosis of parathyroid carcinoma is made in retrospect when hypercalcemia recurs due to local spread of tumor or distant metastases. Several presenting features of a patient with primary hyperparathyroidism, when present, should suggest a malignant rather than a benign etiology. There is no association of gender with parathyroid carcinoma. The ratio of affected women to men is 1:1 in most series, compared to primary hyperparathyroidism, where there is a marked female predominance (3-4:1). Most investigators have noted the average age of the patient with parathyroid carcinoma to be in the fifth decade, approximately 10 years younger than typical patients with primary hyperparathyroidism, who most often present in their riffles or sixties. In contrast, a review of the Mayo Clinic experience (30) and that of the National Cancer Data Base indicated that the average age of their patients was somewhat greater (26), in the middle fifties. In any case, considerations of gender and age are of little help in evaluating the individual patient. Since the advent of the multichannel autoanalyzer in the late 1960s, the clinical profile of primary hyperparathyroidism due to benign adenomatous or hyperplastic disease has changed. Today, primary hyperparathyroidism usually presents with mild hypercalcemia (within 1 m g / d l above the upper limit of normal) that is frequently asymptomatic and often discovered during a routine evaluation or during the investigation of an unrelated complaint (4-6). In contrast, the serum calcium level of most patients with parathyroid carcinoma is much higher, generally above 14 mg/dl, or 3-4 m g / d l above the upper limit of

517

normal (1-3,11,12,14,15,17,26-31,34). Moreover, this more severe hypercalcemia is almost invariably associated with the typical signs and symptoms of hypercalcemia. The most frequent complaints are fatigue, weakness, weight loss, anorexia, nausea, vomiting, polyuria, and polydipsia. Other common presenting symptoms, characteristic of a severely hyperparathyroid state, include bone pain, fractures, and renal colic. When reported, parathyroid hormone levels have ranged from 3 to 10 times above the upper limit of normal for the assay employed. Extremely high levels of parathyroid h o r m o n e are unusual in primary hyperparathyroidism, for which circulating concentrations are commonly less than twice normal. Alkaline phosphatase is also higher in patients with parathyroid carcinoma than in those with primary hyperparathyroidism, for which levels are generally in the vicinity of the upper limit of the normal range (7). Patients with parathyroid carcinoma may have elevated levels of ot and [3 subunits of human chorionic gonadotrophin whereas patients with primary hyperparathyroidism do not (65). A palpable neck mass has been reported in from 30 to 76% of patients with parathyroid carcinoma. This important clinical finding constitutes another striking difference between benign and malignant parathyroid disease, because a palpable neck mass is distinctly unusual in primary hyperparathyroidism (66). In addition, recurrent laryngeal nerve palsy in a patient with primary hyperparathyroidism who has not had previous neck surgery is also very suggestive of parathyroid cancer. The classic target organs of parathyroid hormone, kidney and skeleton, are affected with greater frequency and severity in parathyroid carcinoma (1,2,11,12,27,30,32,33) than is commonly observed in the modern presentation of benign primary hyperparathyroidism. Most recent series of primary hyperparathyroidism report the prevalence of renal involvement, including nephrolithiasis, nephrocalcinosis, and impaired glomerular filtration, to be less than 20% (4,7). In contrast, renal colic is a frequent presenting complaint of parathyroid carcinoma. The prevalence of nephrolithiasis was 56% and the prevalence of renal insufficiency was 84% in one recent series (30). These figures are somewhat higher than previous reports in which the prevalence of renal involvement generally has ranged from 32 to 60%. Bone pain and pathologic fractures are also common features of parathyroid cancer. Overt radiologic signs of hyperparathyroid skeletal disease, such as osteitis fibrosa cystica, subperiosteal bone resorption, "salt and pepper" skull, and absent lamina dura, as well as less specific signs such as diffuse spinal osteopenia, are

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commonly seen in parathyroid carcinoma (44-91%). In contrast, patients with benign primary hyperparathyroidism rarely have skeletal complaints and specific radiologic signs are found in less than 5% (4,5,7). It is also important to note the high incidence of concomitant bone and stone disease that occurs in parathyroid cancer, whereas simultaneous renal and overt skeletal involvement is distinctly unusual in primary hyperparathyroidism. In addition to the kidneys and the skeleton, other organs are frequently affected. Recurrent severe pancreatitis, peptic ulcer disease, and anemia occur with greater frequency in patients with malignant disease than in those with benign primary hyperparathyroidism. Parathyroid carcinoma shares many clinical features with acute primary hyperparathyroidism (parathyroid crisis; see Chapter 34). In view of the marked elevations of serum calcium and parathyroid h o r m o n e that are c o m m o n in parathyroid crisis, the diagnosis of parathyroid cancer should be considered. T h o u g h the distinction between these two entities is not possible preoperatively, it is important to bear the diagnosis in mind because the surgical approach differs. A summary of features that might lead one to suspect parathyroid cancer in a patient with hypercalcemia and elevated parathyroid h o r m o n e levels is shown in Table 1. It should be noted however, that some patients with benign primary hyperparathyroidism present with more severe disease than is commonly seen today. In such patients, the distinction between benign and malignant disease may be even more difficult on clinical grounds because profound hypercalcemia, renal disease, and osteitis fibrosa or diffuse osteoporosis may occur and even concomitant kidney and bone disease

TABLE 1

may be present (64). However, it is preferable to have a high index of suspicion for parathyroid carcinoma when these features are present than to miss the opportunity for surgical cure by failing to consider it in the differential diagnosis.

PATHOLOGY Several operative findings have been described; when present, these help to distinguish benign parathyroid adenomas from parathyroid carcinoma. The typical parathyroid a d e n o m a is usually of soft consistency, r o u n d or oval in shape, and of a reddish-brown color. In contrast, parathyroid carcinoma is frequently described as a lobulated, firm to stony-hard mass. In about 50% of cases it is surrounded by a dense fibrous greyish-white capsule that adheres tenaciously to adjacent tissues and makes the tumor difficult to separate from contiguous structures. If there is gross infiltration of adjacent thyroid, nerve, muscle, or esophagus or obvious cervical node metastases, the diagnosis of carcinoma is not difficult. However, any one or all of these operative findings may be absent and the examination of frozen sections is of little value in distinguishing benign from malignant disease. As is the case with many endocrine neoplasms, the histopathologic distinction between benign and malignant parathyroid tumors is difficult. In 1973 Shantz and Castleman, based on an analysis of 70 cases of parathyroid carcinoma, established a set of criteria for the pathologic diagnosis of this malignancy (28). These histologic features are (1) uniform sheets of (usually chief) cells arranged in a lobular pattern separated by

Parathyroid Carcinoma and Benign Primary Hyperparathyroidism" Typical Features

Feature

Parathyroid carcinoma

Female:male ratio Average age (years) Asymptomatic Serum calcium

1:1 48 14 mg/dl

Parathyroid hormone Palpable neck mass Renal involvement a Skeletal involvement b Concomitant renal and skeletal disease

Markedly elevated Common 32-80% 34-91% Common

Primary hyperparathyroidism 3.5:1 55 >80% _< 1 mg/dl above upper limit of normal Mildly elevated Rare 4-18% 20 mg/dl. Serum PTH values measured in several cases were found to be high, although not always as elevated as might be expected. Affected infants also have shown slightly elevated or high-normal serum magnesium concentrations and low urinary calcium excretions. Clinical signs include lethargy, muscular hypotonia, and severe skeletal deformities, including a narrow thorax, spontaneous rib fractures, bowed long bones, craniotabes, and radiographic osteopenia. As often occurs when hyperparathyroidism develops while the epiphyses are open, the radiographic changes of rickets are found. Subperiosteal resorption of bone is observed, and histologic examination of bone may show typical osteitis fibrosa cystica (29). As with rickets, morbidity often derives from respiratory complications of the thoracic deformity. The syndrome is often fatal, especially if parathyroid surgery is delayed in the face of severe muscular weakness or increasing serum calcium values.

Etiology The earliest reports found that the syndrome was associated with parathyroid hyperplasia but not adenoma (30). Marx et al. (29) recognized that many of these patients were members of families affected with FHH. In one of their cases, both parents were affected members of separate FHH kindreds, suggesting that homozygosity for FHH could be a cause of the syndrome. Cooper et al. (31) reported another patient who was apparently homozygous for FHH because of parental consanguinity and demonstrated that the infant's parathyroid glands in vitro showed poor suppresibility by calcium. In other cases, however, only one parent has had FHH, suggesting that another genetic (presumably recessive) or environmental factor must be acting synergistically with the FHH trait to generate the severe degree of hyperparathyroidism. Such factor(s) remain unidentified but must be operative in only a small percentage of FHH births. In some reported cases, including three of the four patients reported by Marx et al. (29), the father was the affected parent, the mother being normocalcemic. Here the usual activity of the placental calcium pump would place the fetal serum calcium concentration below the presumably higher fetal parathyroid set point and produce parathyroid hyperplasia. In this model, however, the hyperplasia and the bone disease that had developed in utero should resolve gradually after birth, leaving the infant with only the usual features of FHH. This situation is analogous to hyperparathyroidism developing in

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the normal fetus of a hypocalcemic mother. Marx et al. (29) found no evidence of excess fetal wastage in FHH pregnancies of normocalcemic mothers, suggesting that this mechanism alone is not the cause of the severe neonatal hyperparathyroid syndrome. How then would one explain the occasional case of severe neonatal hyperparathyroidism in this situation? One might postulate that another factor prevented the placental calcium p u m p from increasing the calcium gradient sufficiently. Studies of placental weight and histology would be of interest but may not be technically feasible, because the syndrome may be recognized hours to days after birth. Another hypothesis would be that some other factor, such as a subclinical form of F H H in the mother, had shifted the fetal parathyroid set point even further from normal. Detailed study of the calcium inhibitory set point of parathyroid glands from severely affected, but apparently heterozygous, neonates and dynamic in vitro studies of maternal parathyroid function would be of great interest. Other causes of severe neonatal hyperparathyroidism appear to be unrelated to FHH. Several reported cases seemed to show recessive transmission or represented the sporadic occurrence of a similar neonatal syndrome (29).

Treatment In neonates with severe hypercalcemia and marked skeletal deformities, aggressive respiratory support and treatment can be lifesaving, because the skeletal deformity may eventually resolve. Parathyroidectomy can be lifesaving in the most severe cases and should not be delayed (32). Repeated parathyroid operations have sometimes been required because of early recurrence of severe hypercalcemia after subtotal parathyroidectomy. Thus, some authors have advocated deliberate total parathyroidectomy (29,31), with the understanding that lifelong treatment of hypoparathyroidism would then be needed. A possible alternative approach would be total parathyroidectomy with parathyroid autogenous grafting. This seemed satisfactory in one case, but only 6 weeks of postoperative follow-up was available, and later graft-dependent recurrence was a concern (31 ). The presence of skeletal hyperparathyroid changes in F H H offspring is itself not an indication for parathyroid exploration, unless accompanied by severe hypercalcemia. At least two patients with FHH-associated neonatal hyperparathyroidism with significant skeletal changes but mild hypercalcemia (11.2 and 11.4 m g / d l ) have been reported to survive and their skeletal deformities to heal without parathyroid surgery (33,34). These patients, however, clearly did not have the full syndrome of severe neonatal primary hyperparathyroidism.

Histology Histologic examination of the parathyroid glands in severe neonatal hyperparathyroidism usually shows chief cell hyperplasia (29), but one patient had water clear cell hyperplasia of four glands (35).

PRIMARY HYPERPARATHYROIDISM IN FAMILIAL SYNDROMES OF RECESSIVE OR UNCERTAIN INHERITANCE Recessive Isolated Familial Parathyroid Adenomas or Hyperplasia Adenomas

Apparently recessive familial parathyroid adenomas were reported in each of three offspring of a nonconsanguineous marriage. There was no other evidence of endocrine tumors and no hypercalcemia or hyperparathyroidism in other members of a large extended family (36). Hyperplasias

Several reports have described recessive syndromes of isolated primary parathyroid hyperplasia with other associations not including endocrine tumors. A familial syndrome of primary parathyroid hyperplasia with nephropathy and neural deafness was reported in five of six children born to a marriage between first cousins (37). Three of the five had primary parathyroid hyperplasia, three had renal failure, and all five had sensorineural deafness. Female patients were equally as severely affected as males, and the absence of hematuria distinguished the syndrome from Alport's syndrome, which is usually an X-linked illness. The hyperparathyroidism was of early onset, being diagnosed at ages 9, 18, and 22 years. A familial syndrome of primary parathyroid hyperplasia with a tendency for intrathyroidal location of one or more parathyroid glands has been reported (38). A mother and two of her six children were affected. The age of onset was early in the two sons, at "~22 years of age, and was uncertain in the mother, who presented with staghorn calculi at age 49 years. In two of the three affected patients, one of the hyperplastic parathyroid glands was located within the substance of the thyroid. A literature review of large series of hyperparathyroidism in which location of the abnormal gland (s) was specified suggested that an intrathyroidal location was more c o m m o n in familial (10.4%) than in sporadic cases (4.2%). The family was not large e n o u g h to allow the mode of inheritance to be determined or to exclude the possibility of an associated endocrinopathy.

FAMILIALPRIMARYHPT Primary parathyroid hyperplasia was associated with polyps and carcinoma of the colon in two brothers (39). The colon malignancy appeared 12 and 36 years before the hypercalcemia. A sister also had u n d e r g o n e resection of a colon carcinoma but refused calcium measurement. Both parents had expired of a malignancy (lung or breast) in the fifth or sixth decade. This association was probably more than a chance occurrence. Data from three large series found that 7% of 244 patients with primary hyperparathyroidism also had a history of colon cancer; matched controls had a 5% incidence of colon cancer, not a significant difference (39). Colonic polyposis is also associated with familial papillary thyroid cancer (40-42), but those families have not been found to have a high incidence of parathyroid hyperplasia. Mallette et al. (43) reported a family a m o n g whom parathyroid hyperplasia occurred in three siblings, without evidence of other endocrine tumors, with one of the siblings also having a parathyroid carcinoma. A pancreatic carcinoma, visualized in the m o t h e r at laparotomy but not biopsied, had followed a rapid clinical course, making it unlikely to have been of islet cell origin. The parathyroid carcinoma presented as a hard nodule fixed to the thyroid, with severe hypercalcemia (14.7 m g / d l ) . In addition to thyroid invasion, it showed fibrosis, a trabecular growth pattern, and venous invasion. Persistent hypercalcemia, which worsened progressively, led to three further operations at which, respectively, were resected (1) a hyperplastic right inferior parathyroid gland, (2) a hyperplastic right superior parathyroid gland displaced to the posterior superior mediastinum, and (3) a large mass of nodules adherent to the trachea at the site of the initial thyroid resection plus multiple nodules studded along the entire left paratracheal area from u p p e r thyroid cornu to u p p e r thymic pole. These nodules microscopically contained numerous mitoses but more importantly had invaded muscle, confirming that this was a local recurrence of a carcinoma rather than parathyromatosis. A similar syndrome of parathyroid hyperplasia and carcinoma has been reported in at least two other families, each with two affected siblings (44,45). The parathyroid carcinoma did not metastasize in any of the five patients, the diagnosis of carcinoma being based on local recurrence of tumor or histologic findings or both. Only one of the five parathyroid carcin o m a patients in these three families died during the reported follow-up period. He succumbed to severe hypercalcemia hyperparathyroidism, the cause of which was presumed to be further recurrence of carcinoma, but neither left-side parathyroid gland was ever identified. The inheritance of this syndrome is uncertain. Two families could not be screened adequately, but in one family both parents and two other siblings

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were normocalcemic (44). Thus a d o m i n a n t trait has not been excluded. The syndrome is rare e n o u g h that it may take many years to track down its genetic basis. It is possible that one or more of these families had a variant of the cystic parathyroid adenomatosisfibro6sseous jaw tumor syndrome, which may also be associated with parathyroid carcinoma (see above). Two of the four patients examined via mandibular radiographs had mandibular tumors (43,44), although biopsy of one lesion showed increased osteoclastic activity that excluded an ossifying fibroma. There was at least one recurrence of hypercalcemia in each patient at a time that would have been compatible either with appearance of a metachronous adenoma or with progression of hyperplasia in a previously minimally affected gland. The published histologic studies of the carcinomas showed mild cystic changes in only one of the five cases (41), but the cysts in the original a d e n o m a might have been lost after a carcinoma developing in situ had spread t h r o u g h o u t the gland. In the future, we should carefully examine all parathyroid tissue for cystic change in such families. Once such a family has been identified, the surgical treatment of this syndrome should probably employ the same approach used for the cystic adenomatosis jaw tumor syndrome. Because any enlarged parathyroid gland might represent or harbor a carcinoma, biopsy of enlarged parathyroids should be avoided and enlarged parathyroid glands handled with extreme caution. Bilateral inferior parathyroidectomy should probably be routine. Autogenous parathyroid grafting of the most normal-appearing superior parathyroid gland might be considered in preference to subtotal parathyroidectomy, even with the increased chance of later malignant change. It is not clear whether metastasis would be more likely to occur from a gland fragment left at the original site in the neck or from a transplant in the forearm, but the forearm site would be more accessible should the parathyroid r e m n a n t develop aggressive behavior.

REFERENCES 1. Mallette LE. Hyperparathyroidism: The spectrum of parathyroid tumors in primary and secondary hyperparathyroidism. In: Mazzaferri EL, Samaan NA, eds. Endocrine tumors. Boston: Blackwell Scientific, in press. 2. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. A n n Intern Med 1987;107:54-60. 3. Jackson CE. Hereditary hyperparathyroidism associated with recurrent pancreatitis. A n n Intern Med 1958;49:829-836. 4. Jackson CE, Boonstra CE. The relationship of hereditary hyperparathyroidism to endocrine adenomatosis. A m J M e d 1967;43:727-734. 5. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990; 108:1006-1013.

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5a. Hobbs MR, Ole AR, Pidwirny GN, Rosen IB, Zarbo RJ, et al. Hyperparathyroidism-jaw tumor syndrome: The HRPT2 locus is within a 0.7 cm region on chromosome lq. Am J Hum Genet 1999;64:519-525. 6. Kennett S, Pollick H. Jaw lesions in familial hyperparathyroidism. Oral Surg 1971;31:502-510. 7. Dinnen JS, Greenwood RH, Jones JH, Walker DA, Williams ED. Parathyroid carcinoma in familial hyperparathyroidism. J Clin Patho11977;30:966-975. 8. Rosen IB, Palmer JA. Fibro6sseous tumors of the facial skeleton in association with primary hyperparathyroidism: An endocrine syndrome or coincidence? A m J S u r g 1981;142:494-498. 9. Warnakulasuriya S, Markwell BD, Williams DM. Familial hyperparathyroidism associated with cementifying fibromas of the jaws in two siblings. Oral Surg 1985;59:269-274. 10. Mallette LE, Malini S, Rappaport MR Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987; 107:54-60. 11. Flye MW, Brennan ME. Surgical resection of metastatic parathyroid carcinoma. Ann Surg 1981;193:425-435. 12. Cohn K, Silverman M, Corrado J, Sedgewick C. Parathyroid carcinoma: The Lahey Clinic experience. Surgery 1985;98:1095-1100. 13. Hobbs MR, Ole AR, Pidwirny GN, Rosen IB, Zarbo RJ, Coon H, Heath III H, Leppert M, Jackson CE. Hyperparathyroidism-jaw tumor syndrome: The HRPT2 locus is within a 0.7 cM region on chromosome lq. A m J H u m Genet 1999;64:519-525. 14. Wassif WS, Farnebo E The BT, Moniz CE Li FY, Harrison JD, Peters TJ, Larsson C, Harris P. Genetic studies of a family with hereditary hyperparathyroidism-jaw tumour syndrome. Clin Endocrinol 1999; 50:191-196. 14a.Haven CJ, Wong FK, van Dam EWCM, van der Luijt R, van Asperen C, Jansen J, Rosenberg C, de Wit M, Roijers J, Hoppener J, Lips CJ, Larsson C, The BT, Morreau H. A genotypic and histopathological study of a large Dutch kindred with hyperparathyroidism-jaw tumor syndrome. J Clin Endocrinol Metab 2000;85:1449-1454. 15. Grevsten S, Grimelius L, Thordn L. Familial hyperparathyroidism. UppsalaJMed Sci 1974;79:109-115. 16. Sandler LM, Moncrieff MW. Familial hyperparathyroidism. Arch Dis Child 1980;55:146-147. 17. Mlo M, Thompson NW. Familial hyperparathyroidism caused by solitary adenomas. Surgery 1982;92:486-490. 18. Jung RT, Davie M, Grant AM, Jenkins D, Chalmers TM. Multiple endocrine adenomatosis (type 1) and familial hyperparathyroidism. Postgrad MedJ 1978;54:92-94. 19. Marx SJ, Attie ME Levine MA, Spiegel AM, Downs RW, Lasker RD. The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine 1981 ;60:397-412. 20. Streeten EA, Weinstein LS, Norton JA, et al. Studies in a kindred with parathyroid carcinoma. J Clin Endocrinol Metab 1992;75: 362-366. 21. Marx sJ. Familial hypocalciuric hypercalcemia. N Engl J Med 1980;303:810-811. 22. Heath III H. Familial benign (hypocalciuric) hypercalcemia. Endocrinol Metab Clin North Am 1989; 18:723-740. 23. Marx SJ, Spiegel AM, Brown EM, et al. Circulating parathyroid hormone activity: Familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. J Clin Endocrinol Metab 1978;47:1190-1197. 24. Law WM,Jr, James EM, CharboneauJW, Purnell DC, Heath III H. High-resolution parathyroid ultrasonography in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). Mayo Clin Proc 1984;59:155-159. 25. Gilbert F, D'Amour P, Gascon-Barns M, et al. Familial hypocalciuric hypercalcemia: Description of a new kindred with emphasis

26.

27.

28. 29.

30.

31.

32.

33.

34. 35.

36.

37.

38.

39. 40.

41.

42.

43.

44. 45.

on its difference from primary hyperparathyroidism. Clin Invest Med 1985;8:78-84. McMurtry CT, Schranck FW, Walkenhorst DA, et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. A m J M e d 1992;93:247-258. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 1993;366:575-580. Brown EM, Pollak M, Seidman CE, et al. Calcium-ion-sensing cellsurface receptors. N EnglJ Med 1995;333:234-239. Marx SJ, Attie ME Spiegel AM, Levine MA, Lasker RD, Fox M. An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N EnglJ Med 1982;306:257-264. Mihlethaler JR Scharer K. Antener 1. Akuter hyperparathyreoidismus bei primarer nebenschilddriisenhyperplasie. Helv Pediatr Acta 1967;22:529-557. Cooper L, Wertheimer J, Levey R, et al. Severe primary hyperparathyroidism in a neonate with two hypercalcemic parents: Management with parathyroidectomy and heterotopic autotransplantation. Pediatrics 1986;78:263-268. Marx SJ, Spiegel AM, Levine MA, et al. Familial hypocalciuric hypercalcemia: The relation to primary parathyroid hyperplasia. N EnglJ Med 1982;307:416-426. Eftekhari E Yousefzadeh DK. Primary infantile hyperparathyroidism: Clinical, laboratory, and radiographic features in 21 cases. Skel Radiol 1982;8:201-208. Page LA, HaddowJE. Self-limited neonatal hyperparathyroidism in familial hypocalciuric hypercalcemia. J Pediatr 1987;111:261-264. Steinmann B, Gnehm HE, Rao VH, Kind HE Prader A. Neonatal severe primary hyperparathyroidism and alkaptonuria in a boy born to related parents with familial hypocalciuric hypercalcemia. Helv Pediatr Acta 1984;39:171-186. Law WM,Jr, Hodgson SE Heath III H. Autosomal recessive inheritance of familial hyperparathyroidism. N Engl J Med 1983;309: 650-652. Edwards BD, Patton MA, Dilly SA, Eastwood JB. A new syndrome of autosomal recessive nephropathy, deafness, and hyperparathyroidism. J Med Genet 1989;26:289-293. Colon-Zorba GE, Aguilo E Jr, Vazquez-Quintana E. A syndrome of familial intrathyroidal primary parathyroid hyperplasia: Case reports and critical review of literature. PR Health Sci J 1986;555-563. Feig DS, Gottesman IS. Familial hyperparathyroidism in association with colonic carcinoma. Cancer 1987;60:429-432. Delamarre J, Capron JP, Armand A, Dupas JL, Deschepper B, Davion T. Thyroid carcinoma in two sisters with familial polyposis of the colon. Case reports and review of the literature. J Clin Gastroenterol 1988;10:659-662. Herrera L, Carrel A, Rao U, Castillo N, Petrelli N. Familial adenomatous polyposis in association with thyroiditis. Report of two cases. Dis Colon Rectum 1989;32:893-896. Reed MW, Harris SC, Quayle AR, Talbot CH. The association between thyroid neoplasia and intestinal polyps. Ann R Coll Surg England 1990;72:357-359. Mallette LE, Bilezikian JP, Ketcham AS, Aurbach GD. Parathyroid carcinoma in familial hyperparathyroidism. Am J Med 1974;57:642-648. Frayha RA, Nassar VH, Dagher F, Salti IS. Familial parathyroid carcinoma. Geb MedJ 1972;25:299-309. Leborgne J, Neel L, Buzelin E Malvy P. Cancer familial des parathyroides. L'angiographie dans le diagnostic des recidives loco- regionales. Considerations a propos de deux cas. J Chir 1975;109:315-326.

CI-IAPXV 3 8

F a m i l •i a"l and

B e n i g•n

Neonatal

Hyp Severe

" " ocalclurlc

Hyp ercalcemla "

Hyperparathyroidism

GHADA EL-HAJJ FULEIHAN Calcium Metabolism and Osteoporosis Program, American University of Beirut Medical Centeg, Beirut 113-6044, Lebanon HUNTER HEATH III United States Medical Division, Eli Lilly and Company, Indianapolis, Indiana 46285

H I S T O R I C A L PERSPECTIVE AND NOMENCLATURE

glands did not normalize the serum calcium. Seventeen other members with hypercalcemia were discovered in the family, spanning three generations. The hypercalcemia seemed to be inherited in an autosomal dominant pattern. The authors reported the disorder as "Hereditary Hypercalcemia without Definite Hyperparathyroidism" and recognized it as an entity different from hyperparathyroidism: "The lack of firm evidence for hypersecretion of parathyroid h o r m o n e in any of the hypercalcemic members of this family suggests that this condition may be a new entity which certainly differs from that observed in the other 5 families with hyperparathyroidism . . . . " Other terms occasionally used to describe the syndrome include familial parathyroid hyperplasia and familial hypercalcemia. However, the terms familial benign hypercalcemia (FBH) and familial hypocalciuric hypercalcemia (FHH) predominate, being about equally used in the literature. In 1989, Heath coined the encompassing term familial benign hypocalciuric hypercalcemia (FBHH) that was subsequently used in several papers (4-6), and which was later suggested by Strewler as a unifying term containing the key features of the syndrome and as a means to end the confusing division in the literature describing this rare syndrome (7). In this chapter, we use the abbreviation FBHH. FBHH is distinct from other inherited hypercalcemic syndromes usually associated with primary hyperparathyroidism, such as multiple endocrine neoplasia (MEN) types 1 and 2A (8,9), isolated familial primary hyperparathyroidism (10,11) and the hyperparathyroidism-jaw tumor syndrome (12). Indeed,

In 1972, T.E Foley and colleagues described in their paper "Familial Benign Hypercalcemia" (FBH) the key features of the syndrome: asymptomatic hypercalcemia inherited in an autosomal dominant pattern, normal serum PTH levels in the presence of hypercalcemia, low or low normal urinary calcium excretion, normal or slightly elevated serum magnesium level, normal or slightly decreased phosphate level, and normal parathyroid gland pathology in the proband (1). The serum PTH level of the proband only decreased by 29% during an intravenous calcium infusion. The authors interpreted the abnormal pathophysiology of the disease as "an inappropriate requirement of an unusually high concentration of calcium to suppress the production of parathormone." A few years later, investigators at the National Institutes of Health reported the same syndrome as "familial hypocalciuric hypercalcemia" (FHH), in recognition of the unexpectedly low urinary excretion of calcium relative to the hypercalcemia of affected persons (2). However, Jackson and Boonstra had described the syndrome without labeling it as such in 1966, when they reported familial hypercalcemia in a kindred wherein the proband was discovered during a routine serum calcium screening survey (3). The patient had hypercalcemia (12.1 mg/dl, or 3.0 mmol/liter) and a hypernephroma. The serum calcium was not normalized by excision of the renal tumor. Neck exploration revealed four-gland hyperplasia, and removal of three and one-half parathyroid The Parathyroids, Second Edition

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FBHH is characterized by lack of cure after subtotal parathyroidectomy, and the absence of other associated endocrine neoplasias. A disorder closely related to FBHH, neonatal severe hyperparathyroidism (NSHPT), was first described by Landon three decades earlier (13). Philips first noted a case of NSHPT arising from a consanguinous marriage (14), and Hillman commented on the familial occurrence (15). Spiegel et al. first linked this entity to FBHH in 1977; an infant with NSHPT was later found to have a n u m b e r of relatives diagnosed with familial hypocalciuric hypercalcemia (16). Most subsequent publications have described the NSHPT syndrome as occurring in infants belonging to kindreds having FBHH (17-23). NSHPT is characterized by severe hypercalcemia, failure to thrive, respiratory distress, and skeletal anomalies. In some cases, total parathyroidectomy was effective in alleviating the symptoms. Years before the demonstration that mutations in the parathyroid calcium receptor gene can cause FBHH and NSHPT, investigators had proposed that NSHPT is the homozygous form of a genetic abnormality in calcium metabolism, whereas the heterozygous state presents only a mild disorder (17,23,24). However, the genetic basis of NSHPT is more complex than originally thought, as outlined in detail below (see Genetics of Neonatal Hyperparathyroidism, Including Neonatal Severe Hyperparathyroidism).

GENETICS OF FBHH Inspection of many pedigrees convincingly demonstrates that FBHH is inherited as an autosomal dominant condition (1-4,25-30) with equal sex distribution and over 90% penetrance. In two studies of five kindreds evaluating a total of 170 individuals, 42% were affected, 39% were unaffected, and in 19% the status was unclear (31,32). The biochemical abnormalities are present at birth in affected children. Reports of new mutations causing FBHH are extremely rare (33) and very difficult to verify in the absence of a family history and biochemical or genetic testing (see below). Because of the paucity of symptoms associated with this bland syndrome, isolated cases may often go undetected, making it less likely to detect new FBHH mutations. In contrast to FBHH, there have been three well-documented cases of NSHPT associated with de novo mutations (see below). An early report suggested an association of FBHH with h u m a n leukocyte antigen (HLA) haplotypes, but this was not validated in other studies (34-37). Several candidate genes involved in calcium metabolism have been examined with negative findings, including genes for the MEN-l, MEN-2, and parathyroid h o r m o n e (PTH) (31,36,38). An FBHH disease gene was first m a p p e d to chromosome 3 (band

q21-24) by Chou et al. (39) using linkage analysis in four large FBHH families (FBHH~q). Subsequent investigations validated these findings in over 90% of families suitable for genetic linkage analyses (32,40). Two additional genetically distinct forms of FBHH have been described in single families, one linked with markers on the short arm of chromosome 19 (FBHH19p) (32). Another FBHH kindred with atypical features (see clinical characteristics below) from Oklahoma was linked to chromosome 19q (FBHHoK) (41,42).

Molecular Basis of FBHHsq: Mutations in the Calcium Receptor At the same time as the linkage of the FBHH trait to chromosome 3, Brown et al. had also cloned the bovine parathyroid gland calcium-sensing receptor (CaSR) (43) and shortly thereafter Pollak et al. demonstrated that the h u m a n h o m o l o g was close to the site of linkage of FBHH (44). This novel receptor is present in many tissues, including intestine, lung, and various regions of the brain, but is most heavily expressed in the parathyroid glands, thyroid C cells, and kidneys (43). The CaSR shares sequence homology with the metabotropic glutamate receptors that are highly expressed in the central nervous system. The CaSR has three major domains: a large 612-amino acid (aa) extracellular amino-terminal region, a 250-aa domain with seven predicted membrane-spanning segments characteristic of the G protein-coupled receptor superfamily, and a 222aa intracytoplasmic sequence. Because of abnormal calcium sensing by the parathyroids and kidneys in FBHH, the newly cloned CaSR was obviously the next candidate gene to pursue. Pollak et al. used molecular probes for the bovine CaSR to demonstrate three distinct missense mutations in the CaSR gene in three separate FBHH families previously shown to have the FBHH trait linked to chromosomal locus 3q13.3-q21 (44). Different single nucleotide base substitutions were present in each kindred: Arg185Gln, GluZ97Lys, and ArgV95Trp. These sequence variations were not found in genomic DNA of 50 normocalcemic unaffected individuals. Several groups have since then identified dozens of point mutations (missense, nonsense) in the CaSR gene that cosegregate with the FBHH trait (40,45-48). Most of the mutations causing FBHH have been missense mutations occurring in three regions of the receptor sequence: (1) about two-thirds occur within the first 300 aa of the extracellular domain (Fig. 1); (2) proximal to the first transmembrane segment; and (3) within the transmembrane segments and intraor extracellular loops. The first point mutation located in the cytoplasmic tail of the receptor was described in a Swedish family with FBHH having some atypical features (see below, Clinical Characteristics of FBHH).

FBHH AND NSHPT /

SP

NH 2

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HS

Pro39Ala Ser53Pro Pro55Leu Arg62Met Arg66Cys Thz138Met GIT143GID

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~X~XX ~rz:2188er P:o2218~1: axg227Zdm ( ~ ) G1u297Lys C,j,s S 8 2 ~ : 8ez'GO7Stop Alu

Ser657Ti, r Gly67 O/ucg (Glu)

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¢) Conserved Q Cysteine

~c: N.glycosylation •

PKC site

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Pro748Arg ArG795Trp Val817Ile Thr876Alu

FIG. 1 Schematic representation of the proposed structure of the extracellular Ca 2+ calcium receptor cloned from human parathyroid gland. SP, Signal peptide; HS, hydrophobic substance. Also shown are 25 inactivating (missense and nonsense) mutations causing familial benign hypocalciuric hypercalcemia, and 14 activating mutations causing autosomal dominant hypocalcemia (ADH). Mutations are indicated using the three-letter amino acid code, with the normal amino acid indicated first and the FBHH or ADH mutation shown after the number of the relevant codon. Adapted from Bai M, Hebert S, Brown EM, with permission.

Whereas most mutations described have been missense ones, several additional types of mutations have been described. Pearce et al. described in one kindred a nonsense mutation just proximal to the transmembrane domain, producing a stop codon resulting in a truncated and presumably inactive receptor (40). The same group identified in another kindred a single base deletion and nucleotide transversion leading to premature termination of the receptor protein (40). Janicic et al. described in a Nova Scotian family the insertion of a repetitive Alu sequence (46), also predicting a truncated receptor. However, only about two-thirds of families demonstrated to have FBHH linked to chromosome 3 have demonstrable mutations in the coding region of the CaSR gene. In the remaining onethird, the syndrome is presumably due to mutations in introns, or upstream or downstream regulatory regions modulating gene function. Finally, apparently silent benign polymorphisms (without associated abnormalities in calcium metabolism) have been described in the

carboxy-terminal part of the receptor in up to one-third of 100 unaffected subjects (47). However, Cole et al. reported subtle changes in serum calcium in members with these "benign" polymorphisms (49). Perhaps normal h u m a n variation in serum calcium concentration results partly from variations in intrinsic calcium-sensing activity related to genetic polymorphisms (49). Some inactivating mutations of the CaSR have been expressed in appropriate cell lines, yielding insights into the mechanisms through which receptor mutations reduce CaSR activity (50). Reduced functional activity of the receptor could potentially be due to several factors: 1. Reduced affinity for the agonist calcium ion 2. Prevention of formation of the fully glycosylated, biologically active calcium receptor 3. Failure of receptor coupling with and activation of the respective G proteins 4. "Dominant negative" interactions of mutated with normal CaSR protein

610

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Whereas two out of three known missense mutations producing truncated CaSR result in an inactive protein, few missense mutations seem to result in full-length nonglycosylated receptor with marked reduction in biologic function. The majority of missense mutations examined have resulted in apparently normal size glycosylated receptors, which have modest reduction in ligand (calcium) binding affinity and receptor activity. One such mutation, RlS5Q, showed a prominent "dominant negative effect" on the coexpressed wild-type receptor, which may account for the unusually marked hypercalcemia in affected members of the FBHH family harboring that mutation (50). The exact mechanism for the negative dominant effect has not been elucidated, although it may be related to receptor dimerization. Electrophoretic evidence suggests the existence of nonglycosylated and glycosylated monomeric CaSR as well as a CaSR dimer formed through disulfide bonds (50,51). The addition of divalent and trivalent cations to solubilized CaSR shifts its electrophoretic mobility from a mainly monomeric to a dimeric form in a manner similar to their rank order of potency for CaSR activation (51). One mechanism for the dominant negative action may be the formation of heterodimers between the wild-type and mutant receptors, which may reduce the activity of the wild-type partner in the heterodimeric complexes, thereby reducing the total n u m b e r of normally functioning receptors on the cell surface.

In summary, the FBHH syndrome is genetically heterogeneous. In >90% of affected families the FBHH trait links to the region of chromosome 3 containing the CaSR gene. In two-thirds of chromosome 3-1inked families, there are discrete inactivating mutations of the CaSR gene that are almost certainly the cause of the hypercalcemia. The remainder may have CaSR gene mutations outside the coding region. Further understanding of the cation-CaSR structure-function relationship will allow insight into the clinical and biochemical heterogeneity of FBHH syndromes (see below).

Gene Knockout Models of FBHH

Key Characteristics

Ho et al. used targeted disruption of the CaSR gene to create mice that were heterozygous for inactivated CaSR (52). There was a 50% reduction of calcium receptor protein expression in the parathyroid gland and kidney of the heterozygous mice compared with the wild-type animals. These mice shared many phenotypic and biochemical features with h u m a n FBHH: they looked and behaved normally, and had normal fertility and life span. They had mild hypercalcemia (mean serum calcium, 10.4 mg/dl, or 2.6 mmol/liter), nonsuppressed serum PTH levels, higher serum magnesium levels compared to the wild-type mice, and reduced urinary calcium compared to the normal mice. In the mutant mice there was a mild (10%) elevation in the apparent set point for calcium-regulated PTH release, similar to what has been reported in FBHH families (see below). Their skeletal films were normal. Thus, at least three different genetic syndromes result from abnormalities in the CaSR. Inactivating mutations of the CaSR gene result in FBHH and NSHPT (see below), and activating mutations cause autosomal dominant hypocalcemia (53) (see Chapter 49).

FBHH is a rare hypercalcemic syndrome inherited in an autosomal dominant pattern, equally distributed between the sexes, the true prevalence of which is unknown. It is characterized by asymptomatic and usually uncomplicated lifelong hypercalcemia (1,2). The hypercalcemia is mild to moderate, usually 0.02 is helpful in excluding FBHH. Reprinted with permission from Heath III H. The familial benign hypocalciuric hypercalcemia syndromes. In: Raisz L, Rodan G, Bilezikian JP, eds. Principles of bone biology. New York: Academic Press, 1996, with permission.

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is not PTH dependent, consistent with an intrinsic abnormality of renal tubular calcium reabsorption. The avid tubular reabsorption of calcium is corrected with loop diuretics such as ethacrinic acid (80,84), pointing to the thick ascending limb as the site of the abnormality in calcium handling. Studies have confirmed expression of the CaSR in all segments of the nephron, with greatest expression of the transcripts in the cortical thick ascending limb (85). It is possible that the spectrum of abnormal renal calcium handling in FBHH may mirror the large variety of mutations; those in the cytoplasmic domain of the CaSR may be least likely to result in hypocalciuria (69).

FIG. 5 Calcium clearance as a function of plasma calcium in (A) patients with familial benign hypocalciuric hypercalcemia and patients with primary hyperparathyroidism under base line conditions (adapted from Stuckey et a/; Fasting calcium excretion and parathyroid hormone together distinguish familial hypocalciuric hypercalcemia from primary hyperparathyroidism. C/in Endocrino/1987;27:525, with permission from Blackwell Science Ltd.) and (B) during an infusion of calcium chloride in two patients with FBHH and three patients with hypoparathyroidism (adapted from Attie et a/. JCI 1983;72:667-676) in comparison to the calcium clearance curve derived from eight healthy men studied during a calcium-PTH infusion (adapted from EI-Hajj Fuleihan et a/, JCEM 1998;83:2366).

Urinary Magnesium FBHH is characterized by relative hypomagnesuria, with increased tubular reabsorption of magnesium (72,82). The CaSR affects both calcium and magnesium handling, with an inactivated receptor enhancing tubular reabsorption of both calcium and magnesium. Close correlation between urinary calcium and magnesium excretion has been noted in several studies of FBHH (72).

Renal Function and Concentrating Ability Renal function (creatinine clearance) decreases as expected with aging in FBHH, and is comparable to

normal (28,30). Marx et al. compared urinary concentrating ability after an 18- to 24-hour dehydration test in 10 patients with FBHH and 40 patients with PHPT having comparable serum calcium levels and renal function. In contrast to patients with PHPT, subjects with FBHH had no obvious impairment in urinary concentrating ability (86). Other Calciotropic Hormones The vitamin D metabolites 25-hydroxyvitamin D [25 (OH) D ] and 1,25-dihydroxyvitamin D [ 1,25 (OH) 2D ] have been measured in several studies and found to be

FBHH AND NSHPT / TABLE 1

Characteristics of Patients with Familial Benign Hypocalciuric Hypercalcemia and Primary Hyperparathyroidism

Variable Age of onset Symptoms Serum levels Calcium Magnesium Phosphorus Intact PTH 1,25(OH)2D Calcitonin Urinary excretion Cyclic AMP Calcium Ca/Cr clearance Magnesium

615

FBHH

HPT

At birth Usually none

Usually >40 years Asymptomatic in 80%; cortical bone loss; urolithiasis 80%

Normal to increased Normal to low Usually 0.02 Normal to elevated

normal in patients with FBHH (27,87-91). This is in sharp contrast to patients with PHPT, wherein serum 1,25(OH)zD is elevated in 40-60% of patients (92). In parallel to elevated calcitriol levels in primary hyperparathyroidism is enhanced intestinal calcium absorption, whereas calcium absorption is normal in FBHH (27,28,89). Thyroid C cells normally express the CaSR and increase calcitonin secretion in response to elevations in serum calcium. The inappropriately normal serum calcitonin concentrations in patients with FBHH might therefore reflect abnormal calcium sensing (27,28,77) and a shift to the right in the calcium-calcitonin curve. However, basal plasma calcitonin levels are also normal in chronic primary hyperparathyroidism, and calcitonin responses to secretagogues are normal in FBHH (77). Table 1 summarizes the major characteristics of FBHH and PHPT.

Dynamic Studies of PTH Regulation Steady-state serum calcium and PTH levels in patients with FBHH are consistent with abnormal calcium sensing by the parathyroid glands, because the "normal" PTH levels are inappropriate in view of the hypercalcemia. Total parathyroidectomy yields hypocalcemia in FBHH, further evidence for the central role of the parathyroid glands in the hypercalcemia of FBHH. In their original description of the syndrome, Foley et al. suggested that "the primary defect is an abnormally high parathyroid gland reference input value for extracellular fluid calcium ion concentration (1). The responsiveness of the parathyroid glands to changes in

Normal to elevated Normal

extracellular ionized calcium is preserved in FBHH, although somewhat altered. In the limited number of subjects studied, EDTA-induced hypocalcemia resulted in PTH increments that were similar in FBHH patients and healthy controls (93,94), and calcium infusions suppressed PTH variably (1,27,77,94,95). The regression curve relating PTH to calcium in these infusion studies was shifted to the right in FBHH compared to control subjects, suggesting an altered set point (94,95). However, the definition of a set point depends on the determination of C a / P T H curve derived over a spectrum of calcium values, for example, derived from consecutive EDTA and calcium infusions in the same patient, which was not done in previous studies. Our group is systematically evaluating PTH dynamics in FBHH patients with such a protocol, and there is the predicted shift to the right in the C a / P T H (Fig. 6), reflecting partial resistance of the parathyroid gland to extracellular calcium.

Parathyroid Glands in FBHH There is some controversy as to the histologic findings in the parathyroid glands of FBHH patients. Most case and single-family reports have described grossly and histologically unremarkable parathyroid glands (1,3,60,79), findings that were corroborated in larger studies (27-29). With high-resolution parathyroid ultrasonography, there is uniform absence of parathyroid gland enlargement in patients with FBHH, in contrast to patients with PHPT, whose gland enlargement is generally detectable by this technique (96). However, systematic histologic examination of FBHH parathyroid

616

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CHAPTER38 • Normal(N=24) • FBHH pt KV

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FIG. 6 Inverse sigmoid curve in response to consecutive citrate and calcium infusions in patient KV with FBHH (Haden ST, Brown EM, EI-Hajj Fuleihan G, unpublished observation, 1997) in comparison to the Ca/PTH curve derived from 48 healthy subjects (adapted from Haden ST, et aL, Clin Endocrinol 2000;52:329-338). There is a clear abnormality of PTH dynamics in the patient with FBHH compared to normal controls: a shift in the Ca/PTH curve to the right, increased PTH levels in response to hypocalcemia, and decreased suppression in response to hypercalcemia. The set point, the calcium concentration at which there was 50% suppression in PTH levels, was 4.89 mg/dl in the curve derived from 24 young healthy controls and 5.65 mg/dl in the patient with FBHH.

glands has revealed some deviation from normal (97,98). Twenty-eight glands from 23 patients were compared with 82 glands from 47 control patients; whereas the majority of FBHH glands were within extreme normal limits for weight, 15-20% exceeded normal size (97). However, the parenchymal area in FBHH glands was slightly but significantly less than normal, 30 _+ 3% versus 21 + 2% percent, respectively, and the percent fat was higher in FBHH glands (97). Those authors concluded that parathyroid hyperplasia is not a feature of FBHH. In contrast, another study reported that variable degrees of parathyroid hyperplasia were c o m m o n in FBHH (98). In 55 parathyroid glands from 18 patients with FBHH, the average parathyroid parenchymal area was three times that in normal subjects and 13 of

18 patients had one enlarged gland or more (98). The reason for the discrepancies in the histologic findings of the two large studies above is unclear (97,98), but may relate to the different populations studied, with different inactivating mutations in the CaSR having divergent effects parathyroid cell cycle and proliferation. Specifically, alterations in the cytoplasmic tail of the CaSR may promote parathyroid cell proliferation (see earlier, Phenotype-GenotypeAssociations).

DIAGNOSIS AND MANAGEMENT OF FBHH Diagnosis The genetic and molecular bases of FBHH are now clear in most cases. However, the diagnosis of FBHH must still be based on clinical judgment. Genetic linkage studies are impractical because of cost and the necessity for family sampling. It is also impractical to consider specific molecular diagnosis, for several reasons. The n u m b e r of known and suspected CaSR mutations is large, and technology to screen for them is not available; at least a third of FBHH3q kindreds do not have detectable mutations in the coding region of the CaSR gene; and a given family might have one of the rare chromosome 19 variants of FBHH. The diagnosis of FBHH can be straightforward in an asymptomatic hypercalcemic patient with a family history of hypercalcemia, a personal or family history of failed neck exploration, normal serum PTH, and low urinary calcium excretion (1.5 g / d a y with calcium carbonate, calcium citrate, or calcium lactate (114). However, other evidence implicates the dosage of calcium carbonate with the presence of vascular calcifications ( Vide infra) (139,140).

Phosphate-BindingAgents Because dietary phosphate restriction alone cannot control the hyperphosphatemia that exists in almost all patients undergoing hemodialysis, the intake of phosphate-binding agents is required in 90-95% of dialysis patients. The aluminum-containing gels, aluminum hydroxide and aluminum carbonate, were used in the past to reduce phosphate absorption and to control the serum phosphorus levels in patients with far advanced renal failure and in those treated with dialysis (115). However, it was recognized that the ingestion of aluminum-containing gels is a major risk factor for the development of aluminum intoxication, particularly that causing osteomalacia and other symptomatic "lowturnover" disorders of bone (116-121). Kaehny et al. (122) and Recker et al. (123) found that small amounts of aluminum were absorbed and excreted in the urine following the ingestion of large doses of aluminumcontaining gels by normal men. When aluminum is absorbed by patients with renal failure, it cannot be excreted, and it accumulates in the body. Thus both dialysis encephalopathy and aluminum-related bone disease have been reported prior to the initiation of dialysis in azotemic adults and children who were ingesting aluminum gels (116-121). It was shown that plasma aluminum levels correlated with the amount of oral aluminum intake from the

646

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CHAPTER40

phosphate-binding agents (121). Such observations implied a role of aluminum absorption from the gastrointestinal tract as a source of aluminum loading and toxicity. The observations that plasma aluminum levels fall strikingly (84,112) and that aluminum-related bone disease will improve or reverse after the total withdrawal of aluminum gels (84) provide proof that the oral intake of aluminum gels can be responsible for aluminum intoxication. Guidelines for "safe" doses of aluminum hydroxide were proposed, with safe maximum doses considered to be 30 m g / k g / d a y for children (124) and 4-6 tablets/day of aluminum hydroxide for adult patients treated with hemodialysis (125). However, when this r e c o m m e n d e d dose was prospectively evaluated in pediatric patients undergoing peritoneal dialysis, there was a progressive increase in the body burden of aluminum as judged by increments in plasma aluminum levels, increases in plasma aluminum levels after a desferrioxamine infusion test, and by histologic evidence of aluminum deposition in bone (126). Thus, aluminum-containing drugs should be avoided in the vast majority of patients with renal failure. In some dialysis patients, if aluminum gels are needed to control hyperphosphatemia, the gels can be combined with the calcium salts, with the "recomm e n d e d dosages" of aluminum gels not exceeded in such cases, and should be given for short period of time, i.e., few months. In patients receiving aluminum gels, there should be caution concerning the intake of medications that can augment aluminum absorption. Among the factors that enhance aluminum absorption, the most potent is citrate, as citric acid or a salt (127). The simultaneous ingestion of citrate with an aluminum-containing gel markedly augments aluminum absorption, e.g., produces a 20- to 50-fold increase (128). Fatal cases of acute aluminum toxicity have occurred in patients with advanced renal insufficiency (129,130) due to the simultaneous intake of aluminum hydroxide for hyperphosphatemia and the prescription of Shohl's solution or Bicitra to control metabolic acidosis. Other sources of citrate should be avoided with advanced renal failure as well. Thus calcium citrate is an effective phosphatebinding agent (131), but calcium citrate should be avoided because of the potential risk for aluminum intoxication if aluminum gels are coincidentally ingested (127,128). Another drug, AlkaSeltzer, which is often ingested by patients with dyspepsia, contains citric acid and led to fatal aluminum toxicity in a hemodialysis patient (132). Calcium carbonate has proved to be an effective phosphate-binding agent in at least 80-90% of adult and pediatric dialysis patients. Calcium carbonate should be ingested together with a meal, both to maximize its phosphate-binding efficiency and to minimize

the absorption of calcium (133). The required dosage of calcium carbonate varies from patient to patient, but the initial doses have averaged 4-7 g/day. In individual patients, the dose is adjusted empirically according to the levels of serum phosphorus (84,111,112,134). Hypercalcemia is the major side effect, occurring either with or without concurrent vitamin D therapy (84,111,112,135,136). The use of dialysate solutions with a calcium concentration of 2.5 mEq/liter has been very useful in patients treated with hemodialysis (135,136). When calcium carbonate was given as the sole phosphate binder in adult continuous ambulatory peritoneal dialysis patients who used dialysate with 3.5 mEq/liter calcium, the "standard" peritoneal dialysate calcium concentration for several years, hypercalcemia occurred in as many as 44% of patients, and many required the addition of aluminum gels (137). Evidence is accumulating, on the other hand, that disturbances in mineral metabolism contribute to the development of cardiovascular disease and to overall mortality in patients with end-stage renal disease (108,138-141). Block et al. reported that elevated serum phosphorus levels were an independent risk factor for death in adults undergoing dialysis even after adjusting for established cardiovascular risks and other comorbid conditions (108). The mechanisms that underlie this association remain uncertain, but hyperphosphatemia has long been recognized as an important determinant of soft tissue and vascular calcification in patients with chronic renal failure. Several studies suggest that vascular calcification, due at least in part to hyperphosphatemia a n d / o r the dosage of calcium carbonate, represents one pathway by which alterations in mineral metabolism can adversely affect clinical outcomes both in adult and in pediatric patients with ESRD (138-140,142,143). In this context, hyperphosphatemia in patients undergoing long-term dialysis should be managed by implementing alternative methods that do not entail the use of very large oral doses of calcium. Dietary calcium intake should be maintained at levels that are sufficient to satisfy daily requirements, but the administration of additional calcium as calcium-containing, phosphate-binding agents should be avoided. The new phosphate-binding agent, sevelamer (Renagel), approved by the Food and Drug Administration (FDA) in the United States, is an ionexchange resin that binds phosphorus in the intestinal lumen and prevents its absorption. It does not contain either calcium or aluminum, and it has been shown to be effective in managing phosphate retention both in short-term and in long-term studies of patients undergoing hemodialysis (144-147). The cholesterollowering properties of this compound also make it an appealing therapeutic alternative for use in a subgroup

RENAL BONE DISEASES / of patients who are known to be at risk for developing cardiovascular disease. In addition to sevelamer, other iron-containing compounds in different phases of drug development, such as stabilized polynuclear iron hydroxide and ferric polymaltose complex, have been shown to be effective in controlling serum phosphorus levels in short-term studies in adults and rats with chronic renal failure (148,149). Another agent, lanthanum chloride hydrate, also decreases intestinal phosphorus absorption in experimental animals, and clinical trials using this compound are currently underway (150). However, the long-term safety of new phosphate-binding agents containing iron or other heavy metals such as lanthanium remain to be established; until then such drugs should be considered only in the experimental phase. We should not forget the consequences of aluminum intoxication in patients with renal failure. Whether the more stringent control of serum phosphorus levels and the avoidance of hypercalcemia using therapeutic strategies, as advocated by Block and Port, will favorably affect the development and progression of vascular calcification and cardiovascular disease in the ESRD population (Fig. 4) remains to be determined (109). Evidence is accumulating, however, that phosphate retention a n d / o r the conventional therapeutic interventions aimed at managing this consequence of chronic renal failure can aggravate soft tissue and vascular calcifications (139,140). As such, maintaining serum calcium and phosphorus levels and values for the Ca × P ion product in serum within the ranges seen in persons with normal renal function, rather than at the higher levels previously considered to be acceptable in those with chronic renal failure,

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647

seems prudent. In addition, the dialysate calcium concentration of 2.5 mEq/liter should be the standard in the majority of patients, particularly for adults ingesting large doses of calcium-containing, phosphate-binding agents (135,136). Active V i t a m i n D Sterols

Despite dietary phosphate restriction, the intake of phosphate-binding agents, the use of an appropriate level of calcium in dialysate, and an adequate intake of calcium, a significant n u m b e r of uremic patients develop progressive osteitis fibrosa. Appropriate therapy with an active vitamin D sterol can halt or retard the progression of the bone disease in patients with overt secondary hyperparathyroidism. Treatments employing vitamin D~ or D 2 in pharmacologic doses (1), dihydrotachysterol (151), 25-hydroxyvitamin D~ (calcifediol) (152,153), lct-hydroxyvitamin D 3 (alfacalcidol) (77), or 1,25(OH)zD 3 (calcitriol) (20,154-156) have all been associated with improved symptoms and correction of certain biochemical and radiologic features of secondary hyperparathyroidism. Certain data suggest that vitamin D~ or D 2 can produce more normal mineralization of bone than can calcium carbonate (157). In doses of 50,000-200,000 IU/day, vitamin D 3 can improve secondary hyperparathyroidism in uremic patients (1). The use of vitamin D, however, can be accompanied by hypercalcemia that persists for weeks after the drug is stopped. Calcifediol has also been employed in the management of renal osteodystrophy. Considerable data exist to indicate that both adult and pediatric renal patients respond favorably to this sterol in doses of 25-100 ~ g / day (152,153). The results from a six-center study of therapy with 25(OH)zD ~ in dialysis patients demonstrated a reversal of bone pain and tenderness, a decline in the serum alkaline phosphatase activity, and a decrease in the extent of osteitis fibrosa. Several clinical trials have documented the efficacy of 1,25 (OH)zD ~for the treatment of patients with symptomatic renal osteodystrophy (20,35,154,155,158). The results of these studies can be summarized as follows. With regard to symptoms and signs, there has been a decrease in bone pain, improvement in proximal muscle weakness, and improvement in gait posture. An increase in growth velocity of uremic children was shown in patients with very severe secondary hyperparathyroidism (35); other groups failed to confirm significant improvement in height velocity in children, although plasma alkaline phosphatase levels and serum PTH levels decreased toward normal (154). Studies of bone histology have demonstrated improved osteitis fibrosa (155,159,160). The doses of oral 1,25(OH)2D ~ used in these trials have ranged from 0.25 to 1.5

648

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CHAPTER40

Ixg/day. The major side effect was the appearance of hypercalcemia. The increments in serum calcium levels were sometimes rapid and marked, and they were more c o m m o n in two situations: after many weeks or months of treatment in patients with osteitis fibrosa who had experienced a favorable response to therapy, and after only a few weeks of calcitriol treatment in patients receiving relatively low doses and with no clinical response to treatment. In the latter group, who developed hypercalcemia while receiving calcitriol in dosages of 0.25-0.5 pg/day, aluminum-related bone disease or low bone turnover of other cause should be suspected (160). In patients with secondary hyperparathyroidism, the development of hypercalcemia can often be anticipated by a decline in serum alkaline phosphatase into or toward the normal range. Thus the response to treatment with 1,25(OH)zD 3 may suggest the type of renal bone disease that is present (60,160). Increments in serum phosphorus or a greater requirem e n t for a phosphate-binding agent are also observed, probably due to the action of 1,25(OH)zD 3 to augment intestinal absorption of phosphate as well as that of calcium (Fig. 5). l e~-Hydroxyvitamin D~, or alfacalcidol, is the active vitamin D sterol that has been widely used for the m a n a g e m e n t of ESRD patients in Europe, Canada, and Japan (77,161). This sterol undergoes hepatic 25-hydroxylation to 1,25(OH)zD3; the effects of alfacalcidol are very similar to those of 1,25(OH)zD3, but the required dosage is generally 50-75% greater. Patients who receive anticonvulsant therapy concomitantly may fail to respond, perhaps due to impaired hepatic 25-hydroxylation (162).

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Froction number FIG. 1 Gel filtration analysis of cytochemical bioactivity in plasma from a patient with primary hyperparathyroidism (A) and from a patient with malignancy-associated hypercalcemia (B). Vertical arrows from left to right denote, respectively, the elution position of the void volume (Vo), labeled intact PTH(1-84), labeled active PTH(1-34), and salt. Note that in contrast to primary hyperparathyroidism, in malignancy-associated hypercalcemia heterogeneous bioactive forms were observed. (From Goltzman D, Bennett HPJ, Koutsilieris M, Mitchell J, Rabbani SA, Rouleau MF. Rec Prog Horm Res 1986;42:665-703, with permission.)

demonstrated (10). These extracts, when infused into parathyroidectomized rats, induced phosphaturia, increased urinary cyclic AMP excretion, and prevented the decrease in serum calcium that occurred after parathyroidectomy. These in vivo bioassays therefore confirmed the PTH-like nature of the material identified first by in vitro assays. It was then possible, using the cytochemical bioassay, to identify PTH-like bioactivity in the conditioned m e d i u m of cultured Xenopus oocytes that had been microinjected with polyadenylated messenger RNA isolated from several different h u m a n and rodent tumors. This observation demonstrated that the bioactive material was indeed a secreted peptide (11). Ultimately, in vitro adenylate cyclase bioassays were successfully employed to monitor the biochemical purification of tumor-derived material, and a short NHz-terminal fragm e n t of PTHrP was isolated and sequenced (7,12,13). The sequence was then employed, using molecular biologic techniques, to clone cDNAs encoding PTHrP

(14,15).

The deduced amino acid sequence of the initial PTHrP that was cloned from PTHrP cDNAs included a leader sequence, a "pro" sequence of approximately 36 amino acids, and a mature peptide of 141 residues. A high degree of homology with PTH was observed within the first 13 NH2-terminal amino acids. This homology appeared to account for the PTH-like bioactivity of PTHrP, resulting in the PTH-like biochemical abnormalities of malignancy-associated hypercalcemia. Further evidence to account for the similar bioactivities of these peptides was provided by the cloning of a comm o n P T H / P T H r P receptor (i.e., type 1 PTH receptor) with which both ligands interact (16). The P T H / P T H r P receptor was found to be a seven-transmembranespanning, G protein-coupled receptor. It is linked to both the adenylate cyclase and the phospholipase C transducdon systems, although to date, the bulk of existing evidence implicates cyclic AMP as the major mediator of the action of PTH and PTHrP. The receptor binds the NH2-terminal regions of both PTH and PTHrP with approximately equal affinity. A second G proteincoupled receptor, termed PTH receptor type, 2 localized predominantly in brain, pancreas, and testis, was subsequently identified (17); it binds the NH2-terminal end of PTH with considerably higher affinity than it does the NH2-terminal domain of PTHrP. More recently a gene encoding a third form of PTH receptor was identified in zebrafish (17a). This receptor appears to bind only PTHrP. The function of these additional receptors is currently uncertain. The P T H / P T H r P receptor has been found in both bone and kidney and is therefore in the appropriate site to transduce the classic biologic functions of the Nterminal domains of these entities in osseous and renal tissues. In bone, however, the receptor has been localized to stromal or preosteoblastic cells as well as to mature osteoblasts. This is consistent with previous hypotheses and in vivo studies examining the binding sites for PTH and PTHrP in bone. The localization of P T H / P T H r P receptors to osteoblastic cells suggests that the capacity of PTHrP and PTH to resorb bone must be indirect, requiring the release of mediators that then stimulate osteoclast formation and action. The tumor necrosis factor (TNF)-like molecule RANK (receptor activation of NF-KB) ligand (RANKL), also called osteoclast differentiation factor, or ODF (18), has been implicated as the likely mediator. This entity, after release from osteoblastic cells in response to PTHrP or PTH stimulation, can bind to its cognate receptor, RANK, on osteoclast precursors and enhance osteoclast development (18,19-21). A naturally occurring decoy soluble receptor, termed osteoprotegerin (OPG) (22,23), can also be released by osteoblastic stromal cells and bind RANKL, preventing its access to preosteoclast receptor sites and thereby inhibiting

PTHrP AND HYPERCALCEMIA / osteoclastic bone resorption. This system appears also to be involved in mediating the excessive bone resorption associated with hypercalcemia. Finally, evidence that the P T H / P T H r P receptor is responsible for transducing the majority of the bioactivity of PTH and PTHrP in humans was provided by the demonstration that an activating mutation of the receptor results in Jansen's metaphyseal chondrodystrophy (24), a disorder characterized by hypercalcemia and hypophosphatemia typical of hyperparathyroidism, as well as by osteochondrodystrophy typical of a developmental anomaly associated with excess PTHrP in the fetus. Despite the similar bioactivities of the NHz-terminal domains of PTH and PTHrP, differences between the manifestations of primary hyperparathyroidism and of malignancy-associated hypercalcemia are apparent. Osteoclastic bone resorption is enhanced in both disorders, but diminished bone formation, resulting in "uncoupling" of resorption and formation, is seen in malignancy-associated hypercalcemia (25-27). Additionally, in h u m a n subjects with malignancy-associated hypercalcemia, plasma 1,25(OH)203 concentrations are often decreased (4), whereas in primary hyperparathyroidism 1,25 (OH) 203 concentrations are in the upper range of normal or are actually elevated (28). The NHz-terminal bioactive region of PTHrP can, however, increase l e~-hydroxylase activity when infused into human subjects (29). To account for these observations, it has been suggested that other regions of the PTHrP molecule may modify the capacity of the NH zterminal region to increase l~-hydroxylase activity and that other factors released by the tumor may inhibit le~hydroxylase activity (30). Finally, the severe hypercalcemia associated with the malignancy itself may inhibit 1ot-hydroxylase activity. Cloning of cDNAs encoding rat PTHrP, and subsequently other species of PTHrP, disclosed considerable amino acid sequence homology with the h u m a n form of PTHrP up to approximately residue 110 (31-33). The highly conserved sequence between residues 13 and 110 may therefore be of functional importance, although the precise biologic role or roles of this region are at present uncertain. A midregion domain has been implicated in transplancental calcium transport. Additionally, a functional nuclear localization sequence has been identified within the (87-107) region that may direct PTHrP into the nucleolus (34,35) and that may account for the ability of PTHrP to inhibit apoptosis in target cells. Furthermore, carboxyl-terminal fragments induce calcium transients in hippocampal neurons (36), and a small carboxyl-terminal pentapeptide of PTHrP, PTHrP(107-139), also called osteostatin, has been reported to act as a potent inhibitor of osteoclastic bone resorption in vitro

673

(37-39). Whether such a small peptide acts as a systemic factor or is produced a n d / o r metabolized locally by bone cells from a larger precursor is unknown. In subsequent work, the gene structure of PTHrP in several species was elucidated (40-43) and revealed several important features. The gene was found to be a complex transcriptional unit spanning over 15 kb of DNA. The PTHrP (12p) and PTH (11p) genes share a common exonic organization, with separate exons encoding corresponding functional domains such as the leader and pro sequence. Together with other observations this organizational format provided evidence that the PTHrP and PTH genes probably arose from a common ancestral gene. The human PTHrP gene consists of at least seven exons and is driven by several promoter sequences at the 5' end. At the 3' end, alternative splicing may occur, resulting in three potential peptide isoforms of 139, 141, and 173 amino acids, each with a different COOH-terminal sequence. Consequently, peptide heterogeneity and messenger RNA transcript heterogeneity may result from both alternative splicing and alternative promoter utilization. These studies therefore defined the precise chemical features of PTHrP, an essential step in the development of immunologic methods of measurement. They proceeded from careful analysis of the biologic issues related to malignancy-associated hypercalcemia, through the development of bioassays for in vivo and in vitro measurement of the pathogenetic entity, to the application of biochemical and molecular biologic techniques for final identification of the novel mediator.

METABOLISM OF PTHrP Considerable effort has been made to define the molecular forms of PTHrP that may be produced, secreted, and metabolized.

In Vitro Studies: PTHrP Synthesis, Processing,

and Secretion

In addition to alternative splicing, which may lead to the production of three isoforms of h u m a n PTHrP, there is evidence to suggest that heterogeneous forms of PTHrP may also arise from posttranslational processing. Extracts of tumors contain multiple PTHrP molecules of different sizes (44,45). Parathyroid tissue appears to produce a single immunoreactive PTHrP species that migrates with PTHrP(1-84) (46). The lactating mammary gland also produces PTHrP, which (47), although not generally detected in the plasma of nursing mothers (48,49), is secreted in high levels into milk (48), from which it has been purified. The NH zterminal forms of PTHrP in milk have been found to

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CHAPTER42

have molecular masses ranging from 9 to 21 kDa (50,51), which may represent different species ranging from the full-length molecule to smaller fragments. A detailed analysis of the predicted amino acid sequences of the three major PTHrP isoforms reveals multiple potential sites of proteolysis. There are also two potential amidation consensus sites (52) as well as regions rich in serine and threonine residues that are potential sites for 0-glycosylation, as reported in h u m a n keratinocytes (53). Soifer et al. studied (54) the posttranslational processing of PTHrP in human renal carcinoma and rat insulinoma cells that were stably transfected with a h u m a n PTHrP cDNA encoding amino acids 1-141. Both cell lines produced at least three immunoreactive PTHrP species containing (1-36), (37-74), and (1-74) epitopes plus a novel midregion fragment starting at amino acid 38 (approximate molecular mass, 7 kDa) and distal cleavage sites at around amino acids 96-98 and 102-106. This midregion fragment was also shown to be secreted by normal h u m a n keratinocytes. Over 80% of the secreted material described by Soifer et al. was composed of the NH zterminal and midregion fragment whereas only a small fraction contained (1-74) immunoreactivity. These results are in good agreement with experiments examining the processing of endogenous, internally labeled PTHrP in rat Leydig tumor cells in culture (55). The latter studies demonstrated the presence of three molecular forms of PTHrP comigrating with PTHrP(1-36), PTHrP(1-86), and PTHrP(1-141) on high-pressure liquid chromatography (HPLC), and comprising approximately 63, 30, and 7% of newly synthesized PTHrP, respectively. These studies demonstrate that the half-life of intact PTHrP(1-141) is extremely short and suggest the presence of multiple secretory forms ranging from short NHz-terminal fragments to the intact molecule. Whether the multiple forms described are generated in a tissue-specific fashion is not yet known. However, this extraordinarily intricate processing can potentially generate many different forms of PTHrP, and may confound the development and interpretation of specific immunoassays. To add to this complexity, it is also likely that peripheral metabolism of PTHrP occurs and gives rise to additional forms. Not studied yet is the issue of metabolic clearance, which may also be highly variable for each of these different forms of PTHrE

In Vivo Studies: Circulating Forms of PTHrP There appear to be major differences in the circulating forms of PTH and PTHrE Considerably more is known about the secretion and metabolism of the PTH molecule that is uniquely produced by parathyroid glands. The principal form of PTH is PTH(1-84),

which is also believed to be the major if not the only bioactive form in the circulation. However, in contrast to PTH, secretion and metabolism of PTHrP is much less well understood and potentially far more complicated. First, in contrast to the human PTH gene, which encodes a single mature peptide, the h u m a n PTHrP gene has the potential to express three different isoforms. Each of these isoforms in turn has the potential to undergo complicated posttranslational processing and subsequently may be subject to further breakdown and metabolic clearance in sites outside the cell of synthesis. Additionally, in contrast to PTH, which is synthesized exclusively in parathyroid cells, PTHrP is produced by a wide variety of normal and malignant cells, each of which may exhibit tissue-specific expression and unique posttranslational processing. To add to this inordinately complex scheme, it is not known to what degree tumors of the same cell type express and process similar molecular forms. Initial in vivo studies in cancer patients used the cytochemical bioassay to demonstrate elevated plasma levels of PTH-like bioactivity in the absence of detectable immunoreactive PTH (5). Gel filtration analysis of bioactivity revealed a heterogeneous profile, suggesting the presence of multiple bioactive fragments (56) (Fig. 1). Immunoassays specific for selected epitopes of the molecule have confirmed this apparent heterogeneity. Burtis et al. (57), using region-specific immunoassays, identified both NH 2- and COOH-terminal moieties in the circulation of cancer patients. Using a two-site immunoradiometric assay (IRMA), a protein that concomitantly reacts with both PTHrP(1-36) and PTHrP(37-74) antisera was detected together, along with a COOH-terminal (109-136) fragment that was present in equimolar concentrations. The full-length molecule, PTHrP(1-141), appeared to be absent. Henderson et al. (58) reported the presence of PTHrP entities of approximately 6-7 kDa with a radioimmunoassay based on an NHz-terminal antibody raised against PTHrP(1-34). In addition, this study and that of Burtis et al. reported species larger than the predicted full-length protein, suggesting that fragments or the intact form may aggregate in complexes with each other or with other proteins in the circulation, resulting in species of abnormally high molecular weight. Studies of gel filtration patterns of circulating PTHrP u n d e r denaturing conditions may help to resolve this issue. Carboxyl-terminal fragments of PTHrP are metabolized by the kidney (59) and circulating levels of the carboxyl-terminal PTHrP sequence 109-136 have been found to be elevated in patients with renal insufficiency (57). Because malignancy-associated hypercalcemia is characterized by the presence in the circulation of bioactive forms of the hormone, and in view of the fact

PTHrP AND HVeWRCaLCEMIA / that structure-function studies of synthetic fragments indicated such material must contain the aminoterminal region of the molecule, two-site, noncompetitive immunoradiometric assays have been developed for P T H r E This technique increases specificity for the intact molecule and improves sensitivity. However, it must be r e m e m b e r e d that other regions of the molecule may exhibit yet undefined biologic actions that could contribute to the biochemical manifestations of malignancy-associated hypercalcemia or that could carry out noncalcemia related changes and would therefore be of interest to measure. Furthermore, an unidentified subset of tumors may secrete forms of the peptide with novel properties, because histologically identical tumors may or may not be associated with hypercalcemia. Production of other non NHz-terminal molecular forms of PTHrP may also in theory provide useful information concerning the origin of a particular cancer or for monitoring response to treatment, i.e., as a tumor marker. Indeed, radioimmunoassays (RIAs) measuring midregion and inert COOH-terminal fragments of PTH have provided useful information in the past regarding the overall secretory activity of the parathyroid gland as well as the status of renal clearance mechanisms for PTH. These assessments therefore point to the presence of heterogeneous forms of PTHrP in extracellular fluids; they will no doubt be refined as increased knowledge of the chemical nature of these forms becomes available.

R E G U L A T I O N OF PTHrP P R O D U C T I O N IN VITRO In contrast to PTH, which is expressed only in parathyroid tissue, PTHrP is expressed in a wide variety of normal and neoplastic tissues. Using N o r t h e r n blot hybridization and in situ hybridization techniques, mRNA encoding PTHrP has been identified in normal h u m a n keratinocytes (60,61), normal h u m a n cervical epithelial cells (62), normal islet cells (63), lactating m a m m a r y glands (47,64), rat (65) and h u m a n (66) m a m m a r y epithelial cells, brain, fetal liver (60), normal h u m a n melanocytes (67), fetal parathyroid (68), a variety of smooth muscle tissues (69-71), urinary bladder (72), and stromal cells of the spleen and other organs (73). PTHrP is also expressed in a wide variety of neoplastic tissues (46,60,74) and in epithelial tumor cell lines (75), including squamous cell cancers, renal carcinoma (68,76), skin cancers (77), breast cancers (78), m e l a n o m a (67), and parathyroid adenomas (46), as well as in HTL V-l-transformed lymphocytes (79), h u m a n osteosarcoma cells (80), and n e u r o e n d o c r i n e tumor cells (44). This wide tissue distribution is compatible with an a u t o c r i n e / p a r a c r i n e role for the pep-

675

tide, a function that is likely to predominate over its endocrine role in normal tissues. Such a role has been suggested by experiments in h u m a n epithelial cells in which PTHrP acted as a potent antiproliferative (62,81) and prodifferentiating (82) factor. In normal animals, it appears to play no role as an endocrine factor in mediating calcium homeostasis after birth, (83) although it may subserve such a function during fetal life. In adults it would appear that only during an extraordinary situation, such as cancer development and progression, does PTHrP play an endocrine role leading to hypercalcemia. The development of RIAs for PTHrP greatly facilitated the study of its regulation in both normal and cancer cells in culture. The control of PTHrP released into conditioned m e d i u m by normal and neoplastic cell lines was initially monitored using bioassays. RIAs have also been used in conjunction with the traditional bioassay systems to monitor the PTHrP response to various stimuli (61,65,66,83-85). Positive regulation of PTHrP expression and secretion by mitogenic stimuli such as epidermal growth factor (EGF) and fetal bovine serum (FBS) (61,75), as well as by inhibition by 1,25(OH)zD 3, has been demonstrated in h u m a n keratinocytes using these methods (Fig. 2). Studies have shown that the p r o m o t e r of the PTHrP gene contains a vitamin D response element (VDRE) (86) that inhibits gene transcription when it interacts with the vitamin D receptor 1,25(OH)2D 3 complex. Dexamethasone and testosterone (83) have also been shown to reduce PTHrP secretion. In addition, as opposed to the inhibitory influence of calcium on PTH secretion by parathyroid glands, PTHrP secretion by keratinocytes is e n h a n c e d by extracellular calcium. Calcium also stimulates PTHrP production in transformed h u m a n keratinocytes (75), in rat Leydig tumor cells (87), and in a lung carcinoma cell line (88), but inhibits PTHrP expression and secretion in a rat parathyroid cell line (89). A variety of cell lines have been shown to increase PTHrP secretion in vitro in response to a n u m b e r of factors, including phorbol esters (44,80) cyclic AMP (90), calcitonin (91,92), the product of the Tax gene (93), transforming growth factor-[~ (TGF-[3) (04), and angiotensin II (95). It should be emphasized that peptide growth factors are generally positive regulators of PTHrP production. This is exemplified in normal h u m a n keratinocytes, in which EGF is essential for growth and is a potent positive stimulus for PTHrP production. In normal h u m a n m a m m a r y epithelial cells, in sulin-like growth factor-I (IGF-I), rather than EGF, is an absolute r e q u i r e m e n t for cell growth and is also more potent than EGF in stimulating PTHrP production (66). In view of the fact that PTHrP is p r o d u c e d by many normal cells, its secretion by many histologic types of

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tumor cells undoubtedly represents eutopic overproduction of PTHrP in the cells as they undergo malignant transformation rather than ectopic production. The mechanism whereby PTHrP is produced in excess by tumor cells is, however, not well understood at the present time but may be a function of abnormal gene regulation. This p h e n o m e n o n has recently been studied in a model of tumor progression (75), in which PTHrP gene regulation and secretion were analyzed in the transition from the normal to the malignant phenotype. Normal human keratinocytes were established as a keratinocyte cell line following infection with h u m a n papilloma virus type 16 (HPKIA). This cell line

FIG. 2 In vitro regulation of PTHrP secretion in normal and malignant states. The upper panel depicts sites of regulation of PTHrP production that may be targets for intervention to diminish PTHrP release from tumor cells. Growth factors (GF) act via the Ras pathway to stimulate mitogen-activated protein kinase (MAPK) and increase PTHrP gene transcription. 1,25-Dihydroxyvitamin D (1.25) can bind to the vitamin D receptor (VDR) to inhibit transcription. After transcription and translation, preproPTHrP is converted to proPTHrP, which must then be processed to the active form of PTHrP before it is secreted. The lower panel compares the regulations of PTHrP in immortalized keratinocytes (HPK1A) versus malignant keratinocytes (HPK1Aras). (A) The time course of PTHrP secretion in the absence of exogenous mitogenic growth factors, demonstrating that HPK1Aras cells secrete far more PTHrP than do HPK1A cells. (B) The effects of 1,25(OH)2D 3 on EGF-stimulated PTHrP secretion in HPK1A and HPK1Aras cells, showing that 1,25(OH)2D3 is much more effective in inhibiting PTHrP secretion in HPK1A than in HPK1Aras cultures. Significar,t difference from HPK1A (*p < 0.01). (Redrawn from Henderson J, Sebag M, Rhim J, Goltzman D, Kremer R. Cancer Res 1991 ;51:5621-5628.)

was subsequently converted to the malignant phenotype (HPKIAras) using an activated r a s oncogene (75,96,97). In contrast to the established cells (HPKIA), which continued to produce PTHrP in a regulated manner, HPKIAras cells expressed and secreted PTHrP in a constitutive fashion, i.e., in the absence of exogenous mitogenic factors such as EGF, and displayed resistance to the inhibitory effect of 1,25 (OH) 2D~ (Fig. 2). This resistance p h e n o m e n o n was shown to be secondary to phosphorylation of the retinoid X receptor (RXR) on a specific MAP kinase consensus sequence by ras-raf-MAP kinase activation pathway (98). Whether the unregulated production of

PTHrP AND H~F~kCALCWMIA / PTHrP demonstrated in vitro by cultured tumor cells occurs in vivo and can explain the elevated circulating concentrations of PTHrP noted in patients with squamous carcinoma remains to be elucidated. Significant progress has been made from in vitro studies in examining the regulation of PTHrP production by both normal and malignant cells in culture. Studies of this nature have the potential of disclosing critical mechanisms that might also operate in vivo.

nephrogenous cAME Similar results obtained by Henderson et al. (113) using the rat Leydig cell model also demonstrated the important role played by the renal action of PTHrP in contributing to hypercalcemia of malignancy. These studies therefore strongly implicated PTHrP as the humoral factor responsible for hypercalcemia in these models and provided convincing evidence for a pathogenetic role for PTHrP in the syndrome of hypercalcemia associated with malignancy (Fig. 3).

STUDIES OF MALIGNANCY-ASSOCIATED HYPERCALCEMIA IN ANIMALS

Several animal models of malignancy-associated hypercalcemia have been developed over the years in an effort to define the pathogenesis of the human syndrome. These models include both spontaneous and induced tumors in rodents and dogs as well as h u m a n tumors transplanted into athymic mice. One of the most widely used, and therefore best defined model, is the Fischer rat bearing the Rice H500 Leydig cell tumor. This tumor, from which the rat PTHrP cDNA was cloned (32), arises spontaneously in aged Fischer rats but can be successfully passaged by subcutaneous transplantation in younger animals. The nonmetastatic tumor grows rapidly in association with hypercalcemia, hypophosphatemia, increased urinary cAMP, renal phosphate wasting, and suppressed immunoreactive PTH (99-102). This constellation of biochemical abnormalities therefore closely reproduces the syndrome of h u m a n malignancy-associated hypercalcemia. The Walker 256 carcinosarcoma, originating from a rat mammary gland, has also been shown to secrete PTHlike bioactivity, in the absence of PTH immunoreactivity, into conditioned medium when maintained in culture (103-105). Adenocarcinoma of the anal sac in dogs (106) is also associated with hypercalcemia in vivo and appear to produce a factor with PTH-like bioactivity in vitro. Tumors of h u m a n and animal origin that have been transplanted into athymic mice include h u m a n squamous carcinomas (107,108) and a n u m b e r of h u m a n renal carcinomas (109,110), and a melanoma cell line (67). Although all of these models demonstrated to some degree the biochemical abnormalities associated with malignancy-associated hypercalcemia, direct measurement of circulating PTHrP has been accomplished in only some (67,84,108,111). Once antisera directed against the human (h) PTHrP molecule had been developed for use in PTHrP RIAs they were also applied to passive immunization studies in rodents. Using athymic mice beating human squamous carcinomas, Kukreja et al. (107) infused an antiserum directed against NH2-terminal hPTHrP, which quickly reversed the hypercalcemia and the elevation of

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FIG. 3 Effect of passive immunization with PTHrP and PTH antisera on plasma calcium of normal rats and rats with hypercalcemia of malignancy. In normal rats (A) a moderate and transient decrease in plasma calcium was observed after injection of PTH antiserum (/k) but not after injection of PTHrP antiserum (o) or normal preimmune rabbit serum (©). In contrast, hypercalcemic rats implanted with the Leydig cell tumor H500 (B) sustained a prolonged reduction in plasma calcium after injection of PTHrP antiserum (°) but not of PTH antiserum (/k) or normal preimmune rabbit serum (©). Consequently PTH but not PTHrP appears to be the major modulator of plasma calcium homeostasis in the normal animals, whereas PTHrP is the major pathogenetic mediator in the hypercalcemia ofmalignancy. (From Henderson J, Bernier S, D'Amour P, Goltzman D. Effects of passive immunization against parathyroid hormone (PTH) -like peptide on PTH in hypercalcemic tumor bearing rats and normocalcemic controls. Endocrinology, Vol. 127, pp. 1310-1318, 1990. © The Endocrine Society.)

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Animal models have also b e e n useful for examining the therapeutic efficacy of various agents in malignancyassociated hypercalcemia. We have taken advantage of our previous in vitro studies demonstrating the inhibitory effect of 1,25(OH)zD ~ and vitamin D analogs on PTHrP gene expression and secretion (61,75,112) to study their potential usefulness in reducing serum calcium levels in animal models in vivo. A 1,25(OH)2D 3 analog, EB1089, was found to have very low calcemic potency relative to 1,25 (OH)zD s when infused into control animals (84,108,114). In addition, when analyzed in vitro, this analog was 10-100 times more potent than 1,25(OH)2D 3 in inhibiting PTHrP production in cancer cells (112) and was therefore chosen as a candidate for in vivo studies with hypercalcemic, tumor-bearing rats. After continuous infusion into Fischer rats bearing the Rice Leydig cell tumor H500, a significant reduction in circulating PTHrP concentrations was demonstrated using an NHz-terminal PTHrP RIA, with a concomitant reduction in plasma calcium levels (Fig. 4). These studies were further extended in a nude mouse model of h u m a n squamous cancer producing PTHrP in which established hypercalcemia was reversed by infusion of EB1089 directly into the tumor (108). Other approaches have targeted additional loci known to be important for PTHrP production. In view of the critical

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stimulatory role of growth factors in PTHrP production, and in view of the important role of the Ras oncogene signaling pathway, Ras inhibitors have been employed. The inhibitor we used prevents farnesylation of Ras, a necessary step in facilitating binding to cell membranes and therefore permitting signal transduction. We thus showed that transplantation of a cell line overproducing PTHrP into nude mice could be inhibited from producing hypercalcemia after treatment of the mice with a small-molecule organic inhibitor of Ras farnesylation (115). In another approach, stable transfection of H500 Leydig tumor cells with antisense PTHrP was used to diminish intracellular PTHrP production. When these cells were implanted into normal Fischer rats, hypercalcemia did not occur (115). Finally, inasmuch as proper processing of proPTHrP to PTHrP has been shown to be critical for production of fully bioactive PTHrP, and in view of the fact that this process appears to be mediated by the convertase furin, an antisense furin cDNA was stably transfected into H500 Leydig cells. Implantation of these modified cells into Fischer rats resulted in reduced hypercalcemia, diminished tumor growth, and prolonged survival relative to implantation of native cells (116). In the future these and other approaches may yield useful therapies against hypercalcemia of malignancy.

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PTHrP AND HYPERCALCEMIA /

HYPERCALCEMIA D U E T O P T H r P P R O D U C T I O N IN H U M A N S With the development of accurate immunologic techniques for the measurement of PTHrP in humans, considerable progress was made in defining the pathogenetic mechanisms underlying malignancy-associated hypercalcemia as well as assessment of the therapeutic modalities to be used in its treatment.

P T H r P in Cancer Patients Classification of Cancer Patients

One criterion commonly used to define subgroups of patients with malignancy-associated hypercalcemia has been the presence or absence of bone metastases (4). One group, whose underlying mechanism was presumably local osteolytic hypercalcemia (LOH), was composed mainly of patients harboring hematologic malignancies or metastatic solid tumors (e.g., breast cancer). A second group, whose underlying mechanism of hypercalcemia was believed to be secretion by the tumor of a humoral factor with hypercalcemia activity (HHM), was composed mainly of patients with epithelial and renal cancers (3,4,25,117-119). Although this distinction was initially useful in the conceptual approach, which ultimately resulted in the purification and identification of PTHrP, it is less useful in the pathogenetic and clinical sense. The occurrence of bone metastases (i.e., LOH) does not exclude a pathogenetic role for PTHrP that might act systemically (i.e., endocrine) or locally (i.e., paracrine). In fact, animal studies (120,121) have demonstrated a role for PTHrP as a paracrine mediator of bone resorption in metastatic cancer. Thus, it seems likely that all malignancyassociated hypercalcemia, whether associated with a systematic circulating factor or not, is humoral in origin and could be called HHM. Additionally, several studies have reported a poor correlation between the presence or extent of bone metastases and the occurrence of hypercalcemia (122). The terminology "LOH" may also be inaccurate because it is uncertain whether hypercalcemia is ever due solely to osteolysis in the absence of altered renal handling of calcium. A more relevant pathogenetic consideration of malignancy-associated hypercalcemia (MAH), therefore, may be the presence or absence of elevated n e p h r o g e n o u s cyclic AMP, as a reflection of the overproduction and systemic action of PTHrP by these tumors. In this respect it has been estimated that approximately 80% of unselected patients with malignancy-associated hypercalcemia (4) have such an abnormality. Using two-site immunometric assays, several groups have confirmed that between 50

679

and 90% of these patients with solid tumors (57,123,124) and 20 and 60% of patients with hematological tumors (125-127) had elevated circulating concentrations of PTHrP. Similar results were obtained with a midregion PTHrP assay (128). The most frequent solid tumors associated with increased circulating levels of PTHrP are squamous cell cancers of lung, head, and neck, as well as cancer of the kidney and ovary. With the advent of sensitive and specific immunoassays for PTHrP it became possible to redefine the classification of hypercalcemic cancer patients. This is particularly true for patients with breast cancer and hypercalcemia, who often have elevated PTHrP levels, even in the presence of extensive bone metastatic lesions (129), indicating that the humoral and local osteolytic mechanisms frequently occur simultaneously. Indeed, the "humoral" nature of the hypercalcemia due to breast cancer had previously been predicted on the basis of studies of urinary phosphate and cyclic AMP excretion (130,131). It has been suggested that PTHrP may enhance the ability of breast cancer cells to invade bone, and that PTHrP production by metastatic tumor cells is increased by cytokines produced locally in the bone microenvironment. Thus, there occurs a vicious cycle of mutually activating factors (120). Additionally, hematologic malignancies may not readily fall u n d e r an L O H classification. Thus certain types of lymphomas have been shown to produce both PTHrP (79,125) and 1,25(OH)zD ~ (28,132), adding to the complexity of potential underlying mechanisms of hypercalcemia in this condition. As the sensitivity and specificity of PTHrP immunoassays improve, it will be possible to classify MAH patients more quickly and more accurately as PTHrP related or PTHrP unrelated, based on measu r e m e n t of the causative agent per se. As improved methods of treating PTHrP overproduction are developed, such improved diagnostic accuracy should have therapeutic as well as pathogenetic importance.

Relationship of Skeletal Metastasis to PTHrP Concentrations in Patients with Solid Tumors and Hypercalcemia Employing RIAs for the NH2-terminal assay of PTHrP, analyses of patients with malignancy-associated hypercalcemia revealed no more than a modest increase in the percentage of patients with elevated values when patients with bone metastases were excluded from the series studies (Table 1). Thus, in the study of Budayr et al. (133), 51% of patients with MAH had elevated PTHrP values regardless of the presence of bone metastases. The n u m b e r rose to about 85% when patients with bone metastases were excluded. In the study of H e n d e r s o n et al. (58), 50% of unselected

680

/

CHAPTER42 PTHrP Levels in Different Histologic Types of Malignancies

TABLE 1

Occurrence of elevated PTHrP in cancer patients (%) Hypercalcemic subjects Tumor type

BM ( - and +)

BM (+)

BM (-)

Normocalcemic subjects

Ref.

51

N/A a

53 48 60 85 c 100

N/A 33 65 b 0 N/A

85 N/A 53 100 N/A N/A

10 18 0 0 9 N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134) Grill et aL (135) Burtis et aL (57) Ratcliffe et aL (123)

85 50 100

N/A N/A N/A N/A

N/A N/A 66 100

N/A 10 N/A N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134) Grill et al. (135)

60 100 N/A ~

N/A N/A N/A

N/A N/A 25

N/A N/A N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134)

50 60 N/A d

N/A N/A 16

N/A N/A 100

N/A 29 N/A

Budayr et aL (133) Henderson et aL (58) Kao et al. (134)

33

N/A d

N/A d

8

Henderson et aL (58)

0 80 62.5

N/A N/A N/A

N/A N/A N/A

N/A N/A 23

Budayr et al. (133) Kao et al. (134) Kremer et al. (61)

33 16

N/A N/A

N/A N/A

N/A N/A

Budayr et al. (133) Kao et al. (134)

Solid tumors Mixed

Squamous cancers

N/A d

Renal cell cancer

Breast cancer

Hematologic tumors Mixed Lymphoma

Multiple myeloma

aN/A, Information not available. bSolid tumors other than breast. dHypercalcemia of malignancy with I"NcAMP. Patients with (+) or without ( - ) bone metastases (BM).

patients with MAH, harboring a wide variety of histologic tumor types, had elevated PTHrP values. Grouping patients according to the presence or absence of bone metastases did not significantly alter those results. In the study of Kao et al. (134), approximately 48% of unselected patients with MAH had elevated PTHrP values, a percentage that increased to about 53% when patients with bone metastases were

excluded. Finally, in the study of Grill et al. (135), 100% of a group of patients with various solid tumors, excluding breast cancers, having no evidence of bone metastases, had elevated values of PTHrP (ranging from 2.8 to 51.2 pmol/liter). In a second group that included patients with solid tumors of the same type, but having radiologic evidence of metastases, roughly 60% had elevated PTHrP levels ranging from 4.9 to 47.5 pmol/liter.

PTHrP AND HYPERCALCEMIA / In this study all patients with squamous cancer, with or without bone metastases, had elevated PTHrP levels, whereas approximately 60% of hypercalcemic patients with breast cancer, almost all of whom had bone metastases (19/20), had elevated PTHrP values (ranging from 3.9 to 61.6 pmol/liter). Results are somewhat more conflicting when two-site IRMAs are employed. Burtis et al. (57), using a two-site assay specific for PTHrP (1-74), found that 85% of 30 hypercalcemic cancer patients classified as HHM on the basis of elevated nephrogenous cyclic AMP had elevated PTHrP values (mean level, 20.9 -+ 21.8 pM/liter). In contrast, a group of patients represented by four breast cancers, three multiple myelomas, and one undefined lung cancer were classified as LOH on the basis of normal nephrogenous cAMP and extensive bone involvement, and had normal PTHrP levels. On the other hand Ratcliffe et at (123), using a two-site assay specific for PTHrP (1-86), found that all patients in an unselected group with MAH of various histologic types and with advanced metastatic disease had elevated PTHrP levels. These studies indicate that a larger n u m b e r of tumors produce PTHrP than was predicted, and that PTHrP may contribute to the development of hypercalcemia regardless of the presence or absence of skeletal metastases (Table 1). Whether different NHz-terminal species of PTHrP are produced by tumors that differ histologically or have varying metastatic behavior is currently unknown.

associated biochemical abnormalities strongly indicate the presence of a circulating factor with PTH-like bioactivity (138). The demonstration of elevated PTHrP expression by T lymphocytes in culture after infection with HTLV-1 supports this hypothesis (79). Henderson et al. (58) reported 33% of patients with hematologic malignancies had elevated PTHrP values, whereas Kao et al. (134) found elevated levels of PTHrP in four out of five patients with lymphoma and one out of six patients with multiple myeloma. Burtis et al. (57) showed that two patients with lymphomas and elevated nephrogenous cAMP had increased PTHrP levels whereas one patient with multiple myeloma and normal nephrogenous cAMP had a normal PTHrP value. Another group has reported elevated concentrations of PTHrP in two patients with hypercalcemia and nonHodgkin's lymphoma without bone metastases (139). As is the case with breast cancer, evaluation of patients with various hematologic malignancies reveals that PTHrP is frequently elevated in these conditions (Table 1). Another study has confirmed and extended these observations in patients with diverse hematologic malignancies (125). In this study, which included a large n u m b e r of patients with non-Hodgkin's lymp h o m a classified according to disease stage and grade, elevated PTHrP levels were most often found in patients with late-stage disease and high-grade pathology (Table 2). After chemotherapy in several patients

TABLE 2 Distribution of PTHrP in Non-Hodgkin's Lymphoma, According to Disease Stage and Gradea

P T H r P Concentrations in Hematologic Malignancies Associated with Hypercalcemia

Hematologic malignancies that frequently cause hypercalcemia include lymphoma, chronic myeloid and lymphoblastic leukemia, multiple myeloma, and adult T cell leukemia. Extensive bone destruction is common in multiple myeloma, and over 30% of patients develop hypercalcemia (136). The mechanism that has been postulated to explain the hypercalcemia associated with this disorder is production by the plasma cells of a group of cytokines collectively termed "osteoclast-activating factor" (OAF). These include interleukin-one (IL-1) and tumor necrosis factors ~ and [3 (TNFe~ and TNF[3), all of which are potent stimulators of osteoclastic bone resorption. Hypercalcemia is less common in lymphomas (both Hodgkin's and nonHodgkin's). Circulating 1,25(OH)2D ~ is elevated in a number of lymphoma patients who are hypercalcemic without skeletal metastases, and likely plays a pathogenetic role (28,137). By contrast, there is a high incidence of hypercalcemia in patients with adult T cell leukemia/lymphoma. This disorder is caused by infection of T cells with the h u m a n T cell lymphotropic virus type 1 (HTLV-1). In addition to hypercalcemia, other

681

Lymphomas Stage IV

I to III

PTHrP (1")

[PTHrP] (pmol Eq/Liter)

Grade

n

n

Mean _+ SD

H

14

8

I

7

2

52.5 _ 22.5 2 5 _+ 15

L

9

I

H

9

2

13_+5

I

5

0

Ca2+

Ref.

0-2.5 < 15 2-5 1.5 mg/dl). Renal function improved or remained stable in 25 patients, but renal function deteriorated transiently in 8 patients (55). In all 8 patients, the rise in serum creatinine concentration preceded pamidronate administration or could be attributable to other causes. Bisphosphonates have been administered to patients on dialysis without ill effects (56,57). However, bisphosphonates should be administered slowly (over 24 hours) in patients with renal insufficiency, and a reduced dose should be considered (30-45 mg, rather than 60 mg).

MANAGEMENT OF H~F.kCALCV.MZA /

Calcitonin A relative disadvantage of all bisphosphonates is the time it takes (1-2 days) to reduce hypercalcemia. In many patients, it is desirable to begin to lower the serum calcium concentration more rapidly. The advantage of calcitonin is that it acts much more rapidly than any of the bisphosphonates. In combination with rehydration, it is useful for the initial m a n a g e m e n t of severe hypercalcemia. The speed of calcitonin's action may be more related to its effect to facilitate urinary calcium excretion (58) than to its antiosteoclast properties. The recomm e n d e d dose is 4-8 MRC units/kg administered subcutaneously every 6-12 hours for 2-3 days. Serum calcium concentrations may decrease within 2 hours of administration (59). The calcemic nadir is reached within 12-24 hours but is often followed by a return toward initial hypercalcemic levels within 24-72 hours despite continued administration (60,61). Thus, the major disadvantage of calcitonin is that its effects are short-lived and relatively weak. Moreover, many patients develop tachyphylaxis to repeated dosing (62). Because of its limited duration of action, calcitonin is most effective when combined with hydration and a bisphosphonate (Fig. 4). The combination of calcitonin and a bisphosphonate results in a rapid decrease in serum calcium concentration (calcitonin effect), which is sustained for a more prolonged period (bisphosphonate effect) (63-65). Calcitonin is a safe antihypercalcemic agent. Side effects include mild, transient nausea, abdominal cramps, and flushing. True allergic reactions to salmon calcitonin, the preparation that has been used most widely, are quite rare. H u m a n calcitonin, which is less potent than salmon calcitonin, has seldom been stud-

g

3.5

,m

o

0

E~o 0- E ~D .i-i

o

ied in the m a n a g e m e n t of hypercalcemia. Thus, shortterm use of calcitonin should be considered in the cancer patient with severe hypercalcemia because of its rapid onset of action and its safety profile. Simultaneous administration of a bisphosphonate would make likely more prolonged control of the hypercalcemic state. Another feature of calcitonin that should be borne in mind is the suggestion that it has potent analgesic properties (66). It has been reported to provide impressive relief in some patients with painful skeletal metastases.

Glucocorticoids Glucocorticoids can be effective calcium-lowering agents in limited groups of patients with sarcoidosis, vitamin D toxicity, hematologic malignancies associated with increased circulating 1,25-dihydroxyvitamin D [1,25 (OH) zD] , and certain other malignant states (i.e., some breast cancers). For this purpose, 200-300 mg of hydrocortisone, or its equivalent, is given intravenously daily for 3-5 days. Glucocorticoids inhibit gastrointestinal calcium absorption, but their efficacy to reduce hypercalcemia may relate to other properties. They may act to inhibit directly the growth of neoplastic lymphoid tissue (67). In patients with lymphomas associated with increased 1,25(OH)2 O, glucocorticoids reverse hypercalcemia by lowering the concentration of the vitamin D metabolite (68). The same is true in sarcoidosis (69). In vitamin D intoxication, they may act on the target organ. Because excessive bone resorption also occurs in patients with vitamin D toxicity, bisphosphonates additionally may be of benefit in decreasing calcium concentrations in such patients. In general, patients with nonhematologic cancers do not respond to glucocorticoids (70). Primary hyperparathyroidism also is classically unresponsive to glucocorticoid administration (71).

3.0

Plicamycin (Mithramycin) 2.5

I.. L.

o 0

737

Calcitonin 2.0

01234

6

9

14

Days FIG. 4 Mean corrected serum calcium concentrations (_+ SEM) in two groups of patients with hypercalcemia of malignancy treated with either a single infusion of pamidronate alone (©) or with a single infusion of pamidronate combined with suppositories of salmon calcitonin (O). *p < 0.01, **p < 0.005 (from Ref. 63; D Thiebaud, F Jacquet, P Burckhardt. Fast and effective treatment of malignant hypercalcemia. Arch Intern Med 1990;150:2125-2128. Copyrighted 1990, American Medical Association).

Plicamycin, an inhibitor of osteoclast RNA synthesis, is a potent therapy for hypercalcemia that has been in clinical use for more than 25 years (72-75). It is given intravenously in a dose of 15-25 Ixg/kg of body weight over 4-6 hours. The dose can be repeated several times, although a single dose may normalize the serum calcium concentration. The serum calcium concentration begins to decrease as early as 6 hours after administration of the drug. The maximal reduction occurs in 48-72 hours. In one study, 45% of patients randomized to plicamycin, compared to 86% of patients randomized to pamidronate (60 mg), achieved normocalcemia within 7 days (76). The duration of normocalcemia after a single dose of plicamycin is usually a few days and depends on the rate of ongoing bone resorption.

738

/

CHAPTER45

Plicamycin has several adverse side effects. Nausea is c o m m o n and can be minimized by slow intravenous infusion. Care should be taken to avoid local extravasation of the drug, because irritation and cellulitis can result. Hepatic toxicity, manifested most often as transiently elevated serum aminotransferase activity, occurs in approximately 20% of patients (77). Nephrotoxicity (increased blood urea concentration, creatinine, and proteinuria) and thrombocytopenia can also occur (78), the latter especially in patients who have received previous chemotherapy or radiotherapy. Because of the availability of bisphosphonates, which have greater efficacy and fewer side effects, plicamycin is rarely used now for the treatment of hypercalcemia. One situation in which plicamycin may be useful is for the treatment of patients who have hypercalemia that is refractory to therapy with bisphosphonates. Contraindications to the use of plicamycin are overt hepatic or renal dysfunction, thrombocytopenia, or any coagulopathy. G a l l i u m Nitrate Gallium nitrate binds to bone mineral surface and may reduce hydroxyapatite crystal solubility, thus inhibiting bone resorption (79-81). In addition, a direct inhibitory action of gallium on osteoclasts has been observed (82). After gallium nitrate administration, reductions in urinary calcium and hydroxyproline excretion are found, confirming its action as an inhibitor of bone resorption (83). Gallium is administered as a continuous intravenous infusion, 200 m g / m 2 in 1 liter of fluid daily for 5 days. It has been demonstrated to normalize serum calcium concentration in some patients with hypercalcemia of malignancy (84,85). However, the mean duration of normocalcemia in patients who received gallium was only 8 days, which is much shorter than the duration of normocalcemia in patients treated with pamidronate. In addition, the rate of decline of the serum calcium was slow, with the nadir calcium values occurring 8-10 days after the initiation of the gallium infusion. A potential toxicity of gallium nitrate is impaired renal function, manifested by an increase in serum creatinine, which is especially concerning in patients with underlying renal disease or dehydration. Thus, gallium is contraindicated in renal insufficiency. Because of its slow onset and short duration of action, as well as its potential renal toxicity, gallium is no longer used for the treatment of hypercalcemia.

Miscellaneous Therapy

Phosphate Intravenous administration of sodium or potassium phosphate can produce a profound and rapid reduction in serum calcium concentrations. This treatment, however, is potentially very dangerous because of

the possibility of deposition of calcium phosphate complexes in blood vessels, lungs, and kidneys. Precipitation of these complexes has produced severe organ damage and even fatal hypotension in patients rapidly infused with high doses (86-88). The use of intravenous phosphate should be restricted, therefore, to patients with extreme, life-threatening hypercalcemia who are hypophosphatemic and in whom all other measures have failed. Oral phosphate is of little value in the emergency therapy of hypercalcemia, because its calcium-lowering activity is modest, and amounts >2 g daily are often associated with diarrhea. Oral phosphate should be reserved for settings of mild to moderate hypercalcemia associated with serum concentrations of phosphate that are frankly low or in the lower range of normal. The best rationale for its oral use is in patients with mild hypercalcemia due to primary hyperparathyroidism or in special circumstances surrounding the management of the secondary hyperparathyroidism of renal insufficiency (see Chapters 29 and 40).

Dialysis Hypercalcemia may occur in patients with either acute or chronic renal failure. A careful investigation for the cause of hypercalcemia is necessary in all patients with renal failure. Patients with hypercalcemia and renal failure pose a unique therapeutic dilemma. In general, hydration facilitates the renal excretion of calcium. However, in patients with renal failure, enhancing the renal excretion of calcium is not possible. In patients with significant renal failure, dialysis may be very effective in lowering the serum calcium concentration (89,90). Utilizing a low-calcium dialysate, either peritoneal dialysis or hemodialysis may be successfully performed. Reducing the dose of calcium-containing phosphate-binding agents may also be effective for long-term m a n a g e m e n t (91). Pamidronate and clodronate have been used successfully to treat hypercalcemia in a handful of patients on chronic dialysis (56,57). Some patients with advanced secondary hyperparathyroidism and severe hypercalcemia require parathyroidectomy for definite treatment of hypercalcemia.

Mobilization Bed rest is associated with a significant increase in the rate of bone resorption as well as reduced bone formation. Therefore, patients should be encouraged to ambulate as soon as possible so that this contribution to the hypercalcemic state can be prevented.

Choice of Agent The wide clinical spectrum of acute hypercalcemia prevents the use of a single therapeutic regimen for all

MANAGEMENT OF HYPF~RCALCENIA /

hypercalcemic patients. It is necessary to tailor the therapy based on a consideration of the cause of the hypercalcemia, the clinical symptomatology of the patient, and the m o d e of action and potential side effects of the various agents. With mild hypercalcemia (serum calcium concentration 16 m g / d l ) and is associated with clear symptomatology, more vigorous therapy is required along with saline. In this situation, the most rapidly acting osteoclast inhibitor, calcitonin, becomes a valuable drug. Because calcitonin alone seldom fully reverses hypercalcemia, immediate concurrent therapy should be considered. Based on safety profiles and efficacy, pamidronate is the treatment of choice. If the hypercalcemic state is likely to be sensitive to steroids, the concurrent administration of glucocorticoids is worthy of consideration. There are times when, despite the presence of marked hypercalcemia, the clinical appraisal does not lead to the same urgency to treat as in other situations. For example, in a patient whose serum calcium is high, > 1 4 m g / d l , but who has only modest signs or symptoms of hypercalcemia and is otherwise stable, one might use a bisphosphonate, along with modest saline administration. Finally, there is the rare patient in whom the serum calcium concentration is >20 m g / d l . Such a patient requires the most aggressive approach, with high rates of saline infusion, a bisphosphonate, calcitonin, and perhaps hydrocartisone as well if the patient has a hematologic malignancy.

T H E R A P Y OF T H E UNDERLYING DISORDER In most hypercalcemic patients, successful managem e n t of acute hypercalcemia is followed by reappearance of hypercalcemia if definitive therapy of the underlying disorder is not possible. The availability of potent bisphosphonates now allows more long-term control of hypercalcemia even if definitive treatment fails. This is of considerable importance in that the patient whose serum calcium is now normal is still very much subject to the same pathophysiologic mechanisms that originally p r o d u c e d the hypercalcemia. In patients with primary hyperparathyroidism, parathyroidectomy is nearly always successful in preventing recurrent hypercalcemia. Because most hypercalcemic cancer patients have advanced disease, a successful outcome for cancer therapy is much less likely. Nevertheless, satisfactory m a n a g e m e n t of acute hypercalcemia allows time to plan a more definitive approach to the underlying disease.

739

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24. Douglas DL, Russell RGG, Preston CJ, et al. Effect of dichloromethylene diphosphonate in Paget's disease of bone and in hypercalcaemia due to primary hyperparathyroidism or malignant disease. Lancet 1980;1043-1047. 25. Siris ES, Sherman WH, Baquiran DC, et al. Effect of dichloromethylene diphosphonate on skeletal mobilization of calcium in multiple myeloma. N E n g l J M e d 1980;302:310315. 26. Delmas PD, Charhon S, Chapuy MC, et al. Long-term effects of dichloromethylene diphosphonate (CIZMDP) on skeletal lesions in multiple myeloma. Metab Bone Dis Relat Res 1982;4:163-168. 27. Jung A, Chantraine A, Donath A, et al. Use of dichloromethylene diphosphonate in metastatic bone disease. N EnglJ Med 1983;308: 1499-1501. 28. Elomaa I, Blomqvist C, Porkka L, et al. Diphosphonates for osteolytic metastases. Lancet 1985;1:1155-1156. 29. Ryzen E, Martodam RR, Troxell M, et al. Intravenous etidronate in the management of malignant hypercalcemia. Arch Intern Med 1985;145:449-452. 30. Hasling C, Charles P, Mosekilde L. Etidronate disodium in the management of malignancy-related hypercalcemia. Am J Med 1987;82(Suppl. 2A) :51-54. 31. Kanis JA, Urwin GH, Gray RES, et al. Effects of intravenous etidronate disodium on skeletal and calcium metabolism. Am J Med 1987;82(Suppl. 2A):55-70. 32. Meunier PJ, Chapuy M-C, Delmas P, et al. Intravenous disodium etidronate therapy in Paget's disease of bone and hypercalcemia of malignancy: Effects on biochemical parameters and bone histomorphometry. A m J M e d 1987;82(Suppl. 2A):71-78. 33. Jacobs TP, Gordon AC, Silverberg SJ, et al. Neoplastic hypercalcemia: Physiologic response to intravenous etidronate disodium. A m J M e d 1987;82(Suppl. 2A):42-50. 34. Singer FR. Role of the bisphosphonate etidronate in the therapy of cancer-related hypercalcemia. Semin Oncol 1990;2 (Suppl. 5) :34-39. 35. Singer FR, Ritch PS, Lad TE, et al. Treatment of hypercalcemia of malignancy with intravenous etidronate. Arch Intern Med 1991; 151:471-476. 36. Flores JE Rude RK, Chapman RA, et al. Evaluation of a 24 hour infusion of etidronate disodium for the treatment of hypercalcemia of malignancy. Cancer 1994;73:2527-2534. 37. Ringenberg QS, Ritch PS. Efficacy of oral administration of etidronate disodium in maintaining normal serum calcium levels in previously hypercalcemic cancer patients. Clin Ther 1987;9:1-8. 38. Schiller JH, Rasmussen P, Benson AB, et al. Maintenance etidronate in the prevention of malignancy-associated hypercalcemia. Arch Intern Med 1987;147:963-966. 39. Mautalen C, Gonzalez D, Blumenfeld EL, et al. Spontaneous fractures of uninvolved bones in patients with Paget's disease during unduly prolonged treatment with disodium etidronate. Clin Orthop 1986;207:150-155. 40. Coukell AJ, Markham A. Pamidronate: A review of its use in the management of osteolytic bone metastases, tumor-induced hypercalcaemia and Paget's disease of bone. Drugs Aging 1998;12: 149-168. 41. Nussbaum SR, Younger J, VandePol CJ, et al. Single dose intravenous therapy with pamidronate for the treatment of hypercalcemia of malignancy: Comparison of 30-, 60-, and 90 mg dosages. A m J Med 1993;95:297-304. 42. Mannix KA, Carmichael J, Harris AL, et al. Single high-dose (45 mg) infusion of aminohydroxypylidene diphosphonate for severe malignant hypercalcemia. Cancer 1989;64:1358-1361. 43. Ralston SH, Gallagher SJ, Patel U, et al. Comparison of three intravenous bisphosphonates in cancer-associated hypercalcaemia. Lancet 1989;2:1180-1182. 44. Gucalp R, Ritch P, Wiernik PH, et al. Comparative study of pamidronate disodium and etidronate disodium in the treatment of cancer-related hypercalcemia. J Clin Oncol 1992;10:134-142.

45. Jansson S, Tisell LE, Lindstedt G, et al. Disodium pamidronate in the preoperative treatment of hypercalcemia in patients with primary hyperparathyroidism. Surgery 1991;110:480-486. 46. Mark S. Hypercalcaemia in an immobilized patient with pneumonia. B r J Clin Pract 1995;49:327-329. 47. McIntyre, Cameron DE Urquhart SM, et al. Immobilization hypercalcaemia responding to intravenous pamidronate sodium therapy. Post Grad MedJ 1989;65:244-246. 48. Selby PL, Davies M, Marks JS, et al. Vitamin D intoxication causes hypercalcaemia by increased bone resorption which responds to pamidronate. Clin Endocrino11995;43:531-536. 49. Gibb cJ, Peacock M. Hypercalcaemia due to sacoidosis corrects with bisphosphonate treatment. Postgrad MedJ 1986;62:937-938. 50. Hortobagy GN, Theriault RL, Porter L, et al. Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. N E n g l J Med 1996;335: 1785-1837. 51. Berenson JR, Lichtenstein A, Porter L, et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. N E n g l J Med 1996;334:488-493. 52. Pecherstorfer M, Herrmann Z, Body JJ, et al. Randomized phase II trial comparing different doses of the bisphosphonate ibandronate in the treatment of hypercalcemia of malignancy. J Clin Oncol 1996;14:268-276. 53. Body JJ, Lortholary A, Romieu G, et al. A dose-finding study of zoledronate in hypercalcemic cancer patients. J Bone Miner Res 1999; 14:1557-1561. 54. Bounameaux HM, Schifferli J, Montani JP, et al. Renal failure associated intravenous diphosphonates. Lancet 1983;1:471 (letter). 55. Machado CE, Flombaum CD. Safety of pamidronate in patients with renal failure and hypercalcemia. Clin Nephro11996;45:175-179. 56. Yap AS, Hockings GI, Fleming SJ, et al. Use of aminohydroxypropylidene bisphophonate (AHPrBE "APD") for the treatment of hypercalcemia in patients with renal impairment. Clin Nephrol 1990;34:225-229. 57. Hamdy NAT, McCloskey EV, Brown CB, et al. Effects of clodronate in severe hyperparathyroid bone disease in chronic renal failure. Nephron 1990;56:6-12. 58. Hosking DJ, Gilson D. Comparison of the renal and skeletal actions of calcitonin in the treatment of severe hypercalcaemia of malignancy. Q J Med 1984;53:359-368. 59. Silva O, Becker KL. Salmon calcitonin in the treatment of hypercalcemia. Arch Intern Med 1973;132:337--339. 60. Binstock ML, Mundy GR. Effect of calcitonin and glucocorticoids in combination on the hypercalcemia of malignancy. Ann Intern Med 1980;93:269. 61. Warrell RP, Israel R, Frisone M, et al. Gallium nitrate for acute treatment of cancer-related hypercalcemia: A randomized, doubleblind comparison to calcitonin. Ann Intern Med 1988;108:669-674. 62. Ralston SH. Medical management of hypercalcaemia. B r J Clin Pharmacol 1992;34:11-20. 63. Thiebaud D, Jacquet E Burckhardt E Fast and effective treatment of malignant hypercalcemia. Arch Intern Med 1990;150:2125-2128. 64. Ralston SH, Alzaid AA, Gardner MD, Boyle IT. Treatment of cancer associated hypercalcemia with combined aminohydroxypropylidene diphosphonate and calcitonin. Br MedJ 1986;292:1549-1550. 65. Fatemi S, Singer FR, Rude RK. Effect of salmon calcitonin and etidronate on hypercalcemia of malignancy. Calcif Tissue Int 1992;50:107-109. 66. Wisnecki LA. Salmon calcitonin in the acute management of hypercalcemia. Calcif Tissue Int 1990;46(Suppl.):526-530. 67. Goodwin JS, Atluru D, Sierakowski S, et al. Mechanism of action of glucocorticosteroids: Inhibition of T cell proliferation and interleukin 2 production by hydrocortisone is reversed by leukotriene B4. J Clin Invest 1986;77:1244.

1VIANAGEMENT OF HYPERCALCEMIA 68. Breslau NA, McGuire JL, Zerwekh JE, et al. Hypercalcemia associated with increased serum calcitriol levels in three patients with lymphoma. Ann Intern Med 1984;100:1-7. 69. Sandler LM, Winearls CG, Fraher LJ, et al. Studies of the hypercalcemia of sarcoidosis: Effect of steroids and exogenous vitamin D, on the circulating concentrations of 1,25-dihydroxyvitamin D. Q J Med 1984;53:165-180. 70. Percival RC, Yates AJP, Gray RES, et al. The role of glucocorticoids in the management of malignant hypercalcemia. Br Med J 1984;289:287. 71. Bilezikian JP. Hypercalcemic states. In: Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism. New York:Raven, 1992: 1493-522. 72. Stewart AF. Therapy of malignancy-associated hypercalcemia. Am J M e d 1983;74:475. 73. Perlia CP, Gubisch NJ, Cootter J, Edelberg D, Dederick MM, Taylor SG. Mithramycin treatment of hypercalcemia. Cancer 1970;25:389. 74. Minkin C. Inhibition of parathyroid hormone stimulated bone resorption in vitro by the antibiotic mithramycin. Calcif Tissue Res 1973;13:249-257. 75. Kiang DT, Loken MK, Kennedy BJ. Mechanism of the hypocalcemic effect of mithramycin. J Clin Endocrinol Metab 1979;48:341. 76. Thurlimann B, Waldburger R, Senn HJ, et al. Plicamycin and pamidronate in symptomatic tumor-related hypercalcemia: A prospective randomized crossover trial. Ann Oncol 1992;3: 619-622. 77. Green L, Donehower RC. Hepatic toxicity of low doses of mithramycin in hypercalcemia. Cancer Treat Rep 1984;68: 1379-1381. 78. Slavik M, Carter SK. Chromomycin AZ, mithramycin and olivomycin: Antitumor antibiotics of related structure. Adv Pharm Chem 1975;12:1-15. 79. Warrell RP, Jr, Bockman RS, Coonley CJ, et al. Gallium nitrate inhibits calcium resorption from bone and is effective treatment for cancer-related hypercalcemia. J Clin Invest 1984;73: 1487-1490.

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80. Bockman RS, Boskey AL, Alcock N, et al. Gallium nitrate increases bone calcium and crystallite perfection of hydroxyapatite. Calcif Tissue Int 1986;39:376--381. 81. Warrell RP, Bockman RS. Gallium in the treatment of hypercalcemia and bone metastases. In: Important advances in oncology. Philadelphia:Lippincott, 1989:205-220. 82. Hall TJ, Chambers TJ. Gallium inhibits bone resorption by a direct action on osteoclasts. Bone Miner 1990;8:211-216. 83. Warrell RP, Alcock NW, Buckman RS. Gallium nitrate inhibits accelerated bone turnover in patients with bone metastases. J Clin Onco11987;5:292-298. 84. Warrell RP, Israel R. Gallium nitrate for acute treatment of cancer-related hypercalcemia. Ann Intern Med 1988;108: 669-674. 85. Warrell RP, Murphy WK, Schulman P, et al. A randomized doubleblind study of gallium nitrate compared with etidronate for acute control of cancer-related hypercalcemia. J Clin Oncol 1991;9: 1467-1475. 86. Shackney S, Hasson J. Precipitous fall in serum calcium, hypotension and acute renal failure after intravenous phosphate therapy for hypercalcemia. Ann Intern Med 1967;66:906-916. 87. Vernava AM, O'Neal LW, Palermo V. Lethal hyperparathyroid crisis: Hazards of phosphate administration. Surgery 1987;102: 942-948. 88. Carey RW, Schmitt GW, Kopald HH. Massive extraskeletal calcification during phosphate treatment of hypercalcemia. Arch Intern Med 1968;122:150-155. 89. Cardella CJ, Birkin BL, Rapoport A. Role of dialysis in the treatment of severe hypercalcemia: Report of two cases successfully treated with hemodialysis and review of the literature. Clin Nephro11979; 12:285-290. 90. Heyburn PJ, Selby PL, Peacock M, et al. Peritoneal dialysis in the management of severe hypercalcaemia. Br MedJ 1980;280:525-526. 91. Goodman WG, Coburn JW, Slatopolsky E, et al. Renal osteodystrophy in adults and children. Flavus MJ, ed. Primer of the metabolic bone diseases and disorders of mineral metabolism. New York:LippincottRaven, 1996:341-360.

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CHAPTER 4 6

Primary Hyperparathyroidism and Other Causes of Hypercalcemia in Children and Adolescents

EMILY L. GERMAIN-LEE AND MICHAEL A. LEVINE Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

the child, and degree of hypercalcemia. Children may be more adaptive to hypercalcemia c o m p a r e d to adults, and infants and children with mild hypercalcemia (11-13 m g / d l or 2.75-3.25 m m o l / l i t e r ) may not have any symptoms (8). With m o d e r a t e to severe hypercalcemia, symptoms such as anorexia, vomiting, and constipation (rarely diarrhea) may occur. If hypercalcemia is chronic, there may be failure to thrive, which in the infant may be the only physical sign. Dehydration can occur rapidly because of the small size of an infant or child, and renal complications such as nephrocalcinosis, nephrolithiasis, or h e m a t u r i a may be the earliest clinical manifestation of hypercalcemia. Hypertension and increased cardiovascular tone may develop, as well as heart block and shortening of the ST segment. The neurologic symptoms can range from drowsiness or irritability to confusion; in the extreme cases, stupor and coma can ensue. T r e a t m e n t is aimed at the specific etiology, and is often complicated by the n e e d to consider the impact of therapy on growth and development. For infants with mild hypercalcemia, a low-calcium diet or low-calcium formula for the infant may be all that is necessary or practical. If emergency intervention is necessary because of severe hypercalcemia, the usual methods of t r e a t m e n t of hypercalcemia in adults are also used in children, although large, controlled studies showing efficacy and safety are often lacking (9-11). In this chapter, we place primary hyperparathyroidism within the context of the other disorders that cause hypercalcemia in children. We distinguish the neonate and infant from the older child and adolescent because

Hypercalcemia is far less c o m m o n l y detected in children than in adults. This is due in part to the relative infrequency with which serum calcium levels are measured in otherwise well children, but also because of the lower incidence of malignancy and primary hyperparathyroidism in the young. O n the other hand, the failure of many laboratories to report age-adjusted normal values for serum total and ionized calcium concentrations may lead to overdiagnosis of true hypercalcemia, because the u p p e r limit of normal calcium levels is slightly higher in children c o m p a r e d to adults (Table 1) (1-7). Hypercalcemia in neonates and early infancy is defined as a total serum calcium concentration consistently greater than 11.3 m g / d l ; ages 1-4 years, greater than 10.8 m g / d l ; ages 6-12 years, greater than 10.3 m g / d l ; and thereafter equivalent to adult normal ranges. The mechanisms of hypercalcemia in children are similar to those that occur in adults, but the smaller size and relative immaturity of the skeleton and kidney make children particularly sensitive to factors that affect renal handling of calcium and bone remodeling. An increase in net calcium mobilization from the skeleton is most often the cause of hypercalcemia, although excess intestinal absorption of calcium can also lead to hypercalcemia. Regardless of the underlying pathophysiology, hypercalcemia will occur when excessive transport of calcium from the skeleton a n d / o r gut into the extracellular fluid exceeds the ability of the kidney to excrete the increased filtered load. The clinical features are d e p e n d e n t on the underlying disorder, age of The Parathyroids, Second Edition

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TABLE 1 Representative Normal Values for Concentrations of Serum Total Calcium a Group Infants Children Men

Women

Age (years)

Serum total calcium (mg/dl)

0-0.25 1-5 6-12 20 50 70 20 50 70

8.8-11.3 9.4-10.8 9.4-10.3 9.1-10.2 8.9-10.0 8.8- 9.9 8.8-10.0 8.8-10.0 8.8-10.0

aRevised from Portale, AA, Chapter 19, Blood Calcium, Phosphorus, and Magnesium, p. 116 in Primer on the

Metabolic Bone Diseases and Disorders of Mineral Metabolism, Fourth Edition, Editor-in-Chief: Favus, MJ, 1999, Lippincott, Williams, & Wilkins.

the differential diagnoses of hypercalcemia in children are age dependent. Because of the strong association of genetic defects or epigenetic factors during pregnancy with childhood hypercalcemia, a thorough evaluation of hypercalcemia in the neonate, infant, and older child will generally require evaluation of mineral metabolism status in the biologic parents and siblings. Hypercalcemia that is chronic or inadequately treated can lead to special problems in children, including defects in growth and intellectual development, which can have a profound impact on the rest of the child's life. Thus, failure to recognize hypercalcemia in infants and children can lead to significant morbidity, even mortality, and accurate definition of the etiology of the hypercalcemia is critical in determining the prognosis and treatment.

DIAGNOSIS OF HYPERCALCEMIA IN NEONATES AND INFANTS The differential diagnosis of hypercalcemia in neonates and infants up to age 2 years is listed in Table 2 (10,12). Hypercalcemia in the neonatal period or during infancy not only leads to significant morbidity, but can also be life-threatening (10,13). Marked dehydration can occur rapidly in infants who develop polyuria as a consequence of renal resistance to vasopressin. The direct vasoconstrictive effect of calcium can lead to hypertension. Severe hypercalcemia can affect the nervous system and cause lethargy and seizures. In addition, there can be damage to the kidneys from nephrocalcinosis (8,10,12). The urinary excretion of calcium in children differs significantly from that in adults (14). On a diet con-

TABLE 2 Differential Diagnosis of Hypercalcemia in Neonates and Infants (up to 2 Years of Age) latrogenic Phosphate depletion Premature infants on human milk or standard formula Parenteral nutrition Hyperparathyroidism Congenital parathyroid hyperplasia Maternal hypoparathyroidism Inactivating mutations in Ca2+-sensing receptor gene Familial hypocalciuric hypercalcemia (familial benign hypercalcemia) Neonatal severe hyperparathyroidism Jansen's metaphyseal chondrodysplasia Persistent PTHrP Hypervitaminosis D Subcutaneous fat necrosis Williams syndrome/idiopathic infantile hypercalcemia Other inborn metabolic disorders Blue diaper syndrome Lactase deficiency Bartter syndrome Hypophosphatasia IMAGe Down syndrome Severe congenital hypothyroidism Maternal hypercalcemia Vitamin A intoxication

taining approximately 1400 m g / d a y calcium, the average daily calcium output of a child u n d e r 4 years of age is approximately 25-50 mg/day, and for ages 5-14 approximately 75-100 mg/day. On a weight basis this corresponds to a urinary calcium excretion of 2-4 m g / k g / d a y . In infants and younger children it is usually very difficult to obtain a 24-hour urine collection. In these cases a random urine sample can be collected and the calcium:creatinine ratio is determined. These values are age d e p e n d e n t and decline gradually in the first several years of life (15). The u p p e r limits of normal for ratios that have been calculated from molar (gravitometric) concentrations of calcium and creatinine are as follows: less than 7 months, 2.42 (0.86); 7-18 months, 1.69 (0.60); 19 months to 6 years, 1.18 (0.42); and adults, 0.61 (0.22). In neonates, symptoms and signs of hypercalcemia are difficult to detect, and hypercalcemia is typically discovered when a chemistry panel is obtained to evaluate failure to thrive. Hypercalcemia is frequently secondary to iatrogenic causes, such as excessive calcium supplementation or use of extracorporeal membrane oxygenation in the critically ill infant. Phosphate deple-

HVeVgCaLCWMtAIN CHILDREN / tion can result from human milk feeding in preterm, very-low-birth weight infants (10,12,16). This can cause not only mild hypercalcemia, but may also lead to an unusual condition termed "breast milk-induced rickets of prematurity" (10,12,17). This has become rare since the introduction of breast milk fortifiers that contain 30-40 m g / k g / d a y of phosphorus as disodium phosphate (18). Feeding preterm infants a regular-term infant formula also causes hypophosphatemia. Finally, a common cause of phosphate depletion in the hospitalized neonate is from inappropriately supplemented parenteral nutrition (19). Phosphate deficiency activates bone resorption and impairs bone formation. In addition, hypophosphatemia stimulates synthesis of 1,25-dihydroxyvitamin D [ 1,25 (OH) 2o ], which increases intestinal absorption of calcium.

NEONATAL HYPERPARATHYROIDISM Neonatal hyperparathyroidism is quite rare. It is usually due to parathyroid chief cell hyperplasia rather than to parathyroid adenoma as in older patients (20). Hyperparathyroidism may be sporadic or inherited, and both autosomal dominant and autosomal recessive patterns of transmission have been reported (21,22). Infants with this condition have very high serum levels of parathyroid hormone (PTH) and calcium along with low serum levels of phosphate and either normal or elevated alkaline phosphatase levels. At birth there may be evidence of severe bone deformities secondary to inadequate mineralization, as well as multiple fractures. Respiratory difficulties may arise if the rib cage is affected. There may be destructive lesions of metaphyseal ends of long bones as well as poor mineralization of the lateral ends of the clavicles. Hepatosplenomegaly and anemia may be present. The conventional treatment of neonatal hyperparathyroidism is subtotal parathyroidectomy, and failure to normalize the serum calcium level can have profound developmental implications (23). Infants with neonatal hyperparathyroidism secondary to maternal hypocalcemia (most commonly from maternal hypoparathyroidism) usually are not as hypercalcemic as those infants with primary hyperparathyroidism. In fact, only 25% of the cases have hypercalcemia (24). Transient neonatal hyperparathyroidism has also been reported in association with maternal pseudohypoparathyroidism (25) as well as maternal renal tubular acidosis (26,27). Treatment of secondary or adaptive neonatal hyperparathyroidism usually consists of no more than providing an appropriate calcium and phosphorus supply in the milk. Hyperparathyroidism typically resolves within a few weeks.

745

The most common cause of neonatal hyperparathyroidism is related to genetic defects that cause familial hypocalciuric hypercalcemia (FHH) [also termed familial benign hypercalcemia (FBH)], an autosomal dominant trait characterized by moderate hypercalcemia and relative hypocalciuria (see Chapter 38). There is virtually 100% penetrance for hypercalcemia among heterozygotes for the FHH gene, and hypercalcemia can occur as early as the first week of life. By contrast, other autosomal dominant syndromes associated with parathyroid hyperplasia, such as multiple endocrine neoplasia (MEN) type 1 or MEN type 2 (see Chapters 35 and 36), are unlikely to cause neonatal hypercalcemia because of the later age of onset of hyperparathyroidism in these disorders. Inactivating mutations in the CaZ+-sensing receptor gene, localized to chromosome 3q, can lead to both neonatal severe hyperparathyroidism (NSHPT) and FHH (28-34). In many families, NSHPT and FHH are the respective homozygous and heterozygous manifestations of the same genetic defect (28,30,32,33). NSHPT can also result from heterozygous offspring born to affected fathers but unaffected normocalcemic mothers, or in neonates with an apparent de n o v o heterozygous muta2+ tion in the Ca -sensing receptor gene (35). Mutations that inactivate the CaZ+-sensing receptor in patients with NSHPT and FHH have been found scattered throughout the gene, and most families have private mutations. Genetic testing is now available, but requires molecular analysis of the entire gene. Loss of 50% of CaZ+-sensing receptors decreases the sensitivity of the parathyroid cells to extracellular Ca 2+, and leads to mild parathyroid hyperplasia and elevated circulating levels of PTH. Decreased receptor activity in the kidney is thought to account for relative hypocalciuria, the hallmark of the disorder. Although FHH and NSHPT have been linked to the CaZ+-sensing receptor gene on 3q in nearly all families, the disorder has been also linked to the long (36) and short (37) arms of chromosome 19, suggesting genetic heterogeneity for this disorder. In NSHPT, the PTH is quite high and the associated hypercalcemia is severe enough to be life-threatening. In addition, affected infants have hypophosphatemia and osteopenia at birth (34) and may die within a few days if the hypercalcemia is not treated aggressively (38). Children who survive NSPHT but who remain hypercalcemic are at risk of significant impairment in cognitive development (23). Jansen's metaphyseal chondrodysplasia resembles primary hyperparathyroidism in many respects, but levels of circulating PTH are suppressed. This unusual syndrome is caused by heterozygous mutations in the P T H / P T H r P receptor (39), found in the kidney, bone, and growth plate, that lead to constitutive (i.e., ligand independent) activation of the receptor

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(see Chapter 44). The activation of P T H / P T H r P receptor signaling increases bone resorption, leading to hypercalcemia, and impairs chondrocyte differentiation, leading to growth plate defects that cause postnatal short-limbed dwarfism. The skeletal abnormalities are seen on radiologic evaluation from the time of birth and include irregularities of the metaphyses of the long bones and rachitic changes. However, the physical appearance of infants with Jansen's syndrome is normal, including body length. During late childhood, however, the characteristic features gradually appear, including hypertelorism, mandibular hypoplasia, and short-limbed dwarfism. The pathophysiology of hypercalcemia in Jansen's syndrome shares many features with primary hyperparathyroidism (40), but the suppressed serum PTH levels, metaphyseal defects, and growth delay are conclusive distinguishing characteristics. Persistent expression of PTHrP has been reported as a cause of neonatal hypercalcemia (38,41), but description of additional cases will be required to confirm this pathophysiology.

common clinical sign associated with subcutaneous fat necrosis, which is associated with a surprisingly high 15% mortality (44). Williams syndrome is a sporadic disorder associated with hypercalcemia in approximately 15 % of cases. The hypercalcemia typically occurs during infancy and resolves between 2 and 4 years of age (47,48). There are cases, however, of older children and adults who have persistent hypercalcemia (49). There may also be associated nephrocalcinosis and soft-tissue calcifications. Children with Williams syndrome have a characteristic appearance at or soon after birth, which first suggested that there was a problem during intrauterine development (8). The physical features consist of an "elfin" facies (secondary to poor development of the facial bones) with epicanthal folds, hypertelorism, strabismus, bitemporal depressions, periorbital prominence, full cheeks, prominent nasal tip, long philtrum, prominent lips and mouth, and dolicocephaly (Fig. 1). They also have clinodactyly of the fifth fingers and hypoplas-

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N O N P A R A T H Y R O I D CAUSES OF HYPERCALCEMIA Neonates whose mothers ingested excessive amounts of vitamin D a n d / o r its derivatives during pregnancy can develop hypercalcemia (8,25). This usually occurs during treatment of the mother for a hypocalcemic disorder, but can occur by self-medication. Rarely, an infant will be given excessive vitamin supplements over a long period of time that leads to hypervitaminosis D. Approximately 2000 units/kg body weight per day as vitamin O 2 o r vitamin D~ over a period of many months will cause hypercalcemia and hypercalciuria in most patients, and 20,000-40,000 units per day has led to fatal hypercalcemia in infants (8). The earliest evidence of vitamin D intoxication may be the development of renal complications such as polyuria, hematuria, or nephrocalcinosis. Subcutaneous fat necrosis is common in neonates with a complicated delivery and may lead to hypercalcemia within days or weeks of birth. Hypercalcemia results from excess circulating 1,25(OH)zD that is produced by macrophages present within the granulomatous reaction to the necrotic fat. The hypercalcemia is also compounded by calcium release from fat tissues and increased prostaglandin E activity (42-44). The macrophages express ectopic 25 (OH) D~-l-0t-hydroxylase activity that is not regulated by PTH, calcium, phosphorus, or 1,25(OH)zD but that is responsive to glucocorticoids (45,46). Subcutaneous fat necrosis is found in areas of direct trauma that occur during a difficult birth process, such as with forceps or vacuum extraction. These infants often have a history of birth asphyxia as well. Failure to thrive is the most

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FIG. 1 Williams syndrome. An 8-year-old boy with characteristic facial features including epicanthal folds, hypertelorism, bitemporal depressions, periorbital prominence, full cheeks, prominent lips and mouth, and long nasal philtrum. Photograph from Aarskog D and Harrison H, Chapter 17, Disorders of Calcium, Phosphate, PTH, and Vitamin D, p. 1087 in Wilkins' the diagnosis and treatment of endocrine disorders in childhood and adolescence, Fourth edition, Editors: Kappy M, Blizzard RM, and Migeon CJ; 1994, Charles C. Thomas.

HYPERCALCEMIA IN CHILDREN

tic nails. They may have kyphoscoliosis, pecmm excavatum, and an abnormal gait. There are associated cardiac abnormalities, the most typical of which is supravalvular aortic stenosis (30% of affected patients), as well as valvular aortic stenosis, pulmonic stenosis, atrial septal defect, and ventricular septal defect. Many children with Williams syndrome grow poorly from very early in infancy and have general developmental delay. Interestingly, they are quite sociable ("cocktail personality") and do not appear delayed in linguistic abilities (50-53). Williams syndrome has been associated with loss of genetic material at 7q11.13, and likely represents a contiguous gene deletion that typically includes the elastin gene (ELN) (54-56). Hemizygosity of the E L N gene likely accounts for the associated cardiac defects, but cannot explain the hypercalcemia or phenotypic features (57,58). Williams syndrome has also been associated with other chromosomal abnormalities, including an interstitial deletion of chromosome 6(q22.2q23) (59), a terminal deletion of chromosome 4146,XX,del(4)(q33)] (60), as well as chromosomal translocations (61,62). However, the definitive basis remains unknown (63). In most cases, fluorescent in situ hybridization (FISH) using E L N is diagnostic (55,64). Patients with this syndrome have an exaggerated response to pharmacologic doses of vitamin D 2 (65) and a blunted calcitonin response to calcium loading (50). Elevated plasma concentrations of 1,25(OH)zD have been reported in some patients despite circulating levels of PTH that are low or normal (66,67). However, studies have failed to show any consistent abnormality in the metabolism of vitamin D that might explain these features. Some children with hypercalcemia show similar disturbances in vitamin D sensitivity but lack other phenotype features of Williams syndrome and do not have a 7q11.13 deletion. This condition, which may be familial, has been termed idiopathic infantile hypercalcemia (IIH) (68,69). The hypercalcemia in IIH usually resolves within the first few years of life, but persistent hypercalciuria is common. Clinical evaluation and genetic testing provide the ability to differentiate between Williams syndrome and IIH in more than 90% of cases.

I N B O R N ERRORS OF METABOLISM THAT CAUSE HYPERCALCEMIA Many inborn disorders of metabolism are associated with hypercalcemia. Blue diaper syndrome is caused by a defect in tryptophan metabolism (70). The block in tryptophan metabolism leads to urinary excretion of excessive amounts of indole derivatives, including a derivative called "indican" that gives the urine-soaked diaper a blue tint. The mechanism of hypercalcemia in this disorder is unknown.

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Congenital lactase deficiency can cause hypercalcemia during the first few months of life. Seven out of ten infants evaluated with this condition had hypercalcemia, and five of the seven had medullary nephrocalcinosis. The hypercalcemia resolved after initiation of a lactose-free diet, but later in childhood (ages 2 to 10 years) one of the patients still had hypercalciuria and three of the patients had nephrocalcinosis (71). The etiology of the hypercalcemia is unclear, but is thought to be related to metabolic acidosis a n d / o r an increase in intestinal calcium absorption secondary to increased gut lactose (71). Bartter syndrome, due to homozygous inactivation in the gene for either the furosemide-sensitive Na/K/2C1 cotransporter NKCC2 (SLC12A1) or the inwardly rectifying potassium channel ROMK (KCNJ1) (72,73), is a rare cause of neonatal hypercalcemia, but is more commonly associated with hypercalciuria (74,75). The neonate typically presents with vomiting, diarrhea, fever, and resultant failure to thrive. The mothers of these infants are often found to have intrauterine polyhydramnios, thought secondary to fetal polyuria, and deliver prematurely. Hypophosphatasia, an inherited condition that results from deficient bone alkaline phosphatase activity, can also cause hypercalcemia (76-80). Hypophosphatasia is classified into four forms: perinatal hypophosphatasia is the most severe form, and can be lethal in utero or shortly after birth because of inadequate thorax and skull formation. Infants live for a few days at most. Infantile hypophosphatasia presents before age 6 months and can cause pronounced hypercalcemia. Calcium is deposited inadequately into bone, which leads to hypercalcemia with hypercalciuria and nephrocalcinosis. Severe rickets ensues. The diagnosis can be confirmed by finding a very high level of phosphoethanolamine in the urine as well as radiographic evidence of severe bone demineralization and endochondral ossification defects. Although there is no known treatment for this condition, one patient improved after serum transfusions, presumably secondary to a circulating factor that activates alkaline phosphatase at the posttranscriptional level (80). The IMAGe syndrome consists of intrauterine growth retardation, metaphyseal dysplasia, and adrenal hypoplasia congenita (81). The three patients reported with this syndrome also had hypercalcemia a n d / o r hypercalciuria that led to nephrocalcinosis in one patient and prenatal liver and spleen calcifications in another. DAX1 gene mutations, which are associated with congenital adrenal hypoplasia, were not found in these three patients, however. Neither the molecular defect nor the basis for hypercalcemia in this newly recognized syndrome has been identified. There are several reports of hypercalcemia, hypercalciuria, and nephrocalcinosis in infants and toddlers with

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Down syndrome (81a-d). The etiology of the hypercalcemia was initially thought to be secondary to overingestion of cow's milk. However, this has not been found in all cases. The hypercalcemia is now thought to be specifically associated with the genetic defect(s) of Down syndrome, although the mechanism remains unclear.

O T H E R CAUSES OF H Y P E R ~ C E M I A THE NEONATE AND INFANT

IN

Severe congenital hypothyroidism (athyreotic cretinism) is rarely seen in the United States because of the initiation of newborn screening programs. The hypercalcemia in severe congenital hypothyroidism is associated with hypercalciuria, and renal calculus formation has been described. Radiologic studies do not reveal skeletal demineralization, which suggests that the hypercalcemia is secondary to hyperabsorption of calcium. The growth failure of these infants may contribute to the hypercalcemia as well because there is little skeletal uptake of bone mineral. Maternal hypercalcemia can result in transient neonatal hypercalcemia. Unusual causes of maternal hypercalcemia include medications such as lithium or thiazide diuretics as well excessive use of calcium supplements, thyroid hormone, or vitamins D or A. Vitamin A intoxication is usually the result of vitamin supplementation but can also arise from the ingestion of fish oils with a high concentration of vitamin A (82). There have been cases of vitamin A intoxication with resulting hypercalcemia in infants fed chicken liver (83).

HYPERCALCEMIA IN OLDER CHILDREN The differential diagnosis of hypercalcemia in children and adolescents (Table 3) is similar to, but broader than, the differential diagnosis for infants. Because children and adolescents are growing rapidly, the first sign of the hypercalcemia may be poor weight gain a n d / o r poor linear growth. Children with medical conditions requiring calcium supplementation may have excessive calcium intake. Phosphate depletion can occur in any child with an acute or chronic illness and can occasionally lead to hypercalcemia. Children on parenteral nutrition are also at a greater risk for develo p m e n t of hypophosphatemia.

HYPERPARATHYROIDISM IN CHILDREN AND ADOLESCENTS Primary hyperparathyroidism is usually acquired in the child or adolescent, and is nearly always due to a

TABLE 3 Hypercalcemia in Children (over 2 Years of Age) and Adolescents Excessive calcium intake Phosphate depletion Parenteral nutrition Hyperparathyroidism Aquired primary Adenoma Hyperplasia Genetic primary Autosomal dominant/recessive Familial MEN types 1 and 2A (2B) Familial hypocalciuric hypercalcemia Autonomous (tertiary) Hypervitaminosis D Excessive intake Granulomatous diseases: cat scratch fever, sarcoidosis, tuberculosis, histoplasmosis, coccidiomycosis, leprosy Chronic inflammatory disorders Williams syndrome/idiopathic infantile hypercalcemia Immobilization Malignancy-associated hypercalcemia Primary bone tumors Metastatic tumors with osteolysis Tumors secreting PTHrP, prostaglandins, cytokines, growth factors Hepatic disease Hyperthyroidism Adrenal insufficiency Pheochromocytoma Vasoactive intestinal polypeptide-secreting tumor Drugs (thiazides, lithium, systemic retinoid derivatives, theophylline, acetosalicylic acid) Milk-alkali syndrome/calcium gluttony

single a d e n o m a of the parathyroid glands (see Chapter 20). The age range is from 3 to 15 years with a mean of 12.8 years and an equal sex incidence (20,50). Primary hyperparathyroidism is far less c o m m o n in children and adolescents than in adults. From a review of several studies, only 7 out of 514 patients with primary hyperparathyroidism were less than 19 years old (84). Primary hyperparathyroidism may also be genetic, and is often the presenting manifestation of MEN type 1 and less commonly MEN type 2 (see Chapters 35 and 36). Older children with asymptomatic hypercalcemia may have FHH. Hypercalcemia can develop in patients with chronic renal failure who have developed autonomous (so-called tertiary) hyperparathyroidism. This can also occur in children with hypophosphatemic rickets who have been treated with phosphate supplements and inadequate calcitriol (85).

HYPERCALCEMIA IN CHILDREN /

N O N P A R A T H Y R O I D CAUSES OF HYPERCALCEMIA IN O L D E R CHILDREN AND ADOLESCENTS Hypervitaminosis D can result from ingestion of excessive amounts of vitamin D (or its metabolities) for medical conditions such as hypoparathyroidism and rickets, or for nutritional supplementation. Granulomatous disease is associated with ectopic expression of 25(OH) D~-l-ot-hydroxylase activity in activated macrophages (see above, neonatal subcutaneous fat necrosis) (46,86). Infectious diseases such as cat scratch fever (87) as well as histoplasmosis, coccidiomycosis, leprosy, and tuberculosis have all been associated with hypercalcemia in children. Tuberculosis is showing a resurgence in the United States and is especially important to consider in children who live in or emigrate from countries with higher incidences. The usual source of infection is someone close to the children in their home, but there have been recent outbreaks of childhood tuberculosis in schools (nursery, elementary, and secondary), day care centers, school buses, and sports teams. Sarcoidosis occasionally has its onset in childhood, although hypercalcemia is very unusual in the younger age groups (50). Chronic inflammatory diseases such as collagen vascular diseases, including systemic lupus erythematosus and rheumatoid arthritis, can lead to hypercalcemia, as can the h u m a n immunodeficiency virus.

IMMOBILIZATION Immobilization is a very important and c o m m o n cause of hypercalcemia in children and adolescents. Immobilization of a rapidly growing child will lead to a marked decrease in osteoblastic bone formation and a dramatic increase in osteoclastic bone resorption. This imbalance in bone remodeling causes increased movement of calcium (and phosphorous) out of the skeleton with a consequent net loss of bone mass that is termed disuse osteoporosis (88). Hypercalciuria can develop within a few days of immobilization, and hypercalcemia may follow within 1 to 3 weeks. The most typical scenario is the active adolescent boy who has suffered a femur fracture. The sudden transition from an active physical life to complete immobilization, especially if both extremities are immobilized, can cause severe hypercalcemia. In one study, 6 of 12 children who were immobilized following fracture of a single weight-bearing bone developed hypercalcemia (89). Infants and children who have any disorder causing limited mobility, especially those who are wheelchairb o u n d or bedridden, are at high risk for developing immobilization hypercalcemia. As continued improve-

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ments in chronic care now make long-term survival possible for more children with spinal cord injuries or severe head trauma, neuromuscular disorders, cerebral palsy, and spina bifida, it is likely that immobilizationinduced skeletal problems will be more easily recognized. For now, however, it is all too c o m m o n for hypercalcemia to be overlooked in an immobilized child. The child's anorexia, nausea, weight loss, lethargy, and depression might be attributed to hospitalization and immobilization rather than hypercalcemia. The recent demonstration that bisphosphonates can rapidly reverse the hypercalcemia and hypercalciuria of immobilization provides additional justification to monitor young patients for the development of these complications (90,91).

MALIGNANCY-ASSOCIATED HYPERCALCEMIA Malignancy associated hypercalcemia occurs in less than 1% of children with cancer (92). Hypercalcemia has been associated with many kinds of cancer in children, including leukemia, lymphoma, myeloma, neuroblastoma, hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma, and brain and ovarian tumors (92-99). There are two mechanisms that account for the development of hypercalcemia with nonparathyroid tumors: (1) direct invasion of the skeleton by tumor cells and (2) tumor secretion of humoral factors such as PTHrP (see Chapter 42), prostaglandins, interleukin-1 and interleukin-6, transforming growth factor-e¢, tumor necrosis factor, or calcitriol, which activate osteoclastic activity.

O T H E R CAUSES OF HYPERC&LCEMIA Liver diseases such as hepatitis and hepatic failure can cause hypercalcemia in children (100-102). Although this can occur in the infant, it is more comm o n in the older child and adolescent. The basis of the hypercalcemia in these conditions is not clear. Endocrine disorders, including hyperthyroidism (50), adrenal insufficiency, pheochromocytomas, and vasoactive intestinal polypeptide-secreting tumors, have also been d o c u m e n t e d to cause hypercalcemia in children, but are more c o m m o n in adults (see Chapter 41). A variety of drugs other than calcium and vitamin D (and its metabolites) can cause hypercalcemia in older children. Most importantly, systemic retinoic acid derivatives used to treat acne in the adolescent can lead to vitamin A intoxication and hypercalcemia. Theophylline and acetylsalicylic acid can also raise serum calcium levels (46,103). Milk-alkali syndrome and calcium gluttony, most commonly due to the

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excessive intake of calcium-containing antacids or calcium supplements, can lead to hypercalcemia, hypercalciuria, and systemic alkalosis.

TREATMENT The principles of treatment of hypercalcemia in children are similar to those used in the m a n a g e m e n t of adults. Parathyroid surgery, the primary treatment modality for primary hyperparathyroidism in adults, is also the preferred treatment for symptomatic primary hyperparathyroidism in infants and young children. Indeed, the younger age of pediatric patients provides an even more compelling justification for recommending surgery for most patients. Of course, hypercalcemic children with FHH will rarely require any intervention unless they have neonatal severe hyperparathyroidism, in which event urgent parathyroidectomy may be necessary. Due to the low frequency of hypercalcemia in children, comprehensive clinical trials on the safety and efficacy of new medical treatments such as the bisphosphonates are lacking, although recent small studies have reported promising results (9,11,91,102,104,105). Medical therapy of hypercalcemia in children requires special consideration of the long-term effects of many of these agents (such as glucocorticoids and bisphosphonates) on growth and development of the skeleton, as well as on other organ systems. Calcitonin tends to be used more frequently in children because it has no long-term sequelae, whereas steroids are used less frequently than in adults because they result in poor linear growth and osteoporosis. Children with Williams syndrome or idiopathic infantile hypercalcemia have mildly elevated serum levels of 1,25-dihydroxyvitamin D, and a low-calcium formula in the infant or reducedcalcium diet in the older child may be all that is needed to treat the hypercalcemia a n d / o r hypercalciuria, particularly when long-term treatment will be necessary. CalciloXD (Ross Laboratories, North Chicago, IL), a low-calcium infant formula without vitamin D, is commonly used. As the hypercalcemia improves, the CalciloXD can be gradually mixed with regular formula or breast milk. The infants and children on the lowcalcium diet need to be followed closely, however, for the possible development of hypocalcemia and rickets. In children, unlike adults, growth is a very valuable clinical parameter to monitor for efficacy of treatment.

SUMMARY Primary hyperparathyroidism and other causes of hypercalcemia occur far less commonly in children than in adults. Hypercalcemia can have a subtle clinical

presentation, with failure to thrive as the only sign. The etiology of hypercalcemia in children is age d e p e n d e n t and includes a broad differential diagnosis. Although these conditions are not common, it is nevertheless important not to overlook them, as untreated hypercalcemia can have a p r o f o u n d impact on a child's growth and development.

ACKNOWLEDGMENTS This work has been supported in part by grants from the National Institutes of Health (DK-34281 and DK56178 and GCRC M01-RR00052).

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50. Aarskog D, Harrison HE. Disorders of calcium, phosphate, PTH and vitamin D. In: Kappy MS, Blizzard RM, Migeon CJ, eds. Wilkins' the diagnosis and treatment of endocrine disorders in childhood and adolescence. Springfield, Illinois:Thomas, 1994:1068-1069. 51. Dilts CV, Morris CA, Leonard CO. Hypothesis for development of a behavioral phenotype in Williams syndrome. AmJMed Genet Suppl 1990;6:126-131. 52. Jones W, Bellugi U, Lai Z, Chiles M, ReillyJ, Lincoln A, Adolphs R. Ii. Hypersociability in Williams Syndrome. J Cogn Neurosci 2000;12(Suppl. 1):30-46. 53. Morris CA, Carey JC. Three diagnostic signs in Williams syndrome. A m J Med Genet Supp11990;6:100-101. 54. Ewart AK, Morris CA, Atkinson D, Jin W, Sternes K, Spallone P, Stock AD, Leppert M, Keating MT. Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome. Nat Genet 1993;5:11-16. 55. Lowery MC, Morris CA, Ewart A, Brothman LJ, Zhu XL, Leonard CO, CareyJC, Keating M, Brothman AR. Strong correlation of elastin deletions, detected by FISH, with Williams syndrome: Evaluation of 235 patients. Am J Hum Genet 1995;57:49-53. 56. Meng X, Lu X, Li Z, Green ED, Massa H, Trask BJ, Morris CA, Keating MT. Complete physical map of the common deletion region in Williams syndrome and identification and characterization of three novel genes. Hum Genet 1998;103: 590-599. 57. Urban Z, Peyrol S, Plauchu H, Zabot MT, Lebwohl M, Schilling K, Green M, Boyd CD, Csiszar K. Elastin gene deletions in Williams syndrome patients result in altered deposition of elastic fibers in skin and a subclinical dermal phenotype. Pediatr Dermato12000; 17:12-20. 58. Zhang J, Kumar A, Roux K, Williams CA, Wallace MR. Elastin region deletions in Williams syndrome. Genet Test 1999;3: 357-359. 59. Bzduch V, Lukacova M. Interstitial deletion of the long arm of chromosome 6(q22.2q23) in a boy with phenotypic features of Williams syndrome [letter]. Clin. Genet. 1989;35:230-231. 60. Jefferson RD, Burn J, Gaunt KL, Hunter S, Davison EV. A terminal deletion of the long arm of chromosome 4 [46,XX,del(4) (q33) ] in an infant with phenotypic features of Williams syndrome. J Med Genet 1986;23:474-477. 61. Telvi L, Pinard JM, Ion R, Sinet PM, Nicole A, Feingold J, Dulac O, Pompidou A, Ponsot G. De novo t(X;21)(q28;ql 1) in a girl with phenotypic features of Williams-Beuren syndrome. J Med Genet 1992;29:747-749. 62. von Dadelszen P, Chitayat D, Winsor EJ, Cohen H, MacDonald C, Taylor G, Rose T, Hornberger LK. De novo 46,XX,t(6;7) (q27;qll;23) associated with severe cardiovascular manifestations characteristic of supravalvular aortic stenosis and Williams syndrome. Am J Med Genet 2000;90:270-275. 63. Peoples R, Franke Y, Wang YK, Perez-Jurado L, Paperna T, Cisco M, Francke U. A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome---deletion region at 7ql 1.23. A m J H u m Genet 2000;66:47-68. 64. Dewan K, Borgaonkar DS, Bartoshesky LE, Tuttle D. Micro-deletion detected by fluorescent in situ hybridization for Williams syndrome. Del MedJ 1999;71:467-469. 65. Taylor AB, Stern PH, Bell NH. Abnormal regulation of circulating 25-hydroxyvitamin D in the Williams syndrome. NEnglJMed 1982;306:972-975. 66. Garabedian M, Jacqz E, Guillozo H, Grimberg R, Guillot M, Gagnadoux ME Broyer M, Lenoir G, Balsan S. Elevated plasma 1,25-dihydroxyvitamin D concentrations in infants with hypercalcemia and an elfin facies. N Engl J Med 1985; 312:948-952.

67. KnudtzonJ, Aksnes L, Akslen LA, Aarskog D. Elevated 1,25-dihydroxyvitamin D and normocalcaemia in presumed familial Williams syndrome. Clin Genet 1987;32:369-374. 68. Martin ND, Snodgrass GJ, Cohen RD, Porteous CE, Coldwell RD, Trafford DJ, Makin HL. Vitamin D metabolites in idiopathic infantile hypercalcaemia. Arch Dis Child 1985;60:1140-1143. 69. McTaggart SJ, Craig J, MacMillan J, Burke JR. Familial occurrence of idiopathic infantile hypercalcemia. Pediatr Nephrol 1999;13:668-671. 70. Drummond KN, Michael AF, Ulstrom RA, Good RA. The blue diaper syndrome: Familial hypercalcemia with nephrocalcinosis and indicanuria. Am J Med 1964;37:928-948. 71. Saarela T, Simila S, Koivisto M. Hypercalcemia and nephrocalcinosis in patients with congenital lactase deficiency. J Pediatr 1995;127:920-923. 72. Amirlak I, Dawson KP. Bartter syndrome: An overview. QJMed 2000;93:207-215. 73. Bettinelli A, Ciarmatori S, Cesareo L, Tedeschi S, Ruffa G, Appiani AC, Rosini A, Grumieri G, Mercuri B, Sacco M. et al. Phenotypic variability in Bartter syndrome type I. Pediatr Nephrol 2000; 14:940-945. 74. Seyberth HW, Rascher W, Schweer H, Kuhl PG, Mehls O, Scharer K. Congenital hypokalemia with hypercalciuria in preterm infants: A hyperprostaglandinuric tubular syndrome different from Bartter syndrome. JPediatr 1985; 107:694-701. 75. Shoemaker L, Welch TR, Bergstrom W, Abrams SA, Yergey AL, Vieira N. Calcium kinetics in the hyperprostaglandin E syndrome. Pediatr Res 1993;33:92-96. 76. Fedde KN, Blair L, SilversteinJ, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR, et al. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 1999;14:2015-2026. 77. Mornet E. Hypophosphatasia: The mutations in the tissuenonspecific alkaline phosphatase gene. Hum Mutat. 2000;15: 309-315. 78. Mochizuki H, Saito M, Michigami T, Ohashi H, Koda N, Yamaguchi S, Ozono K. Severe hypercalcaemia and respiratory insufficiency associated with infantile hypophosphatasia caused by two novel mutations of the tissue-nonspecific alkaline phosphatase gene. EurJ Pediatr 2000;159:375-379. 79. Teree TM, Klein LR. Hypophosphatasia: Clinical and metabolic studies. J Pediatr 1968; 72:41-50. 80. Whyte ME Magill HL, Fallon MD, Herrod HG. Infantile hypophosphatasia: Normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J Pediatr 1986;108:82-88. 81. Vilain E, Le Merrer M, Lecointre C, Desangles E Kay MA, Maroteaux P, McCabe ER. IMAGe, a new clinical association of intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies. J Clin Endocrinol Metab 1999;84:4335-4340. 81a. Cobefias C, Spizzirri F, Zanetta D. Another toddler with Down Syndrome, nephrocalcinosis, hypercalcemia, and hypercalciuria. Pediatr Nephrol 1998;12:432. 8lb. Manz E A toddler with Down Syndrome, hypercalcaemia, hypercalciuria, medullary nephrocalcinosis and renal failure. Pediatr Nephrol 1996;10:251. 81c. Andreoli SP, Revkees S, Bull M. Hypercalcemia, hypercalciuria, medullary nephrocalcinosis and renal insufficiency in a toddler with Down syndrome. Pediatr Nephro11995;9:673. 81d. Proesmans W, DeCock P, Eyskens B. A toddler with Down Syndrome, hypercalcaemia, hypercalciuria, medullary nephrocalcinosis and renal failure. Pediatr Nephro11995;9:112-114.

HYVERCALCE~IA IN CHILDREN 82. Doireau V, Macher MA, Brun P, Bernard O, Loirat C. Vitamin A poisoning revealed by hypercalcemia in a child with kidney failure. Arch Pediatr 1996;3:888-890. 83. Mahoney CP, Margolis MT, Knauss TA, Labbe RF. Chronic vitamin A intoxication in infants fed chicken liver. Pediatrics 1980;65:893-897. 84. Mundy GR. Primary hyperparathyroidism. In: Mundy GR, ed. Calcium homeostasis: hypercalcemia and hypocalcemia. London: Martin Dunitz, 1990;137-167. 85. Rivkees SA, Hajj-Fuleihan G, Brown EM, Crawford JD. Tertiary hyperparathyroidism during high phosphate therapy of familial hypophosphatemic rickets. J Clin Endocrinol Metab 1992;75: 1514-1518. 86. Fuss M, Pepersack T, Gillet C, Karmali R, Corvilain J. Calcium and vitamin D metabolism in granulomatous diseases. Clin Rheumatol 1992;11:28-36. 87. Bosch X. Hypercalcemia due to endogenous overproduction of active vitamin D in identical twins with cat-scratch disease. JAMA 1998;279:532-534. 88. Stewart AF, Adler M, Byers CM, Segre GV, Broadus AE. Calcium homeostasis in immobilization: An example of resorptive hypercalciuria. N EnglJ Med 1982;306:1136-1140. 89. Rosen JF, Wolin DA, Finberg L. Immobilization hypercalcemia after single limb fractures in children and adolescents. AmJDis Child 1978;132:560-564. 90. Kedlaya D, Brandstater ME, Lee JK. Immobilization hypercalcemia in incomplete paraplegia: Successful treatment with pamidronate. Arch Phys Med Rehabi11998;79:222-225. 91. Massagli TL, Cardenas DD. Immobilization hypercalcemia treatment with pamidronate disodium after spinal cord injury. Arch Phys Med Rehabi11999;80:998-1000. 92. McKay C, Furman WL. Hypercalcemia complicating childhood malignancies. Cancer 1993;72:256-260. 93. Arase Y, Endo Y, Hara M, Kumada H, Ikeda K, Yoshiba A. Hepatic squamous cell carcinoma with hypercalcemia in liver cirrhosis. Acta PatholJpn 1988;38:643-650.

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94. Florell SR, Bruggers CS, Matlak M, Young RH, Lowichik A. Ovarian small cell carcinoma of the hypercalcemic type in a 14 month old: The youngest reported case. Med Pediatr Oncol 1999;32:304-307. 95. Giebel SC, Stanhope CR, Malkasian GD,Jr, Schray ME Heath III H, Gaffey TA. Humoral hypercalcemia associated with a dysgerminoma. Mayo Clin Proc 1992;67:966-968. 96. Leone N, Debernardi-Venon W, Marzano A, Massari M, Rizzetto M. Hypercalcaemia secondary to hepatocellular carcinoma. Ital J Gastroenterol Hepatol 1999;31:604-606. 97. Schleef J, Wagner A, Kleta R, Schaarschmidt K, DockhornDworniczak B, Willital G, Jurgens H. Small-cell carcinoma of the ovary of the hypercalcemic type in an 8-year-old girl. Pediatr Surg Int 1999;15:431-434. 98. Yamashita F, Iwao T, Torimura T, Tanaka M, Hirai K, Abe M, Toyonaga A, Sugihara S, Kojiro M, Tanikawa K. Sclerosing hepatocellular carcinoma with hypercalcemia--a case report. Kurume Med J 1992;39:113-116. 99. Yen TC, Hwang SJ, Wang CC, Lee SD, Yeh SH. Hypercalcemia and parathyroid hormone-related protein in hepatocellular carcinoma. Liver 1993;13:311-315. 100. CadranelJE Cadranel J, Buffet C, Ink O, Pelletier G, Bismuth E, Etienne JE Hypercalcaemia associated with chronic viral hepatitis. Postgrad Med J 1989;65:678-680. 101. Ford DJ, Reid IR. Hypercalcaemia associated with viral hepatitis. Lancet 1990;336:181. 102. Attard TM, Dhawan A, Kaufman SS, Collier DS, Langnas AN. Use of disodium pamidronate in children with hypercalcemia awaiting liver transplantation. Pediatr Transplant 1998;2: 157-159. 103. Pont A. Unusual causes of hypercalcemia. Endocrinol Metab Clin North Am 1989; 18: 753-764. 104. Tezer KM. Use of bisphosphonates in hypercalcemia associated with childhood cancer. J Clin Oncol. 1999;17:1960. 105. Lteif AN, Zimmerman D. Bisphosphonates for treatment of childhood hypercalcemia. Pediatrics 1998;102:990-993.

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Hyp oparat hy roldlsm . . in. the. Differential . Diagnosis of Hypocalcemia

W . D O W N S Division of Endocrinology and Metabolism, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298

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INTRODUCTION

for every 1 g / d l change in albumin concentration below the normal range (1):

Calcium is present in extracellular fluid in three forms: b o u n d to protein (40-45%), complexed with inorganic anions (5-10%), and ionized (45-50%). Clinically significant hypocalcemia involves a decrease in the concentration of the physiologically i m p o r t a n t ionized calcium. It is the free calcium ion concentration that is important for normal cellular function, and it is this free calcium ion concentration that is sensed and regulated by the parathyroid glands.

corrected calcium = total calcium + 0.8 (4 - serum albumin). For example, a patient with a measured total serum calcium of 8.0 m g / d l (normal range 8.9-10.5) and a measured albumin of 2.5 g / d l (normal range 3.7-4.9) would have an albumin concentration that is 1.5 g / d l below the normal value of 4.0 g/dl. Multiplying 1.5 by 0.8 provides a correction in the calcium of 1.2 m g / d l , which added to the measured value of 8.0 gives a corrected serum calcium of 9.2 m g / d l [calculated as 8.0 + 0.8(4 - 2.5)]. The corrected serum calcium in this case predicts that the ionized calcium, if measured, would be normal and that the decline in total calcium is attributable to the low concentration of albumin-bound calcium. Unfortunately, these formulas do not always correctly identify patients with true ionized hypocalcemia (2), because other factors, including p H and the concentration of other anions, contribute to changes in ionized calcium. Therefore, for patients who have symptoms that may be caused by hypocalcemia and in critically ill patients in whom ionized calcium is m o r e commonly abnormal (3), direct m e a s u r e m e n t of ionized calcium is advisable if there is any uncertainty about the diagnosis of hypocalcemia. M e a s u r e m e n t of ionized calcium requires attention to careful specimen collection and p r o m p t handling by a qualified laboratory.

D E T E R M I N A T I O N OF H Y P O C A L C E M I A Because total serum calcium is most often measured in clinical practice, rather than the free or ionized fraction, a low total serum calcium concentration can be found in patients who do not have low ionized calcium and who do not have any disorder of calcium metabolism. Albumin is the most a b u n d a n t calcium-binding serum protein, and hypoalbuminemia is responsible for most of the reduced total serum calcium concentration found in chronically ill, malnourished, and hospitalized patients. A n u m b e r of rule-of-thumb correction formulas have been proposed to determine which patients n e e d further evaluation for true hypocalcemia and which are likely to have normal ionized calcium with low serum protein as the cause for a low total serum calcium concentration. The most widely used of these corrections adjusts total serum calcium by 0.8 m g / d l The Parathyroids, Second Edition

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S Y M P T O M S A N D SIGNS OF H Y P O C A L C E M I A The clinical manifestations of hypocalcemia may be quite variable a m o n g patients. Those who have acute decreases in serum ionized calcium from normal can have p r o m i n e n t symptoms, whereas those with chronic hypocalcemia may be relatively symptom free. Most of the symptoms of hypocalcemia are related to the important role of calcium ions in neuromuscular function, and are manifestations of increased neuromuscular excitability (Table 1). Patients with mild hypocalcemia may have some perioral numbness, tingling paresthesias of the distal extremities, and occasional muscle cramping. With moderate hypocalcemia, some patients will notice a general sense of irritability and anxiety, and more severe muscle cramps can occur. Severe hypocalcemia is accompanied by tetanic muscle cramps, carpopedal spasm, and may progress to lifethreatening laryngospasm, seizures, and coma. In addition to the commonly recognized symptoms of skeletal muscle cramping, hypocalcemia can be associated with other manifestations of abnormal neuromuscular function. A prolonged QT interval is often observed on the electrocardiogram, and reversible congestive heart failure has been reported in patients with severe hypocalcemia (4). Nonspecific electroencephalographic changes can occur (5,6). Abnormal smooth muscle function of the gastrointestinal and biliary tracts appears to account for abdominal pain and malabsorptive symptoms in patients rarely (7,8). The classic physical findings of hypocalcemia are also manifestations of enhanced neuromuscular irritability. Chvostek's sign is elicited by tapping over the facial nerve just anterior to the ear, producing contraction of the facial muscles. Slightly positive reactions, such as minimal twitching at the corner of the mouth, occur in some normal individuals (9). However, more marked muscle contraction, including movement of the u p p e r lip, nasal fold, and periorbital muscles,

TABLE 1

Clinical Manifestations of Hypocalcemia

Manifestations of impaired neuromuscular signaling Paresthesias, perioral numbness Prolonged QT interval on EKG Muscle cramps, carpopedal spasm, tetany Laryngeal stridor, convulsions Mental changes Physical signs of hypocalcemia Chvostek's sign Trousseau's sign Calcium precipitation Cataract Basal ganglia calcification

indicates significant hypocalcemia. Trousseau's sign is elicited as carpal spasm after inflation of a blood pressure cuff above systolic pressure for 3-5 minutes. These signs of latent tetany are not present in every patient who has hypocalcemia, however. Chronic hypocalcemia can be accompanied by other signs. Dry skin, coarse hair, and brittle nails are common. Dental abnormalities, including enamel hypoplasia and absence of adult teeth, may indicate that hypocalcemia has been present since childhood (10). Calcification of the basal ganglia occurs in all forms of hypoparathyroidism and can be recognized with computed tomographic imaging even when plain X-rays are normal (11). The reason for calcification in this particular area of the brain is not known. Cataracts can occur with long-standing hypoparathyroidism (12).

PATHOPHYSIOLOGIC MECHANISMS OF HYPOCALCEMIA The parathyroid glands sense and regulate the concentration of ionized calcium by secreting parathyroid h o r m o n e (PTH). PTH has effects on bone, through osteocytes and osteoclasts, to shift calcium from mineral storage sites to the extracellular space. PTH also has effects on the kidney to enhance distal renal tubular calcium reabsorption, proximal renal tubular phosphate excretion, and production of 1,25-dihydroxyvitamin D [ 1,25 (OH) 2o ] , which promotes intestinal calcium absorption and stimulates the differentiation and development of functional osteoclasts. The multiple actions of PTH are reviewed in detail elsewhere in this volume (Chapters 11-17). Defects in any of these processes can result in abnormalities of calcium regulation. The specific causes of hypocalcemia can be divided into those in which parathyroid h o r m o n e secretion is impaired or abnormal and those in which the parathyroid glands are normal (Table 2). Clinical disorders causing hypocalcemia occur as a result of disordered calcium sensing; absence, damage, or dysfunction of the parathyroid glands; and abnormal target organ response to parathyroid hormone. When the parathyroid glands are normal, hypocalcemia caused by other mechanisms (such as deficiency or disordered metabolism of vitamin D) induces parathyroid h o r m o n e secretion and the physiologic effects of parathyroid h o r m o n e can be observed as reactive secondary hyperparathyroidism.

HYPOPARATHYROIDISM In hypoparathyroidism, the clinical findings are those expected as a result of low PTH, including low total and

HYPOCALCEMIA / TABLE 2 Causes of Hypocalcemia Hypoparathyroidism/abnormal PTH secretion Surgical hypoparathyroidism Transient postsurgical hypoparathyroidism Autoimmune (idiopathic) hypoparathyroidism Activating mutations of the calcium-sensing receptor Developmental abnormalities of parathyroid glands (DiGeorge syndrome) Transient neonatal hypocalcemia Infiltrative damage of parathyroid glands (hemochromatosis, Wilson's disease, sarcoidosis, amyloidosis, metastatic cancer) Irradiation Severe magnesium deficiency Hypermagnesemia Pseudohypoparathyroidism/PTH resistance Pseudohypoparathyroidism Type la, with Albright osteodystrophy and multiple hormone resistance Type lb, with resistance confined to PTH target tissues Type 2, defective phosphaturic response to PTH Severe magnesium deficiency Disorders of vitamin D metabolism Acquired vitamin D deficiency Inadequate sunlight and dietary vitamin D Malabsorption Renal insufficiency Anticonvulsants Hereditary disorders of vitamin D metabolism 1oL-hydroxylase deficiency Resistance to vitamin D action Altered bound calcium Hyperphosphatemia (rhabdomyolysis, tumor lysis, phosphate infusion) Massive blood transfusion (citrate) Acute severe illness (pancreatitis, sepsis, etc.) Increased osteoblastic activity Postparathyroidectomy, "hungry bones" syndrome Osteoblastic tumor metastasis Hypocalcemic drugs

ionized calcium, and an increase in serum phosphorus concentration. PTH concentrations are low, but even in modern assays may still remain detectable. Patients with hypoparathyroidism have increased renal calcium clearance because of the absence of a PTH effect to enhance distal tubular calcium reabsorption.

Surgical Hypoparathyroidism Despite the best efforts of experienced endocrine surgeons, hypoparathyroidism occurs in patients who have parathyroid or thyroid surgery and particularly after radical surgery for carcinoma involving structures in the neck. Surgical series have indicated that the incidence of transient hypoparathyroidism after thyroid

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surgery may be as high as 10-15%; the incidence of permanent hypoparathyroidism is about 1-4% (13). Permanent hypoparathyroidism can be caused not only by inadvertent removal of parathyroid tissue, but also by damage to the blood supply to the parathyroid glands. Experienced surgeons are aware of the location of the parathyroid glands in relation to their blood supply and will autotransplant threatened parathyroid tissue when necessary (14).

Transient Postsurgical Hypoparathyroidism Transient hypoparathyroidism occurs when the damage to the parathyroid glands is incomplete, but other reasons for hypocalcemia after thyroid surgery intervene. After surgery for thyrotoxicosis, for example, elevated bone turnover during the hyperthyroid state and increased ionized calcium may cause mild parathyroid gland suppresion. Temporary hypocalcemia after surgery for primary hyperparathyroidism can occur due to suppression of normal parathyroid glands by hypercalcemia. In the mild primary hyperparathyroidism characteristic of the western world, postoperative hypocalcemia has become uncommon. However, if normal parathyroid glands are suppressed, they usually recover quickly and the serum calcium concentration returns to normal within days. More prolonged hypocalcemia suggests damage to normal parathyroid tissue or significant uptake of calcium into remineralizing "hungry bones," a situation that can be distinguished from hypoparathyroidism by a persistently low phosphorus concentration and an appropriately elevated PTH concentration.

Autoimmune (Idiopathic) Hypoparathyroidism Hypoparathyroidism occurs as part of a polyglandular endocrine deficiency syndrome (type 1), characterized most commonly by hypoparathyroidism, adrenal insufficiency, and mucocutaneous candidiasis (15). The syndrome usually presents during childhood. Not all patients express the complete triad, and some affected patients have other autoimmune disorders, such as alopecia, pernicious anemia, thyroid disease, diabetes mellitus, autoimmune hepatitis, and gonadal failure. Patients have circulating cytotoxic antibodies against parathyroid tissue (16), and the parathyroid glands are typically atrophic. The immunologic and genetic aspects of autoimmune hypoparathyroidism are reviewed in detail in Chapter 50. Idiopathic hypoparathyroidism also occurs sporadically in adults and is associated with antiparathyroid antibodies occasionally. Some of these cases may simply be related to incomplete penetrance of the familial polyglandular syndrome type 1. It is also possible that

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some may be related to relatively asymptomatic hypoparathyroidism associated with activating mutations of the calcium-sensing receptor.

Activating Mutations of the Calcium-Sensing Receptor The calcium-sensing receptor is an extracellular G protein-coupled receptor that senses the extracellular ionized calcium concentration, and whose normal action in the setting of an increase in extracellular calcium causes a decrease in PTH gene transcription and PTH secretion (17) (see Chapter 8). Inactivating mutations of this receptor have been recognized to cause autosomal d o m i n a n t hypercalcemia associated with low urinary calcium [familial hypocalciuric hypercalcemia (FHH) ]. It has been recognized that activating mutations of the calcium-sensing receptor will lower the set point for regulation of serum calcium. Serum ionized calcium activity is consequently decreased, and PTH synthesis and secretion are reduced even at relatively low calcium concentrations. Although some patients may be symptomatic of hypocalcemia, these tend to be more mild and intermittent than might be expected. Many affected individuals are asymptomatic and are detected only on family screening once an affected p r o b a n d is identified (18). The disorder is inherited as an autosomal dominant trait. One hallmark of this type of familial hypoparathyroidism is increased urinary calcium excretion, just the opposite of what happens in FHH. Thus, affected individuals who are treated with calcium and vitamin D could be predisposed to marked increases in urinary calcium excretion, nephrocalcinosis, and renal insufficiency when serum calcium is normalized (18). Treatment of asymptomatic individuals with calcium and vitamin D is therefore not advisable, rather only when symptoms are present.

Developmental Abnormalities of the Parathyroid Glands Disorders in which there is abnormal or absent formation of the parathyroid glands are associated with hypocalcemia. The DiGeorge syndrome occurs as a result of abnormal development of the third and fourth branchial pouches, which give rise to the parathyroid glands, and is characterized by a group of findings now clustered u n d e r the term CATCH 22 (cardiac defects, abnormal facies, thymic hypoplasia/aplasia, cleft palate, h_ypocalcemia, and 2 2 q l l chromosomal microdeletion) (19). Because of an associated T cell abnormality, early mortality from infection is common. Other forms of congenital and familial hypoparathyroidism have been described less frequently (20,21). One interesting family appears to have an abnormality

of the preproparathyroid h o r m o n e signal peptide, which results in defective intracellular processing of PTH (22). The molecular genetics of hypoparathyroidism are described in detail in Chapter 49.

Transient Neonatal Hypocalcemia Transient hypocalcemia in the newborn period is much more c o m m o n than the genetic disorders that lead to p e r m a n e n t hypoparathyroidism. There is a n expected decrease in serum calcium concentration after birth, and some infants have an exaggeration of this process with development of hypocalcemia. Neonatal hypocalcemia is self-limited, and tends to occur more commonly in premature and low-birthweight infants, perhaps because of deficient PTH secretion from less mature parathyroid glands. More severe neonatal hypercalcemia occurs in infants born to mothers with primary hyperparathyroidism or other forms of hypercalcemia.

Infiltrative Damage to Parathyroid Glands Parathyroid gland function can be impaired by a variety of disease processes in which there is infiltrative involvement. Hypoparathyroidism is recognized to occur in patients with hemochromatosis and iron overload. In a large study of patients with thalassemia, 3.6% of 1861 patients were found to have hypoparathyroidism (23). Parathyroid gland infiltration can also occur in patients with Wilson's disease, sarcoidosis, tuberculosis, amyloidosis, and metastatic cancer, but there are only a few reports of hypocalcemia in such cases (24-26).

Radiation-Induced Damage to Parathyroid Tissue Exposure to external radiation and to the doses of internal radiation used in the treatment of Graves disease with iodine-131 is only rarely reported in association with hypocalcemia. In those who receive large doses of radioactive iodine for the treatment of thyroid cancer, however, some evidence for diminished parathyroid function after treatment has been documented (27). However, such patients have usually had previous thyroid surgery, which may have contributed to diminished parathyroid reserve.

Magnesimn Deficiency Moderate declines in serum magnesium concentration can stimulate PTH secretion slightly, but severe magnesium deficiency results in a reversible abnormality of PTH secretion (28). This is actually one of the more c o m m o n causes of acute hypocalcemic tetany in malnourished alcoholic patients. The tendency to

HYPO~CFMIA hypocalcemia cannot be treated simply with calcium, but is readily reversible with magnesium replacement. With profound magnesium depletion, there is evidence that cellular resistance to the actions of PTH can occur as well (29).

Hypermagnesemia Hypermagnesemia is associated with declines in serum calcium, usually during magnesium infusion in obstetric practice. Studies have shown suppression of PTH secretion and an increase in urinary calcium excretion during magnesium infusion (30). Hypocalcemia in this setting is rarely symptomatic, perhaps because the excess magnesium tends to blunt neuromuscular irritability.

PARATHYROID HORMONE RESISTANCE Pseudohypoparathyroidism Pseudohypoparathyroidism represents the classic syndrome of resistance to the actions of PTH. Patients with pseudohypoparathyroidism can have symptoms of hypocalcemia, and laboratory studies show hypocalcemia and hyperphosphatemia, just as in primary hypoparathyroidism. However, measurement of PTH reveals elevated rather than decreased h o r m o n e concentrations. The parathyroid glands are hyperplastic. Chase et al. showed that PTH infusion failed to induce an expected increase in urinary cyclic AMP in such patients (31), locating the biochemical defect to the PTH receptor-adenylate cyclase complex. Patients with defective urinary cyclic AMP production are now classified as pseudohypoparathyroidism type 1. Another group of patients with hypocalcemia has been shown to have a normal urinary cyclic AMP response to PTH, but a defective phosphaturic response. This second group, termed pseudohypoparathyroidism type 2 (32), is a heterogeneous group of disorders, and in many patients the abnormal phosphaturic response is reversible with normalization of serum calcium. Pseudohypoparathyroidism type 1 can be further classified by clinical features and biochemical defect. Patients with pseudohypoparathyroidism type la have hypocalcemia and PTH resistance associated with other endocrine deficits, including hypothyroidism and oligomenorrhea, and they have a characteristic body habitus, termed Albright hereditary osteodystrophy, with shortened metacarpals, short stature, round face, and obesity. These patients have a genetic disorder with abnormalities of the G protein signal-transducing protein that couples receptor activation with stimulation of adenylate cyclase (33,34). Because G proteins couple receptor-adenylate cyclase activity for multiple hormones, this explains the multiple hormone resistance.

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Some family members of patients with typical pseudohypoparathyroidism have abnormal G protein, but may have normal serum calcium and a normal urinary cyclic AMP response to PTH infusion; such individuals are said to have pseudopseudohypoparathyroidism (35). Patients with pseudohypoparathyroidism type l b have hypocalcemia and PTH resistance, but hormone resistance is confined to PTH target tissues. In fact, some patients with this form of pseudohypoparathyroidism have significant PTH-induced bone disease even though there is clear renal resistance to PTH. There has been speculation that the biochemical defect in pseudohypoparathyroidism type l b would be in the PTH receptor, but there is now good evidence that the PTH receptor is normal in these patients (36,37). There are some patients who have hypocalcemia, PTH resistance, multiple hormone deficiencies, and Albright hereditary osteodystrophy, who nevertheless appear to have normal G protein function. Some evidence for an adenylate cyclase catalytic unit defect was identified in one such patient (38), but this has not been confirmed by others. If a catalytic unit defect does exist, this would be a separate form of pseudohypoparathyroidism (type lc). The clinical presentation and biochemical mechanisms of pseudohypoparathyroidism are summarized in Chapter 51.

Severe Magnesium Deficiency As noted above, severe magnesium deficiency can cause resistance to the target organ effects of PTH (29), as well as abnormal PTH secretion.

DISORDERS OF VITAMIN D METABOLISM The active metabolite of vitamin D [1,25 (OH) 2D] plays a critically important role in normal calcium homeostasis, by enhancing calcium absorption and by promoting the normal differentiation and development of osteoclasts. Deficiencies in the supply of vitamin D precursors, abnormalities in conversion of precursors to the active metabolite, and resistance to the action of 1,25(OH)zD can all cause hypocalcemia. Inadequate sunlight exposure and inadequate dietary vitamin D are common even in temperate latitudes where sunlight exposure might be considered adequate. Malabsorption of vitamin D because of intestinal or biliary disease also leads to vitamin D deficiency. Some drugs, notably anticonvulsants, accelerate the clearance of vitamin D metabolites. In patients with renal failure, synthesis of active metabolites in the kidney is abnormal. Genetic disorders can also result in deficient renal l e~-hydroxylase activity and inherited resistance to the effects of 1,25 (OH) 2O .

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In all of these disorders, the normal parathyroid glands attempt to compensate for low serum calcium concentrations, and secondary hyperparathyroidism occurs. These disorders, unlike those characterized by hypoparathyroidism or pseudohypoparathyroidism, are likely to be associated with low serum phosphate concentrations because of increased renal phosphate excretion and with evidence for increased parathyroid h o r m o n e action on bone.

OTHER CAUSES OF HYPOCALCEMIA Alterations in Bound Calcium There are a n u m b e r of situations in which increases in organic anions or changes in calcium binding cause a shift in the equilibrium between b o u n d and ionized calcium. For example, an increase in phosphate concentration will bind calcium into complexes and decrease the ionized calcium activity. C o m m o n circumstances in which this occurs include rhabdomyolysis and tumor lysis syndrome. Phosphate infusion can also cause hypocalcemia, and phosphate enemas have been reported to cause hypocalcemia (39), particularly in children. Citrate infused during massive blood transfusion can also complex calcium and decrease the concentration of ionized calcium. Acute illness is associated with a high incidence of hypocalcemia, and it is suspected that release of free fatty acids binds calcium in this situation (3,40).

Increased Osteoblastic Activity Bone resorption and formation are usually very tightly coupled, but in certain situations, bone formation can proceed so briskly that hypocalcemia occurs. This type of "hungry bone syndrome" can occur during the postoperative period after surgical treatment for severe hyperparathyroidism (41,42). Early hypocalcemia in such patients is certainly due to suppression of any remaining normal parathyroid tissue, but healing bone can utilize such a large a m o u n t of calcium that hypocalcemia occurs despite recovery of suppressed parathyroid glands. Affected patients generally have preexisting evidence of hyperparathyroid bone disease, and hypocalcemia persists despite elevated circulating PTH concentrations until the bone is healed. Patients may require treatment with calcium and vitamin D for several days or weeks, and some patients with severe bone disease have required treatment for months. One sign that the bone is healing is a reduction in bone specific alkaline phosphatase activity, a readily available marker for bone formation. Certain cancers can form osteoblastic metastases in bone. Those cancers most commonly reported to be

associated with osteoblastic metastases and hypocalcemia are prostate cancer and breast cancer (43).

Drug-Induced Hypocalcemia A n u m b e r of medications cause hypocalcemia. Some inhibit osteoclast-mediated bone resorption (bisphosphonates, plicamycin, calcitonin, gallium nitrate), some decrease PTH secretion (calcimimetic agents), and some complex calcium and magnesium (phosphate, foscarnet) (44-46).

AN APPROACH TO THE PATIENT WITH H Y P O C A L C E M I A A careful history and examination are important in evaluating and managing patients who have hypocalcemia. Many of the disorders that cause hypocalcemia can be identified with a t h o r o u g h knowledge of the patient's medical and family history. The clinical setting will usually make readily apparent those patients who are likely to have transient hypocalcemia. The examination will determine the severity of hypocalcemia so that therapy can be planned, and will identify other endocrine disorders and skeletal or dental abnormalities. Laboratory evaluation should include measurement of serum proteins to identify those patients who have apparent hypocalcemia with normal corrected calcium, serum creatinine to assess renal function, and serum phosphorus. Magnesium concentrations should also be assessed, particularly in those at risk for magnesium deficiency. PTH measurement is now a routine clinical procedure that will help to narrow the relevant differential diagnosis. Basic clinical and laboratory data will guide the care of the patient who presents with hypocalcemia.

REFERENCES 1. Editorial. Serum calcium. Lancet 1979;1:858-859. 2. Ladenson JH, Lewis JW, Boyd JC. Failure of total calcium corrected for protein, albumin, and pH to correctly assess free calcium status. J Clin Endocrinol Metab 1978;46:986-993. 3. Zaloga GE Hypocalcemia in critically ill patients. Crit Care Med 1992;20:251-262. 4. Rowell WG, Kreisberg RA. Hypocalcemic congestive heart failure. South MedJ 1987;80:396-398. 5. Swash M, Rowan AJ. Electroencephalographic criteria of hypocalcemia and hypercalcemia. Arch Neuro11972;26:218-228. 6. Fukuyama Y, Hayashi M. Sleep electroencephalograms and sleep stages in hypoparathyroidism. Eur Neurol 1979; 18:38-48. 7. Heubi JE, Partin JC, Schubert WK. Hypocalcemia and steatorrhea--clues to etiology. Dig Dis Sci 1983;28:124-128. 8. Reracchi M, Bardella MT, Conte D. Late-onset idiopathic hypoparathyroidism as a cause of diarrhea. Eur J Gastroenterol Hepatol 1998; 10:163-165.

HVeOCALCEMIA / 9. McGreal GT, Kelly JL, Hehir DJ, Brady ME Incidence of false positive Chvostek's sign in hospitalised patients. Ir J Med Sci 1995;164:56. 10. Nikiforuk G, Fraser D. The etiology of enamel hypoplasia: A unifying concept. J Pediatr 1981 ;98:888-893. 11. Posen S, Clifton-Bligh P, Cromer T. Computed tomography of the brain in surgical hypoparathyroidism. Ann Intern Med 1979;91:415-417. 12. Blake J. Eye signs in idiopathic hypoparathyroidism. Trans Ophthalmol Soc UK 1976;46:448-451. 13. Bergamaschi R, Becouarn G, RoncerayJ, ArnaudJP. Morbidity of thyroid surgery. A m J Surg 1998;176:71-75. 14. Shaha AR, Jaffe BM. Parathyroid preservation during thyroid surgery. Am J Otolaryngol 1998; 19:113-117. 15. Betterle C, Greggio NA, Volpato M. Clinical review 93: Autoimmune polyglandular syndrome type 1. J Clin Endocrinol Metab 1998;83:1049-1055. 16. Brandi ML, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA. Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 1986;83:8366-8369. 17. Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999;106:238-253. 18. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N EnglJ Med 1996;335:1115-1122. 19. Sergi C, Serpi M, Muller-Navia J, Schnabel PA, Hagl S, Otto HE Ulmer HE. CATCH 22 syndrome: Report of 7 infants with followup data and review of the recent advancements in the genetic knowledge of the locus 22q 11. Pathologica 1999;91:166-172. 20. Trump D, Dixon PH, Mumm S, Wooding C, Davies KE, Schlessinger D, Whyte ME Thakker RV. Localisation of X linked recessive idiopathic hypoparathyroidism to a 1.5 Mb region on Xq26-q27. J Med Genet 1998;35:905-909. 21. Kelly TE, Blanton S, Saif R, Sanjad SA, Sakati NA. Confirmation of the assignment of the Sanjad-Sakati (congenital hypoparathyroidism) syndrome (OMIM 241410) locus to chromosome 1q42-43. J Med Genet 2000;37:63-64. 22. Sunthornthepvarakul T, Churesigaew S, Ngowngarmratana S. A novel mutation of the signal peptide of the preproparathyroid hormone gene associated with autosomal recessive familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1999;84: 3792-3796. 23. Italian Working Group on Endocrine Complications in Nonendocrine Diseases. Multicentre study on prevalence of endocrine complications in thalassemia major. Clin Endocrinol 1995;42: 581-586. 24. Carpenter TO, Larves DL, Anast CS. Hypoparathyroidism in Wilson's disease. NEnglJ Med 1983;309:873-877. 25. Dill JE. Hypoparathyroidism in sarcoidosis. South Med J 1983; 76:414. 26. Ellis HA, Mawhinney WH. Parathyroid amyloidosis. Arch Pathol Lab Med 1984;108:689-690. 27. Glazebrook GA. Effect of decicurie doses of radioactive iodine 131 on parathyroid function. A m J Surg 1987;154:368-373. 28. Anast CS, Mohs JM, Kaplan SL, Burns TW. Evidence for parathyroid failure in magnesium deficiency. Science 1972; 177:606-608. 29. Estep H, Shaw WA, Watlington C, Hobe R, Holland W, Tucker SG. Hypocalcemia due to hypomagnesemia and reversible

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parathyroid hormone unresponsiveness. J Clin Endocrinol Metab 1969;29:842-848. 30. Cholst IN, Steinberg SF, Tropper PJ, Fox HE, Segre GV, Bilezikian JR The influence of hypermagnesemia on serum calcium and parathyroid hormone levels in human subjects. N Engl J Med 1984;310:1221-1225. 31. Chase LR, Melson GL, Aurbach GD. Pseudohypoparathyroidism: Defective excretion of 3',5'-AMP in response to parathyroid hormone. J Clin Invest 1969;48:1832-1844. 32. Drezner M, Neelon FA, Lebovitz HE. Pseudohypoparathyroidism type II: A possible defect in the reception of the cyclic AMP signal. N EnglJ Med 1973;289:1056-1060. 33. Levine MA, Downs RW, Jr, Moses AM, Breslau NA, Marx SJ, Lasker RD, Rizzoli RE, Aurbach GD, Spiegel AM. Resistance to multiple hormones in patients with pseudohypoparathyroidism: Association with deficient activity of guanine nucleotide regulatory protein. AmJMed 1983;74:545-556. 34. Levine MA. Pseudohypoparathyroidism: From bedside to bench and back. J Bone Miner Res 1999;14:1255-1260. 35. Levine MA, Jap TS, Mauseth RS, Downs RW, Spiegel AM. Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 1986;62:497-502. 36. Jan de Beur SM, Ding CL, LaBuda MC, Usdin TB, Levine MA. Pseudohypoparathyroidism l b: Exclusion of parathyroid hormone and its receptors as candidate disease genes. J Clin Endocrinol Metab 2000;85:2239-2246. 37. BettounJD, Minagawa M, Kwan MY, Lee HS, Yasuda T, Hendy GN, Goltzman D, White JH. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type lb. J Clin Endocrinol Metab 1997;82:1031-1040. 38. Barrett D, Breslau NA, Wax MB, Molinoff PB, Downs RW, Jr. New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase. AmJPhysio11989;257:E277-E283. 39. Campisi P, Badhwar V, Morin S, Trudel JL. Postoperative hypocalcemic tetany caused by fleet phospho-soda preparation in a patient taking alendronate sodium: Report of a case. Dis Colon Rectum 1999;42:1499-1501. 40. Lind L, Carlstedt F, Rastad J, Stiernstrom H, Stridsberg M, Ljunggren O, Wide L, Larsson A, Hellman P, Ljunghall S. Hypocalcemia and parathyroid hormone secretion in critically ill patients. Crit Care Med 2000;28:93-99. 41. Brasier AR, Nussbaum SR. Hungry bone syndrome: Clinical and biochemical predictors of its occurrence after parathyroid surgery. Am J Med 1988;84:654-660. 42. Savazzi GM, Allegri L. The hungry bone syndrome: Clinical problems and therapeutic approaches following parathyroidectomy. EurJ Med 1993;2:363-368. 43. Riancho JA, Arjona R, Valle R, Sanz J, Gonzalez-Macias J. The clinical spectrum of hypocalcaemia associated with bone metastases. J Intern Med 1989;226:449-452. 44. Schussheim DH, Jacobs TP, Silverberg SJ. Hypocalcemia associated with alendronate. Ann Intern Med 1999;130:329. 45. Jacobson MA, Gambertoglio JG, Aweeka FT, Causey DM, Portale AA. Foscarnet-induced hypocalcemia and effects of foscarnet on calcium metabolism. J Clin Endocrinol Metab 1991 ;72:1130-1135. 46. Gearhart MO, Sorg TB. Foscarnet-induced severe hypomagnesemia and other electrolyte disorders. Ann Pharmacother 1993;27:285-289.

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CHAPTER 4 8

Magnesium Deficiency in Parathyroid Function

ROBERT

K. R U D E

University of Southern California School of Medicine, Los Angeles, California 90089

INTRODUCTION

M A G N E S I U M METABOLISM

Magnesium (Mg) is one of the most plentiful elements on earth. In vertebrates it is the fourth most abundant cation and the second most abundant intracellular cation. Therefore, it is not surprising that Mg is involved in numerous biologic processes and is essential for life (1,2). Mg was involved in early evolution as a means harnessing energy from the sun. Chlorophyll is the Mg chelate of porphyrin. T h r o u g h the process of photosynthesis, ATP is formed, providing energy for the synthesis of carbon dioxide and water into carbohydrate and oxygen. In animal cells as well as in plant cells in absence of the sun, stored chemical energy is utilized to maintain life. This chemical energy is released by Mg-dependent oxidative phosphorylation in which ATP is again formed. Mg has evolved to become a required cofactor in literally hundreds of enzyme systems (1-3). Examples of the physiologic role of Mg are shown in Table 1. Mg may be required for substrate formation. For example, all enzymes that utilize ATP do so as the metal chelate, MgATE Free Mg 2+ also acts as an allosteric activator of numerous enzyme systems as well as playing a role in ion currents and in membrane stabilization. Mg is therefore critical for a great n u m b e r of cellular functions, including oxidative phosphorylation, glycolysis, DNA transcription, and protein synthesis.

The normal adult total body Mg content is approximately 25g, of which 50-60% resides in bone. One-third of skeletal Mg is exchangeable and this fraction may serve as a reservoir for maintaining a normal extracellular Mg concentration. Extracellular Mg accounts for about 1% of total body Mg. The normal serum Mg concentration is 0.71-0.91 mmol/liter (1.7-2.2 m g / d l ) . About 70-75% of plasma Mg is ultrafilterable, of which the major portion is ionized. The remainder is protein bound, chiefly to albumin. The concentrations of Mg within cells are on the order of (1-3) X 10 -~ mol/liter, of which 0.5-5% is ionized or free (4,5). The kidney is the principal organ involved in Mg homeostasis (6). During Mg deprivation, the kidney avidly conserves Mg and less than 1 mEq is excreted in the urine per day. Conversely, when excess Mg is taken, it is rapidly excreted into the urine. The major sites of Mg reabsorption in the n e p h r o n are the proximal convoluted tubule (5-15%) and the thick ascending limb of Henle (50-60%), and the distal tubule (10%) (7). The factors that regulate renal Mg homeostasis are unknown. PTH, when given in large doses in humans or other species, will decrease urinary Mg excretion. Patients with either primary hyperparathyroidism or

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CI-IAeTER48 TABLE 1 Physiologic Role of Magnesium

Enzyme substrate (ATP-Mg, GTP-Mg) Kinase (hexokinase, creatine kinase, protein kinase) ATPase or GTPase (Na+/K+-ATPase, Ca2+-ATPase) Cyclases (adenylate cyclase, guanylate cyclase) Direct enzyme activation Phosphofructokinase Creatine kinase 5-Phosphoribosyl-pyrophosphate synthetase Adenylate cyclase Phospholipase C Na+/K+-ATPase Influence membrane properties Nerve conduction Calcium channel activity Potassium transport

hypoparathyroidism usually have normal serum Mg levels, however, suggesting that PTH is not an important physiologic regulator of Mg homeostasis (8). Studies have suggested that the concentration of calcium a n d / o r Mg in the extracellular fluid may regulate absorption of Mg in the thick ascending limb of Henle by activation of the CaZ+-sensing receptor in this segment of the nephron (7,9). Intestinal Mg absorption is inversely proportional to the amount ingested. Under normal dietary conditions in healthy individuals, approximately 30-50% of ingested Mg is absorbed (10). Mg is absorbed along the entire intestinal tract, including the large and small bowel, but the sites of maximal Mg absorption appear to be the ileum and distal jejunum (11). There exists both a passive and active transport system for Mg. A principal factor regulating intestinal Mg transport has not been described. Vitamin D and its metabolites, 25hydroxyvitamin D and 1,25-dihydroxyvitamin D, have been found in some studies to enhance intestinal Mg absorption but to a much lesser extent than they do calcium absorption (12,13).

MAGNESIUM DEFICIENCY Magnesium deficiency is more prevalent than previously appreciated. Approximately 10% of patients admitted to large city hospitals are hypomagnesemic (14). This incidence may increase to as high as 65% in a medical intensive care unit (15). Because Mg is ubiquitous in food, moderate to severe degrees of Mg depletion are most unusual in healthy individuals with a normal caloric intake. Clinically apparent hypomagnesemia a n d / o r Mg deficiency are usually due to losses of Mg from either the gastrointestinal tract or the kidney.

Causes of Mg deficiency are shown in Table 2 (for review see Refs. 3, 5, and 16). The Mg content of upper intestinal tract fluids is approximately 1 mEq/liter. Vomiting and nasogastric suction, therefore, may contribute to Mg depletion. The Mg contents of diarrheal fluids and fistulous drainage are much higher (up to 15 mEq/liter). Consequently, Mg depletion is common in acute and chronic diarrhea, regional enteritis, ulcerative colitis, and intestinal and biliary fistulas. Malabsorption syndrome due to nontropical sprue, radiation injury resulting from therapy for disorders such as Whipple's disease and carcinoma of the cervix, and intestinal lymphangiectasia may result in Mg deficiency, presumably due to intestinal mucosal damage. Steatorrhea may also cause or contribute to Mg malabsorption through formation of nonabsorbable magnesium-lipid salts. Resection or bypass of the small bowel, particularly the ileum, for obesity, enteritis, or vascular infarction, also often results in Mg deficiency.

TABLE 2 Causes of Magnesium Deficiency Gastrointestinal disorders Prolonged nasogastric suction Malabsorption syndromes Extensive bowel resection Acute and chronic diarrhea Intestinal and biliary fistulas Protein-calorie malnutrition Acute hemorrhagic pancreatitis Primary hypomagnesemia (neonatal) Renal loss Chronic parenteral fluid therapy Osmotic diuresis Glucose (diabetes mellitus) Mannitol Urea Hypercalcemia Alcohol Drugs Diuretics (furosemide, ethacrynic acid) Aminoglycosides Cisplatin Cyclosporin Amphotericin B Pentamidine Cardiac glycosides (possible) Metabolic acidosis (starvation, ketoacidosis, alcoholism) Renal diseases Chronic pyelonephritis, interstitial nephritis, and glomerulonephritis Diuretic phase of acute tubular necrosis Postobstructive nephropathy Renal tubular acidosis Postrenal transplantation Primary hypomagnesemia

MAGNESIUM IN PARATHYROIDFUNCTION / Excessive excretion of Mg into the urine underlies the basis of Mg depletion in many patients. Proximal tubular Mg reabsorption is proportional to tubular fluid flow and sodium reabsorption. Therefore, chronic parenteral fluid therapy, particularly with sodiumcontaining fluids and volume expansion states, such as primary aldosteronism, may result in Mg deficiency. Similarly, osmotic diuresis due to glucosuria (diabetes mellitus) (17), mannitol, and urea will result in urinary Mg wasting. Disorders causing an increase in the serum calcium concentration will also lead to renal magnesium wasting, presumably due to activation of the Ca 2+sensing receptor in the thick ascending limb of Henle, which results in a decrease in the transepithelial voltage and subsequent decrease in calcium and magnesium reabsorption (7,9). Exceptions are familial hypocalciuric hypercalcemia, due to an inactivating mutation of the CaZ+-sensing receptor, and lithium ingestion, in which urinary magnesium excretion is decreased. Certain drugs are becoming recognized as common causes of renal Mg wasting and Mg depletion (18). Diuretics acting at the loop of Henle (e.g., furosemide, bumetamide, and ethacrynic acid) have been shown by micropuncture studies and clinical studies to result in marked Mg wasting. The effect of thiazide diuretics is controversial; some studies demonstrate a Mg wasting effect, but others do not. The commonly used aminoglycosides have been shown to cause a reversible renal lesion that results in hypermagnesuria and hypomagnesemia (18). Similarly, amphotericin B therapy has been reported to result in renal Mg wasting. Cisplatin is a chemotherapeutic agent used in the treatment of epithelial neoplasms. Renal Mg wasting resulting in hypomagnesemia has been reported in up to 100% of patients receiving this agent. Cyclosporin is an immunosuppressive agent that also results in nephrotoxicity and renal Mg wasting. Pentamidine has been reported to result in renal Mg loss in AIDS patients (18). A rising blood alcohol level has been associated with renal Mg wasting and is one factor contributing to Mg deficiency in chronic alcoholism. Metabolic acidosis due to diabetic ketoacidosis, starvation, or alcoholism also causes renal Mg wasting. Some renal tubular, glomerular, or interstitial diseases have been associated with renal Mg wasting. There may be other accompanying tubular abnormalities, and reduced glomerular filtration rate may or may not be present. Diabetes mellitus is probably the most common disorder associated with Mg deficiency (17). The incidence of hypomagnesemia in diabetes mellitus has been reported to vary from 25 to 39% (3,5,16). The serum Mg concentration correlates inversely with the serum glucose concentration and the degree of glucosuria. The mechanism for the Mg depletion is probably

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mostly due to the glucosuria (osmotic diuresis). Patients with ketoacidosis may also waste Mg into the urine during the acidosis per se. Hypomagnesemia can be found in association with a number of other endocrine disorders, as shown in Table 3. The mechanism leading to the hypomagnesemia most frequently involves urinary Mg wasting. Phosphate depletion has been shown experimentally in humans and rats to result in urinary Mg wasting and hypomagnesemia (19). Excessive urinary Mg wasting and hypomagnesemia can be seen in severe primary hyperparathyroidism, treated hypoparathyroidism, and thyrotoxicosis; the urinary losses may be due to the hypercalcemia as discussed above a n d / o r hypercalciuria occurring in these states (7,9). The hypomagnesemia that may be seen in primary hyperaldosteronism has been related to plasma volume expansion and subsequent renal Mg wasting. Hypomagnesemia may also accompany the hungry bone syndrome, a phase of rapid bone mineral accretion in subjects with hyperparathyroidism or hyperthyroidism following surgical treatment. Bartter syndrome and Gitelman syndrome are autosomal recessive disorders characterized by hypokalemic alkalosis. Hypomagnesemia due to renal Mg wasting has been reported. Elucidation of the molecular defect of these disorders has clarified the clinical and biochemical classification (20). Gitleman syndrome has been found to be due to a genetic mutation of the thiazide-sensitive NaC1 cotransporter of the distal convulted tubule (chromosome 16) and is characterized by hypokalemic alkalosis, absence of hypertension, hypocalciuria, hypomagnesemia, and presentation after 8 years of age (20,21). Bartter syndrome has been found to be due to a genetic defect of the N a / K / 2 C L cotransporter in the thick ascending limb of Henle (20,22). It usually occurs in the neonatal period to offspring of consanguineous marriage and is characterized by salt wasting, hypovolemia, activation of the renin-antiotensin system, and hypolkalemic alkalosis. TABLE 3 Endocrineand Metabolic Disorders Associated with Magnesium Deficiency Diabetes mellitus Phosphate depletion Primary hyperparathyroidism Hypoparathyroidism Hyperthyroidism Primary aldosteronism Hungry bone syndrome Excessive lactation Gitelman syndrome

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Hypercalciuria occurs in conjunction with the Na loss and nephrocalcinosis often occurs. Hypomagnesemia is not often observed.

MANIFESTATIONS OF M A G N E S I U M DEFICIENCY Because Mg deficiency is usually secondary to another disease process or to a therapeutic agent, the features of the primary disease process may complicate or mask Mg deficiency. A high index of suspicion is therefore warranted. Frequent manifestations of moderate to severe Mg deficiency are shown in Table 4 (for review see Refs. 3, 5, 16, and 23). Neuromuscular hyperexcitability is often the presenting complaint. Latent tetany, as elicited by a positive Chvostek's and Trousseau's sign, or spontaneous carpal pedal spasm may be present. Frank generalized seizures may also occur. Though hypocalcemia may contribute to the neurologic signs, hypomagnesemia without hypocalcemia has been reported to result in neuromuscular hyperexcitability (24). Other signs may include vertigo, ataxia, nystagmus, and athetoid and choreiform movements as well as muscular tremor, fasciculation, wasting, and weakness. Electrocardiographic abnormalities of Mg deficiency in man include prolonged PR interval and QT interval.

TABLE 4

Manifestations of Moderate to Severe Magnesium Deficiency

Neuromuscular Positive Chvostek's and Trousseau's sign Spontaneous carpal-pedal spasm Seizures Vertigo, ataxia, nystagmus, athetoid and choreiform movements Muscular weakness, tremor, fasciculation, and wasting Psychiatric: depression, psychosis Cardiac arrhythmia EKG: prolonged PR interval and QT interval, U waves Atrial tachycardia, premature contractions, and fibrillation Junctional arrhythmias Ventricular premature contractions, tachycardia, fibrillation Sensitivity to digitalis intoxication Torsades de pointes Biochemical Hypokalemia Renal potassium wasting Decreased intracellular potassium Hypocalcemia Impaired PTH secretion Renal and skeletal resistance to PTH Resistance to vitamin D

Mg deficiency may also result in arrhythmias. Supraventricular arrhythmias, including premature atrial complexes, atrial tachycardia, atrial fibrillation, and junctional arrhythmias, have been described (25). Ventricular premature complexes, ventricular tachycardia, and ventricular fibrillation are more serious complications (26). A common laboratory feature of Mg deficiency is hypokalemia. During Mg deficiency there is intracellular potassium depletion as well as an inability of the kidney to conserve potassium. Attempts to replete the potassium deficit with potassium therapy alone are not successful without simultaneous Mg therapy (27-29). This biochemical feature may be a contributing cause of the electrocardiologic findings and cardiac arrhythmias discussed above. Soon after the observation that Mg deficiency may cause neuromuscular hyperexcitability, it was noted that hypocalcemia was also a common finding in moderate to severe Mg deficiency (30). Correction of the hypocalcemia was possible only with Mg therapy. The relationship of Mg with mineral homeostasis in normal physiology as well as the pathophysiologic events leading to hypocalcemia soon followed.

EFFECT OF MAGNESIUM ON PARATHYROID HORMONE SECRETION Calcium is the major regulator of PTH secretion. A n u m b e r of in vitro and in vivo studies, however, have demonstrated that Mg can modulate PTH secretion in a similar manner. Perfusion of isolated parathyroid glands of goats and sheep with varying concentrations of Mg showed that acute elevations of Mg inhibited PTH secretion but that acute reductions stimulated PTH secretion (31). These findings were confirmed in studies of bovine and rat parathyroid glands in vitro in which an increase in media Mg concentration inhibited release of PTH but low-media Mg stimulated PTH release (32,33). Studies have also demonstrated that hypermagnesemia will inhibit PTH secretion in humans (34-36). Mg could therefore be a physiologic regulator of PTH secretion. Mg, however, has only approximately 30-50% of the effect of calcium on either stimulating or inhibiting PTH secretion (35-39). The finding in humans that a 5% (0.03 mM) decrease in serum ultrafilterable Mg did not result in any detectable change in intact serum PTH concentration whereas a 5.5% (0.07mM) decrease in ionized calcium resulted in a 400% increase in serum PTH supports this concept (40). The inhibitory effects of Mg on PTH secretion may vary, depending on the extracellular calcium concentration (40). At physiologic calcium and Mg concentrations, these divalent cations were found

MAGNESIUM IN PARATHYROID FUNCTION

to be relatively equipotent at inhibiting PTH secretion from dispersed bovine parathyroid cells (40). At a low calcium concentration (0.5 mM) however, a threefold greater Mg concentration was required for similar PTH inhibition. Altering the Mg concentration did not diminish the ability of calcium to inhibit PTH secretion. Differences have also been noted in the effect of Mg and calcium on the biosynthesis of PTH in vitro. Changes in calcium over the range of 0 to 3.0 m M resulted in increased PTH synthesis as assessed by amino acid incorporation (41-43) or DNA synthesis (43), whereas changes in Mg over the range of 0 to 1.7 m M had no effect. The modulation of PTH secretion by extracellular calcium appears to be mediated by calcium binding to a specific cell surface receptor, the CaZ+-sensing receptor (44), which leads to activation of phospholipase C and subsequent accumulation of inositol 1,4,5-trisphosphate (45). This leads to release of calcium from intracellular stores (45) and subsequent decrease in PTH secretion. The CaZ+-sensing receptor is activated by other cations, including Mg (46). The affinity of this receptor for Mg is less than calcium, but does result in a rapid increase in intracellular calcium (47). It is probable, therefore, that the effect of Mg on PTH secretion may be mediated through the CaZ+-sensing receptor. It has also been suggested that Mg might alter the influx of extracellular calcium through ion channels (47). Based on the above, it is apparent that acute changes in the serum Mg concentration may modulate PTH secretion and should be considered in the evaluation of the determination of serum PTH concentrations.

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D therapy will not correct the hypocalcemia (for review see Refs. 3, 5, 16, and 30). Even mild degrees of Mg depletion, however, may result in a significant decrease in the serum calcium concentration, as demonstrated in experimental h u m a n Mg depletion (51). One major factor resulting in the decrease in the serum calcium is impaired parathyroid gland function. Low concentrations of Mg in the media of bovine or rat parathyroid cell cultures impair PTH release in response to a low media calcium concentration (52,53). Determinations of serum PTH concentrations in hypocalcemic hypomagnesemic patients have shown heterogeneous results, as shown in Fig. 1. The majority of patients have low or normal serum PTH levels (30,55-59). Normal serum PTH concentrations are thought to be inappropriately low in the presence of hypocalcemia. Therefore, a state of hypoparathyroidism exists in most hypocalcemic Mg-deficient

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EFFECT OF MAGNESIUM DEFICIENCY O N PARATHYROID GLAND FUNCTION Though acute changes in the extracellular Mg concentrations will influence PTH secretion qualitatively similar to that of calcium, it is clear that Mg deficiency markedly perturbs mineral homeostasis (30). Hypocalcemia is a prominent manifestation of Mg deficiency in humans (30). This has also been found to be true in most species, including monkey, cow, sheep, pig, dog, chick, and guinea pig (48). The rat, however, will develop hypercalcemia when Mg depleted while maintained on a normal calcium diet (48-50). On a low-calcium diet, however, the rat will become hypocalcemic (48). In humans, Mg deficiency must become moderate to severe before symptomatic hypocalcemia develops. A positive correlation has been found between the serum Mg and calcium concentrations in hypocalcemic hypomagnesemic patients (30). Mg therapy alone will restore serum calcium concentrations to normal in such patients within days (30). Calcium a n d / o r vitamin

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patients. Some patients, however, have elevated levels of PTH in the serum (30,55-59). The administration of Mg will result in an immediate increase in the serum PTH concentration, regardless of the basal PTH level (30,54,58,59). As shown in Fig. 2, 10 mEq of Mg administered intravenously over 1 minute caused an immediate marked rise in the serum PTH in patients with either low, normal, or elevated basal serum PTH concentrations. This is distinctly different than the effect of a Mg injection in normal subjects, in whom, as discussed above, Mg will cause an inhibition of PTH secretion (51). The ability of Mg to stimulate the rise in PTH appears to be specific for Mg depletion because Mg injection does not result in an increase in PTH in primary or secondary hyperparathyroidism (58). The serum PTH concentration will gradually fall to normal within several days of therapy as the serum calcium concentration normalizes (30,54,57-59), as shown in Fig. 3.

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The i m p a i r m e n t in PTH secretion appears to occur early in Mg depletion. Normal h u m a n subjects experimentally placed on a low-Mg diet for only 3 weeks showed similar, but not as marked, changes in the serum PTH levels (51). In this study there was a decrease in both the serum calcium and the PTH concentrations in 20 of 26 subjects at the end of a 3-week dietary Mg deprivation period. The administration of intravenous Mg at the end of the Mg depletion period resulted in a significant increase in the serum PTH concentration, whereas a similar Mg injection suppressed PTH secretion prior to the low-Mg diet. In this study, as with hypocalcemia hypomagnesemic patients, some subjects had elevations in the serum PTH concentration. The heterogeneous serum PTH values may be explained based on the severity of Mg depletion. As hypomagnesemia develops, the parathyroid gland will react normally with an increase in PTH secretion. As intracellular Mg depletion develops, however, the ability of the parathyroid to secrete PTH is impaired, resulting in a decrease in the serum PTH levels, with a

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resultant decrease in the serum calcium concentration. This concept is supported by the observation that the change in serum PTH in experimental h u m a n Mg depletion is positively correlated with the decrease in red blood cell intracellular free Mg 2+ (51). A slight decrease in red blood cell Mg 2+ resulted in a increase in PTH. However, a greater decrease in red blood cell Mg 2+ correlated with a progressive decrease in serum PTH concentrations. It is conceivable that either PTH synthesis a n d / o r PTH secretion may be affected, given the wide requirem e n t of Mg for energy generation and protein synthesis. The immediate increase in PTH following the administration of intravenous m a g n e s i u m to Mgdeficient patients strongly suggests that the defect is in secretion, because biosynthesis of PTH is estimated to take approximately 45 minutes in vitro (41).

as illustrated in Fig. 4 (30,59). This also suggests skeletal resistance to PTH because exogenous PTH administered to hypoparathyroid patients causes a rise in the serum calcium within 24 hours (60). A n u m b e r of clinical studies, in which exogenous PTH was administered to hypocalcemic Mg-deficient patients, have demonstrated that PTH had little effect in increasing the serum calcium concentration (30,61-64). In one such study, shown in Fig. 5, parathyroid extract did not result in an elevation in the serum calcium concentration or urinary hydroxyproline excretion in hypocalcemic

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FI6.5 The effect of an intravenous injection of 200 units of parathyroid extract (PTE) on the excretion of urinary cyclic AMP in a magnesium-deficient patient before (o--o) and after (°---,) 4 days of magnesium therapy. Urine was collected for four consecutive 1-hour periods, two before and two after the PTE injection. Though Mg deficient, the patient had minimal rise in urinary cyclic AMP in response to PTH, but following Mg therapy the response was normal (taken from Ref. 30).

770

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hypomagnesemic patients (61). Following Mg repletion, however, a clear response to PTH was observed. PTH has also been shown to have a reduced calcemic effect in Mg-deficient dogs, chicks, and rats (65-69). The ability of PTH to resorb bone in vitro is also greatly diminished in the presence of low media Mg (70). In one in vivo study of isolated perfused femur in the dog, the ability of PTH to simulate an increase in the venous cyclic AMP was impaired during perfusion with low-Mg fluid, suggesting skeletal PTH resistance (71). Not all h u m a n studies have shown skeletal resistance to PTH, however (72-75). It appears likely that skeletal PTH resistance may be observed in patients with more severe degrees of Mg depletion. In h u m a n studies, patients in whom a normal calcemic response to PTH was demonstrated were subjects who had been on recent Mg therapy (72-75). Patients who have been found to be resistant to PTH have, in general, not had prior Mg administration (30,61-64). Consistent with this notion is that in the Mg-depleted rat, normal responses to PTH were observed when the serum Mg concentration was 0.95 m g / d l (76); however, in another study rats with a mean serum Mg of 0.46 m g / d l were refractory to PTH (65). In addition, a longitudinal study of Mg deficiency in dogs demonstrated a progressive decline in responsiveness to PTH with increasing degrees of Mg depletion (68). Calcium release from the skeleton also appears to be d e p e n d e n t on physicochemical processes as well as cellular activity (77,78). Low Mg will result in a decrease in calcium release from bone (77,78) and may be another mechanism for hypocalcemia in Mg deficiency. The renal response to PTH has also been assessed by determining urinary excretion of cyclic AMP a n d / o r phosphate (Figs. 4 and 5) in response to exogenous PTH. In some patients, a normal effect of PTH on urinary phosphate and cyclic AMP excretion has been noted (54-56,62,74,75). In general, these were the same subjects in which a normal calcemic effect was also seen (54,74,75). In other studies of more severely Mg-depleted patients, an impaired response to PTH has been observed (30,61,79). Shown in Fig. 5 is the effect of PTH on urinary cyclic AMP in one hypocalcemic Mg-deficient patient prior to and following Mg therapy. As shown, prior to Mg treatment, PTH did not increase urinary cyclic AMP excretion. Following Mg repletion, however, the rise in cyclic AMP was normal. A decrease in urinary cyclic AMP excretion in response to PTH has also been described in the Mg-deficient dog and rat (68,69).

EFFECT OF M A G N E S I U M D E F I C I E N C Y O N VITAMIN D M E T A B O L I S M A N D A C T I O N Mg may also be important in vitamin D metabolism a n d / o r action, as suggested by a n u m b e r of clinical observations(for review see Refs. 30, 80, and 81).

Patients with hypoparathyroidism who have been resistant to therapeutic doses of vitamin D have been reported to become more responsive to vitamin D after Mg therapy (82-84). The treatment of malabsorption syndromes with vitamin D may not be effective until Mg is simultaneously administered (85,86). Rickets, thought to be secondary to vitamin D resistance, have healed with Mg therapy (87). Patients with hypocalcemia and Mg deficiency have been reported to be resistant to pharmacologic doses of vitamin D (85,88,89), lc~-hydroxyvitamin D (90,91), and 1,25dihydroxyvitamin D (92). Similarly, impaired calcemic response to vitamin D has been found in Mg-deficient rats (93-95), lambs (96), and calves (97). Intestinal calcium transport in animal models of Mg deficiency has been found to be reduced in some (98,99) but not all (94,100) studies. Calcium malabsorption was associated with low serum levels of 25hydroxyvitamin D in one study (93), but not another (100), suggesting that Mg deficiency may impair intestinal calcium absorption by more than one mechanism. The exact nature of altered vitamin D metabolism a n d / o r action in Mg deficiency is unclear. Patients with Mg deficiency and hypocalcemia frequently have low serum concentrations of 25-hydroxyvitamin D (101-104) and therefore nutritional vitamin D deficiency may be one factor. Therapy with vitamin D, however, results in high serum levels of 25-hydroxyvitamin D without correction of the hypocalcemia (85), suggesting that the vitamin D deficiency is not the prime reason. In addition, conversion of radiolabeled vitamin D to 25-hydroxyvitamin D was found to be normal in three Mg-deficient patients (105). Serum concentrations of 1,25-dihydroxyvitamin D have also been found to be low or low normal in most hypocalcemic Mgdeficient patients (101-104). Magnesium-deficient diabetic children, when given a low-calcium diet, did not exhibit a normal rise in serum 1,25-dihydroxyvitamin D or PTH (106). The response returned to normal following Mg therapy, supporting the notion of altered vitamin D metabolism in Mg deficiency. Because PTH is a major trophic factor for 1,25-dihydroxyvitamin D formation, the low serum PTH concentrations could explain the low 1,25-dihydroxyvitamin D levels. In support of this is the finding that some hypocalcemic Mgdeficient patients treated with Mg have a rise in serum 1,25-dihydroxyvitamin D to high normal or to frankly elevated levels (see Fig. 6) (101). Most patients, however, do not have a significant rise within 1 week after institution of Mg therapy, despite a rise in serum PTH and normalization of the serum calcium concentration, as shown in Fig. 6 (101). These data suggest that Mg deficiency in humans also impairs the ability of the kidney to synthesize 1,25-dihydroxyvitamin D. This is supported by the observation that the ability of exogenous administration of h u m a n PTH(1-34) to normal sub-

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jects after 3 weeks of experimental Mg depletion resulted in a significantly lower rise in serum 1,25dihydroxyvitamin D concentrations than before institution of the diet (51). It appears, therefore, that the renal synthesis of 1,25-dihydroxyvitamin D is sensitive to Mg depletion. T h o u g h Mg is known to support the 25-hydroxy-lot-hydroxylase in vitro (107,108), the exact Mg requirement for this enzymatic process is not known. The association of Mg deficiency with impaired vitamin D metabolism and action therefore may be due to several factors. In some cases vitamin D deficiency may contribute (80,81,101-104). The major reasons, however, appear to be due to a decrease in PTH secretion with resultant decreased trophic effect on 1,25-dihydroxyvitamin D synthesis as well as a direct effect of Mg depletion on the ability of the kidney to synthesize 1,25-dihydroxyvitamin D (30,54-59,80,81,101-104). In addition, Mg deficiency may impair intestinal calcium absorption by resulting in low levels of vitamin D metabolites (30,101-104) or by a direct mechanism (98,99). Skeletal resistance to vitamin D and its metabolites may also play an important role (90-97). It is clear, however, that restoration of normal serum 1,25-dihydroxyvitamin D concentrations is not required for normalization of the serum calcium level. Most Mgdeficient patients who receive Mg therapy exhibit an immediate rise in PTH followed by normalization of the serum calcium prior to any change in serum 1,25-dihydroxyvitamin D concentrations (101-104). An overall view of the effect of Mg deficiency on calcium homeostasis is shown in Fig. 7.

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FIG. 7 Disturbance of calcium metabolism during magnesium deficiency. Hypocalcemia is caused by a decrease in PTH secretion as well as renal and skeletal resistance to the action of PTH. Low serum concentrations of 1,25-dihydroxyvitamin D may result in reduced intestinal calcium absorption. (From Ref. 135, RK Rude. Disorders of magnesium metabolism. In: RD Cohen, B Lewis, KGMM Alberti, AM Denman, Eds. The metabolic and molecular basis of acquired disease. Copyright © 1990 Bailliere Tindall, London.)

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M E C H A N I S M OF IMPAIRED MINERAL H O M E O S T A S I S IN M A G N E S I U M D E F I C I E N C Y The mechanism for impaired PTH secretion and action in Mg deficiency remains unclear. It has been suggested that a high serum PTH concentration is more important in causing PTH resistance than is the Mg deficiency, because PTH administration has been demonstrated to result in diminishing peripheral effects of PTH (59). In the Mg-deficient patient, however, as Mg deficiency worsens serum PTH concentrations fall (30,54-59). PTH resistance has been demonstrated in patients who had undetectable low PTH levels, suggesting that there are other operative mechanisms (30,61-64). Mg depletion may alter the function of secondmessenger systems. PTH is thought, in part, to exert is biologic effects through the intermediary action of cyclic AMP (109). Adenylate cyclase requires Mg for cyclic AMP generation both as a component of the substrate (Mg-ATP) and as an obligatory activator of enzyme activity (110). There are two Mg 2+ binding sites within the adenylate cyclase complex: one resides on the catalytic subunit and the other on the guanine nucleotide regulatory protein, Gs (111,112). The requisite role which Mg 2+ plays in adenylate cyclase function suggests that factors that would limit the availability of Mg 2+ to this enzyme could have significant effects on the cyclic nucleotide metabolism of a cell and hence on overall cellular function. It is clear that some patients with severe Mg deficiency have a reduced urinary excretion of cyclic AMP in response to exogenously administered PTH (30). In addition, PTH had a blunted effect in causing a rise in cyclic AMP from isolated perfused tibiae in Mg-deficient dogs (71). These observations correspond well with the impaired calcemic and phosphaturic effects of PTH in Mg-deficient patients and animals, as discussed above. Nine isoforms of adenylate cyclase have been identified; their activities are modulated by both Mg '2+ and C a 2+ (113). Though Mg 2+ is stimulatory, Ca 2+ may inhibit or activate enzyme activity (113). In plasma membranes from parathyroid, renal cortex, and bone cells, Ca '~+ was observed to inhibit competitively Mg z+activated adenylate cyclase activity (114-117). Thus, the ambient Mg 2+ concentrations can markedly affect the susceptibility of this enzyme to the inhibitory effects of Ca 2+. Because total intracellular calcium has been observed to rise during Mg depletion (118,119), the combination of higher intracellular C a z+ and increased sensitivity t o C a 2+ inhibition due to Mg depletion could explain the defective PTH action in Mg deficiency. Though cyclic AMP is an important mediator of PTH action, current data do not support an important role in mediating Ca 2+ -regulated PTH secretion (120).

The effect of C a 2+ o n PTH secretion appears to involve the Ca-sensing receptor and G protein activation of the phospholipase C second-messenger system (120,121). PTH activation of phospholipase C leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol1,4,5-triphosphate (IP~) and diacylglycerol. IP 3 binds to specific receptors on intracellular organelles (endoplasmic reticulum, calciosomes), leading to an acute transient rise in cytosolic C a 2+ with subsequent activation of calmodulin-dependent protein kinases. Diacylglycerol activates protein kinase C. Mg depletion could perturb this system via several mechanisms. First, Mg 2+ -dependent guanine nucleotide regulating proteins (e.g., G 8 or Gll ) are also involved in activation of phospholipase C (122,123). Mg 2+ has also been shown to be a noncompetitive inhibitor of IPs-induced Ca 2+ release (124). A reduction of Mg 2+ from 300 to 30 IxM increased C a 2+ release in response to IP~ by two- to threefold in mitochondrial membranes obtained from canine cerebellum (124). In these same studies Mg 2+ also was found to inhibit IP s binding to its receptor. Magnesium, at concentrations of 0.5 mM, decreased maximal IP s binding threefold (124). These Mg 2+ concentrations are within the estimated physiologic intracellular range (200-500 IxM) and therefore Mg 2+ may be an important physiologic regulator of the phospholipase C second-messenger system. Second-messenger systems are widely distributed enzymes in the body and if the above hypothesis were true, the secretion and action of other hormones might also exhibit impaired activity in Mg deficiency. The action of ACTH, TRH, GnRH, and glucagon, however, have been demonstrated to be normal in hypocalcemic Mg-deficient patients (125). It was hypothesized that the adenylate cyclase enzyme in the target tissues of those hormones might have a lower Mg 2+ requirement or be less inhibitable by Ca 2+ compared to the parathyroid, kidney, and bone, which would afford selective protection from the effects of Mg depletion. Specific forms of adenylate cyclase have distinct activities in the presence of Mg 2+(126). Prior investigations have suggested that Mg affinity for adenylate cyclase is higher (lower KaMg ) i n liver (127), adrenal (128), and pituitary (129) than in parathyroid (114). In one study, investigation of Ka Mg and /~ ca in tissues from one species (guinea pig) demonstrated that under agonist stimulation the Ka Mg from liver < thyroid < kidney = bone, and the/q. Ca 2+ for liver > renal > kidney = bone (117) These data suggest that adenylate cyclase regulation by divalent cations varies from tissue to tissue and may explain the greater propensity for disturbed mineral homeostasis in Mg deficiency. It is clear that the effect of Mg depletion on cellular function in terms of the second-messenger systems is

MAGNESIUM IN PARATHYROIDFUNCTION / most complex, potentially involving substrate availability, G protein activity, release of and sensitivity to intracellular Ca 2+, and phospholipid metabolism.

D I A G N O S I S OF M A G N E S I U M DEFICIENCY As m e n t i o n e d above, Mg is principally an intracellular cation. Less than 1% of the body Mg content is in the extracellular fluid compartments. The serum Mg concentration, therefore, may not reflect the intracellular Mg content. A serum Mg concentration that will result in impaired PTH secretion or resistance cannot be predicted. Nevertheless, measurement of the serum Mg concentration is the most available and commonly employed test to assess Mg status. The normal serum Mg concentration ranges from 1.7 to 2.2 m g / d l (0.71-0.91 mM); a value - C) in exon 2 (29), which resulted in the substitution of arginine (CGT) for cysteine (TGT) in the signal pepdde. The presence of this charged amino acid in the midst of the hydrophobic core of the signal pepdde impeded the processing of the mutant preproPTH, as demonstrated by in vitro studies. These revealed that the mutation impaired the interaction with the nascent protein and the translocadon

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machinery, and that cleavage of the mutant signal sequence by solubilized signal peptidase was ineffective (29,30). Ineffective cleavage of the p r e p r o P T H sequence results in a molecule that does not proceed successfully through the subsequent intracellular steps required for ultimate delivery of PTH to secretory granules. The parathyroid cell, therefore, cannot respond to hypocalcemia with the secretion of native, biologically active PTH. A Form o f A u t o s o m a l Recessive Hypoparathyroidism

Another abnormality of the PTH gene, involving a d o n o r splice site at the exon 2 and intron 2 boundary, has been identified in one family with autosomal recessive isolated hypoparathyroidism (31). This mutation involved a single base transition (g + c) at position 1 of intron 2. The effect of this alteration in the invariant gt dinucleotide of the 5' d o n o r splice site consensus on mRNA processing was assessed by an analysis of the non-tissue-specific transcription of the normal and mutant PTH genes. This non-tissue-specific expression of genes has been estimated to be at the rate of one molecule of correctly spliced mRNA per 1000 cells (32,33). Although the physiologic relevance of this low level of non-tissue-specific (or illegitimate) transcription is not known, it is of clinical importance. Easily accessible peripheral blood lymphocytes can be used to

detect abnormalities in mRNA processing, thereby avoiding the requirement for tissue that may be obtainable only by biopsy. Use of these methods revealed that the d o n o r splice site mutation resulted in exon skipping, in which exon 2 of the PTH gene was lost and exon 1 was spliced to exon 3. The lack of exon 2 would lead to a loss of the initiation codon (ATG) and the signal peptide sequence (Fig. 2), which are required, respectively, for the proper c o m m e n c e m e n t of PTH mRNA translation and for the translocation of the PTH peptide. Thus, the patients' parathyroid cells would not contain any translated PTH products.

X-Linked Recessive Hypoparathyroidism Hypoparathyroidism with an X-linked recessive transmission pattern has been reported in two multigenerational kindreds (34-37). Only males were affected, suffering from infantile epilepsy and hypocalcemia. The hypoparathyroidism is due to a defect in parathyroid gland development. Linkage studies utilizing X-linked RFLPs in these two families assigned the mutant gene to chromosome Xq26-q27 (34). A novel approach utilizing mitochondrial DNA analysis established a common ancestry in these two X-linked hypoparathyroid kindreds (35). A c o m m o n ancestry for these two kindreds from eastern Missouri in the United States had been suspected, but it could not be established despite five gen-

MOLECULARGENETICSOF HYPOPARATHYROIDISM / erations of extensive genealogic records. The mitochondrial genes are transmitted through the maternal line exclusively. If relatedness among the two kindreds involved the maternal lines, analysis of mitochondrial genetic markers would reveal common features. The DNA sequence of the mitochondrial (mt) D loop was compared among individuals in both kindreds. The mt DNA sequence was identical among affected males and their maternal lineage for individuals in both kindreds, but differed at three to six positions when compared with the mitochondrial DNA of the fathers. These results demonstrated that the two kindreds with Xlinked recessive hypoparathyroidism are indeed related and that an identical gene defect is likely to be responsible for the disease. Additional studies have refined the location of this gene to be between factor IX (FIX) and DXS 1205 (36). Analysis of a yeast artificial chromosome (YAC) contig of this region (37) indicates that the region is 1.5 million base pairs in size and contains at least three candidate genes. The specific gene defect responsible for this form of X-linked hypoparathyroidism has yet to be identified. The results of this study also demonstrate that the mitochondrial genetic approach may be of importance in detecting common ancestry in other X-linked diseases.

COMPLEX SYNDROMES ASSOCIATED WITH HYPOPARATHYROIDISM Hypoparathyroidism may occur as part of a complex syndrome that may either be associated with a congenital developmental anomaly or with an autoimmune syndrome.

Congenital Syndromes Hypoparathyroidism has been reported to occur in association with the congenital developmental anomalies of the DiGeorge, the Kenney-Caffey, and the Barakat syndromes and also in syndromes associated with either lymphedema, or renal dysplasia and deafness, or with dysmorphic features and growth failure (Table 1). The inheritance of these congenital disorders, which has been reported in a few patients or a single family, has sometimes not been fully established. However, an autosomal dominant inheritance for the DiGeorge syndrome, which has been investigated by the methods of molecular genetics, is established.

DiGeorgeSyndrome Patients with the DiGeorge syndrome (DGS) typically suffer from hypoparathyroidism, immunodefi-

783

ciency, congenital heart defects, and deformities of the ear, nose, and mouth (2). The disorder arises from a congenital failure in the development of the derivatives of the third and fourth pharyngeal pouches with resulting absence or hypoplasia of the parathyroids and thymus. Most cases of DGS are sporadic but an autosomal dominant inheritance of DGS has been observed and an association between the syndrome and an unbalanced translocation and deletions involving 22q11.2 have also been reported (38). In some patients, deletions of another locus on chromosome 10p have been observed in association with DGS (39). Mapping studies of the DGS deleted region on chromosome 22q11.2 have defined a 250-kb minimal critical region (40), and cloning of the translocation breakpoint on 22q11.21 from a DGS patient (41) has revealed that there are probably two genes (rnex40 and nex2.2-nex3), transcribed in opposite directions, that are disrupted by this breakpoint (42). The coding region of one of these genes, rnex40, has homology to the mouse and rat androgen receptors and contains a leucine zipper motif, suggesting that the DGS candidate gene may be a DNA-binding protein. Eleven nucleotides of the rnex40 gene are deleted at the translocation junction, making it likely that loss of function of this gene is responsible for at least part of the DiGeorge phenotype (42). Another partial transcript, referred to as nex2.2-nex3, was also identified from this breakpoint. Both rnex40 and nex2.2-nex3 are deleted in all DGS patients with 2 2 q l l deletions and studies aimed at assessing the presence of hemizygosity and mutations in these two genes in DGS patients who do not have detectable 2 2 q l l deletions are required to demonstrate the role of these genes in the etiology of the DGS. Such studies have been performed for a h u m a n homolog of a yeast gene, referred to as UDF1L, that encodes a protein involved in the degradation of ubiquinated proteins, (43). UDF1L is located on 2 2 q l l and has been found to be deleted in all of 182 patients with the 2 2 q l l deletion syndrome (43), which includes patients with the DGS, the velo-cardio-facial syndrome (VCFS), and the conotruncal anomaly face syndrome (CAFS) (38,40). However, a smaller deletion of approximately 20 kb that removed exons 1 to 3 of UDF1L was detected in one patient (43). This patient, who had a de novo deletion resulting in haploinsufficiency of UDF1L, suffered from neonatal-onset cleft palate, small mouth, low-set ears, broad nasal bridge, an interrupted aortic arch, a persistent truncus arteriosus, hypocalcemia, T lymphocyte deficiency, and syndactyly of her toes (43). These results indicate that abnormalities of the UDF1L gene are likely to contribute to the etiology of early-onset DGS. Patients with late-onset DGS (44,45) develop symptomatic hypocalcemia in childhood or during adolescence with only subtle phenotypic

784

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CHAPTER49

abnormalities. These late-onset DGS patients have similar microdeletions in the 22ql I region, and the molecular definition of these variants of the DiGeorge syndrome may well provide additional insights into the regulation of PTH secretion a n d / o r parathyroid gland development.

Mitochondrial Disorders Associated with Hypoparathyroidism Hypoparathyroidism has been reported to occur in three disorders associated with mitochondrial dysfunction: the Kearns-Sayre syndrome (KSS), the MELAS syndrome, and a mitochondrial trifunctional protein deficiency syndrome. KSS is characterized by progressive external ophthalmoplegia and pigmentary retinopathy before the age of 20 years, and is often associated with heart block or cardiomyopathy. The MELAS syndrome consists of a childhood onset of mitochondrial encephalopathy, lactic acidosis, and strokelike episodes. In addition, varying degrees of proximal myopathy can be seen in both conditions. Both KSS and the MELAS syndrome have been reported to occur with insulin-dependent diabetes mellitus and hypoparathyroidism (46,47). A point mutation in the mitochondrial gene tRNA leucine (UUR) has been reported in one patient with the MELAS syndrome who also suffered from hypoparathyroidism and diabetes mellitus (48). Large deletions, consisting of 6741 and 6903 base pairs and involving >38% of the mitochondrial genome, have been reported in other patients who suffered from KSS, hypoparathyroidism, and sensorineural deafness (47,49). Rearrangements (50) and duplication (51) of mitochondrial DNA have also been reported in KSS. Mitochondrial trifunctional protein deficiency is a disorder of fatty acid oxidation that is associated with peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women who carry an affected fetus. Hypoparathyroidism has been observed in one patient with trifunctional protein deficiency (52). The role of these mitochondrial mutations in the etiology of hypoparathyroidism remains to be further elucidated.

Kenney-Caffey Syndrome Hypoparathyroidism has been reported to occur in over 50% of patients with the Kenney-Caffey syndrome, which is associated with short stature, osteosclerosis and cortical thickening of the long bones, delayed closure of the anterior fontanel, basal ganglia calcification, nanophthalmos, and hyperopia (53,54). Parathyroid tissue could not be found in a detailed postmortem examination of one patient (55) and this suggests that hypoparathy-

roidism may be due to an embryologic defect of parathyroid development. A molecular genetic analysis using PTH gene RFLP analysis revealed no abnormalities (56), and mutations at other locimfor example, in developmental genesmneed to be investigated.

Additional Familial Syndromes Single familial syndromes in which hypoparathyroidism is a component have been reported (Table 1). The inheritance of the disorder in some instances has been established and molecular genetic analysis of the PTH gene has revealed no abnormalities. Thus, an association of hypoparathyroidism, sensorineural deafness, and renal dysplasia has been observed in one British family, in whom an autosomal dominant inheritance of the disorder was established (57). An analysis of the PTH gene in this family revealed no abnormalities. Autosomal recessive inheritance of hypoparathyroidism in association with renal insufficiency and development delay has been reported in one Asian family (58), and a similar analysis of the PTH gene revealed no abnormalities (22). The occurrence of hypoparathyroidism, nerve deafness, and a steroid-resistant nephrosis leading to renal failure, which has been referred to as the Barakat syndrome (59), has been reported in four brothers from one family, and an association of hypoparathyroidism with congenital lymphedema, nephropathy, mitral valve prolapse, and brachytelephalangy has been observed in two brothers from another family (60). Molecular genetic studies have not been reported from these two families. A syndrome in which hypoparathyroidism was associated with severe growth failure and dysmorphic features has been reported in 12 patients from Saudi Arabia (61). Consanguinity was noted in families of 11 of the 12 patients, the majority of which originated from the western province of Saudi Arabia. This syndrome, which is inherited as an autosomal recessive disorder, has also been identified in families of Bedouin origin, and homozygosity and linkage disequilibrium studies have located this gene to chromosome lq42-q43 (62). Molecular genetic investigations of these disorders will help to identify additional genes that regulate the development of the parathyroid glands.

Blomstrand Disease Blomstrand chondrodysplasia is an autosomal recessive h u m a n disorder characterized by early lethality, dramatically advanced bone maturation, and accelerated chondrocyte differentiation (63). Affected infants, who usually have consanguineous unaffected parents (64-68), develop pronounced hyperdensity of the

MOLECULAR GENETICS OF HYPOPARATHYROIDISM / entire skeleton with markedly advanced ossification, which results in extremely short and poorly modeled long bones. Mutations of the P T H / P T H r P receptor that impair its function are associated with Blomstrand disease (69-71). Thus it seems likely that affected infants will, in addition to the skeletal defects, also have abnormalities in other organs, including secondary hyperplasia of the parathyroid glands, presumably due to hypocalcemia.

Pluriglandular Autoimmune Hypoparathyroidism Hypoparathyroidism may occur in association with candidiasis, pernicious anemia, allopecia, vitiligo, and a u t o i m m u n e Addison's disease, and the disorder has been referred to as either the a u t o i m m u n e polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome or the polyglandular autoimm u n e type 1 syndrome (72). This disorder has a high incidence in Finland, and a genetic analysis of 58 patients from 42 Finnish families indicated autosomal recessive inheritance of the disorder (73). In addition, the disorder has been reported to have a high incidence a m o n g Iranian Jews, although the occurrence of candidiasis was less c o m m o n in this population (74). Linkage studies of 14 Finnish families m a p p e d the APECED gene to chromosome 21q22.3 (75). Further positional cloning studies led to the isolation of a novel gene from chromosome 21q22.3. This gene, referred to as MRE (autoimmune regulator), encodes a 545-amino acid protein that contains motifs suggestive of a transcriptional factor and includes a nuclear localization signal, two zinc-finger motifs, a proline-rich region, and three LXXLL motifs (76,77). Six MRE mutations have been reported in the APECED families and a codon 257 (Arg ---> Stop) mutation was the p r e d o m i n a n t abnormality in 82% of the Finnish families (76,77). The identification of the genetic defect causing APECED will not only facilitate genetic diagnosis but will also enhance the elucidation of the mechanisms causing a u t o i m m u n e disease.

CALCIUM-SENSING

RECEPTOR ABNORMALITIES The CaSR is a G protein-compled receptor that is located in the plasma m e m b r a n e of the cell (Fig. 1); this is a critical site to enable the parathyroid cell to recognize changes in extracellular calcium concentration. Thus, an increase in extracellular calcium leads to CaSR activation of the G protein signaling pathway, which in turn increases the free intracellular calcium concentration and leads to a reduction in trascription of the PTH gene. The CaSR is also expressed in the dis-

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tal renal tubule, and activation in response to hypercalcemia can increase renal calcium excretion. CaSR mutations that result in a loss of function are associated with familial benign hypercalcemia with hypocalciuria (FHH) (78-83). However, CaSR missense mutations that result in a gain of function (or added sensitivity to extracellular calcium) lead to hypocalcemia with hypercalciuria (84-88). These hypocalcemic individuals are generally assymptomatic and have serum PTH concentrations that are in the low-normal range, and because of the insensitivities of previous PTH assays in this range, such patients have often been diagnosed to be hypoparathyroid. In addition, such patients may have hypomagnesemia. Treatment with Vitamin D or its active metabolites to correct the hypocalcemia in these patients results in marked hypercalciuria with attendent risk of nephrocalcinosis, nephrolithiasis, and renal impairment. Thus, these patients need to be distinguished from those with true hypoparathyroidism.

PSEUDOHYPOPARATHYROIDISM Patients with pseudohypoparathyroidism (PHP) are characterized by hypocalcemia and hyperphosphatemia due to PTH resistance (2,89-91) (also see Chapter 51). Instead of PTH deficiency these patients have elevated levels of serum PTH that are biochemically and biologically normal. Resistance to PTH, demonstrated by little or no increase in urinary excretion of n e p h r o g e n o u s cyclic AMP and phosphate after PTH infusion, is referred to as PHP type 1. The occurrence of PHP type 1 with the somatic features of Albright's hereditary osteodystrophy (AHO) is referred to as PHP type l a, whereas t h e presence of biochemical features without somatic features is referred to as PHP type lb. The occurrence of somatic features (AHO) without the biochemical abnormalities is referred to as pseudo-pseudohypoparathyroidism (PPHP). The absence of a normal rise in urinary excretion of cyclic AMP excretion after an infusion of PTH in PHP type la and PPHP indicates a defect at some site of the PTH receptor-adenylyl cyclase system. This receptor system is regulated by at least two G proteins, one of which stimulates (Gsot) and another which inhibits (Gi0t) the activity of the m e m b r a n e - b o u n d enzyme that catalyzes the formation of the intracellular secondmessenger cyclic AME Inactivating mutations of the Gsc~ gene (referred to as GNAS1), which is located on chromosome 20q13.2, have been identified in PHP type la and PPHP patients (89-91). A mutational "hot-spot" in exon 7 that consists of a 4-bp deletion (A GACT) of codon 189 and the first nucleotide of codon 190 has been identified (89).

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These mutations, which are heterozygous, lead to an ~ 5 0 % reduction in GsoLexpression or activity, and are thought to explain, at least partially, the resistance to PTH and other hormones [e.g., thyroid-stimulating h o r m o n e (TSH), gonadotrophins, and glucagon] that mediate their actions through G protein-coupled receptors (89-91). However, a similar reduction in GsoL activity/protein is also found in patients with PPHP, who show the same physical features as individuals with PHP-la, but lack the endocrine abnormalities. Thus, GNAS1 mutations do not fully explain the PHP-la or PPHP phenotypes (89-94), and studies of PHP-la and PPHP that occurred within the same kindred revealed that the h o r m o n a l resistance is paternally imprinted. Thus, PHP-la occurred in a child only when the mutation was inherited from a m o t h e r affected with either PHP-la or PPHP (95,96). These findings in humans gained support by observations in heterozygous Gnas knockout mice that lacked exon 2. Mice that had inherited the mutant allele from a female showed undetectable Gsct protein in the renal cortex and decreased blood calcium concentration due to PTH resistance. In contrast, offspring that had inherited the mutant allele, lacking exon 2, from a male showed no evidence of endocrine abnormalities (97). T i s s u e - o r cell-specific GsoLexpression is thus likely to be involved in the pathogenesis of PHP-la and PPHP, and this may also help to explain the dominant phenotype that arises from heterozygous GNAS1 mutations. Expression of the GNAS1 gene has been shown to be further complicated by alternative splicing, which results in several different mRNAs, some of which are derived only from either the paternal or the maternal allele. This complexity of the GNAS1 gene may contribute to the unique phenotypic abnormalities observed in patients with PHP because the alternative splicing gives rise to at least three different gene products that are transcribed either from the maternal or the paternal allele, or from both alleles (98-100). The first of these products is Gset, which is encoded by exons 1 through 13 of the GNAS1 gene and mediates the biologic functions of a large variety of G protein-coupled receptors, including the P T H / P T H r P receptor. The second product is XL~s, which is encoded by a novel first exon (XL) spliced onto exons 2 through 13. The encoded XL~s, which is a ~92-kDA protein, shares considerable amino acid sequence identity with the carboxyl-terminal portion of Gset (101), but does not appear to function as a stimulatory G protein. XL~s expression occurs at numerous sites and is particularly high in endocrine and n e u r o e n d o c r i n e cells (101). Furthermore XL~s appears to be transcribed only from the paternal allele (98-100). The third product of the GNAS1 gene is NESP55 (102), which is transcribed only from the maternal allele (99,100). NESP55 is encoded by yet another exon of the GNAS1 gene, which is located upstream of exon XL and the GsoL-specific exon

1. The NESP55 exon is also spliced to exons 2 through 13, but the NESP55 protein, which is thought to act as a n e u r o e n d o c r i n e secretory protein (102), shares no amino acid sequence homology with either XL~s or Gs0L. The complexity of the GNAS1 gene and its use of different allele-specific promoters makes it plausible to postulate that mutations in the Gsct-specific exons 1 to 13 (89-94) will affect not only the functional properties of GsoL, but also those of XL~s and of NESP55. GNAS1 mutations have not been detected in PHP type l b (PHP-lb), which had originally been considered to be due to a defect of the P T H / P T H r P receptor. However, it is important to note that PHP-lb patients generally have a normal or increased osseous response to PTH, as assessed by bone turnover and osteoclastic resorption (89,90,103,104), and the normal growth plate development in these patients is consistent with a normal chondrocyte response to PTHrE These observations made it unlikely that defects in the P T H / P T H r P receptor were the cause of PHP-lb, and indeed studies of the P T H / P T H r P receptor gene and mRNA in PHP-lb patients have failed to identify mutations (105-108). In order to identify the location of the PHP-lb gene a genome-wide search was therefore undertaken in four unrelated kindreds, and this m a p p e d the PHP-lb locus to chromosome 20q13.3, a location that also contains the GNAS1 gene (109). In addition, paternal imprinting of the genetic defect was observed and this is similar to the findings in kindreds with PHP type la a n d / o r PPHE Two possible explanations for these observations have been proposed. First, PHP-lb may be due to a defect in a tissue-or cellspecific enhancer, or promoter of the GNAS1 gene, and this may affect, directly or indirectly, the expression levels of the Gset-specific transcripts a n d / o r the transcripts encoding XL~s and NESP55; or second, PHP-lb may be caused by a defect in a gene close to the GNAS1 gene that is transcribed only from the maternal allele and affects P T H / P T H r P receptor or Gset expression a n d / o r function in some renal cells.

CONCLUSION Application of the methods of molecular genetics to the study of the hypoparathyroid disorders has resulted in considerable advances that have identified some genes and their encoded proteins that are involved in the regulation of PTH synthesis and secretion, and in mediating PTH actions in different target issues. In addition, the identification of mutations has helped to provide molecular explanations and insights into a variety of familial and sporadic disorders of calcium homeostasis and bone development. Genetic mapping studies have also helped to identify the chromosomal locations of some hypoparathyroid disorders, and

MOLECULARGENETICS OF HYPOPARATHYROIDISM / future positional cloning approaches, which will progress with greater rapidity owing to the Human Genome Sequencing project, will provide additional insights into the mechanisms that regulate PTH action and calcium homeostasis.

ACKNOWLEDGMENTS I am grateful to the Medical Research Council (United Kingdom) for support, to B. Harding for preparing the figures, and to Julie Allen for typing the manuscript and expert secretarial assistance. Note added in proof." Hypoparathyroidism, deafness, and renal dysplasia (HDR) is an autosomal dominant syndrome (57) associated with haploinsufficiency of the GATA3 gene (111), which is located on chromosome 10p15. GATA3 belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development, and these findings (111) in HDR patients indicate an important role for GATA3 in parathyroid development.

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MOLECUI~a~ GENETICS OF HYPOPARATHYROIDISM / 71. Karaplis AC, Bin He MT, Nguyen A, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998; 139:5255-5258. 72. Ahonen P, Myllarniemi S, Sipila I, et al. Clinical variation of autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829-1836. 73. Ahonen E Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED): Autosomal recessive inheritance. Clin Genet 1985;27:535-542. 74. ZlotogoraJ, Shapiro MS. Polyglandular autoimmune syndrome type 1 among Iranian Jews. J Med Genet 1992;29:824-826. 75. Aaltonen J, Bjorses P, Sandkuijl L, et al. A n autosomal locus causing autoimmune disease: Autoimmune polyglandular disease type 1 assigned to chromosome 21. Nat Genet 1994;8:83-87. 76. Nagamine K, Peterson P, Scott HS, et al. Positional cloning of the APECED gene. Nat Genet 1997;17:393-398. 77. The Finnish-German APECED consortium: An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc finger domains. Nat Genet 1997;17:399-403. 78. Pollak MR, Brown EM, Chou YWH, Herbert SC, Marx sJ, Steinmann B, Levi T, Seidman CE, Seidman JG. Mutations in the human CaZ+-sensing receptor gene cause familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-1303. 79. Chou YWH, Pollak MR, Brandi ML, Toss T, Arnqvist H, Brew Atkinson A, Papapoulos SE, Marx S, Brown EM, Seidman JG, Siedman CE. Mutations in the human Ca2+-sensing receptor gene that cause familial hypocalciuric hypercalcaemia. Am J Hum Genet 1995;56:1075-1079. 80. Pearce SHS, Trump D, Wooding C, Besser GM, Chew SL, Grant DB, Heath DA, Hughes IA, Paterson CR, Whyte ME Thakker RV. Calcium-sensing receptor mutations in familial benign hypocalcaemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683-2692. 81. Janicic N, Pausova Z, Cole DEC, Hendy GN. Insertion of an Alu sequence in the CaZ+-sensing receptor gene in familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Am J H u m Genet 1995;56:880-886. 82. Aida K, Koishi S, Inoue M, Nakazato M, Tawata M, Onaya T. Familial hypocalciuric hypercalcaemia associated with mutation in the human CaZ+-sensing receptor gene. J Clin Endocrinol Metab 1995;80:2594-2598. 83. Heath III H, Odelberg S, Jackson CE, The BT, Hayward N, Larsson C, Buist NRM, Krapcho KJ, Hung BC, Capuano IV, Garrett JE, Leppert ME Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcaemia suggest receptor functional domains. J Clin Endocrinol Metab 1996;81:1312-1317. 84. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG. Autosomal dominant hypocalcaemia caused by a calcium-sensing receptor gene mutation. Nat Genet 1994;8:303-307. 85. Finegold DN, Armitage MM, Galiani M, Matise TC, Pandian MR, Perry YM, Deka R, Ferrell RE. Preliminary localisation of a gene for autosomal dominant hypoparathyroidism to chromosome 3q13. Paediatr Res 1994;36:414-417. 86. Perry YM, Finegold DN, Armitage MM, Ferrell RE. A missense mutation in the Ca-sensing receptor causes familial autosomal dominant hypoparathyroidism. Am J Hum Genet 1994;55:A17. 87. Pearce SHS, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, KendallTaylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcaemia with hypocalciuria due to mutations in the calcium-sensing receptor gene. N EnglJ Med 1996;335:1115-1122.

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88. Baron J, Winer KK, Yanovski JA, et al. Mutations in the Ca 2+sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 1996;5:601-606. 89. Yu S, Yu D, Hainline BE, BrenerJL, Wilson KA, Wilson LC, OudeLuttikhuis ME, Trembath RC, Weinstein LS. A deletion hot-spot in exon 7 of the GsoLgene (GNAS1) in patients with Albright hereditary osteodystrphy. Hum Mol Genet 1995;4:2001-2002. 90. van Dop C. Pseudohypoparathyroidism: Clinical and molecular aspects. Semin Nephrol 1989;9:168-178. 91. Weinstein LS. Albright hereditary osteodystrophy, pseudohypoparathyroidism, and Gs deficiency. In: Spiegel AM, ed. G proteins, receptors, and disease. Totowa, New Jersey:Humana,

1998;23-56.

92. Schuster V, Eschenhagen T, Kruse K, et al. Endocrine and molecular biological studies in a German family with Albright hereditary osteodystrophy. E u r J Pediatr 1993; 152:185-189. 93. Miric A, Bechio JD, Levine MA. Heterogeneous mutations in the gene encoding the oL-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 1993;76:1560-1568. 94. Weinstein LS, Gejman PV, Friedman E, et al. Mutations of the Gs o~-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990;87:8287-8290. 95. Davies AJ, Hughes HE. Imprinting in Albright's hereditary osteodystrophy. J Med Genet 1993;30:101-103. 96. Wilson LC, Oude-Luttikhuis MEM, Clayton PT, et al. Parental origin of GsoLgene mutations in Albright's hereditary osteodystrophy. J Med Genet 1994;31:835-839. 97. Yu S, Yu D, Lee E, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein a-subunit (GsO0 knockout mice is due to tissue-specific imprinting of the GsOL.Proc Natl Acad Sci USA 1998;95:8715-8720. 98. Hayward B, Kamiya M, Strain L, et al. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc Natl Acad Sci USA 1998;95:10038-10043. 99. Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998;95:15475-15480. 100. Peters J, Wroe SF, Wells CA. A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2. Proc Natl Acad Sci USA 1999;96:3830-3835. 101. Kehlenbach RH, MattheyJ, Huttner WB. XLoLsis a new type of G protein. Nature 1994;372:804-809. 102. Ischia R, Lovisetti-Scamihorn P, Hogue-Angeletti R, et al. Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem 1997;272:11657-11662. 103. Murray T, Gomez Rao E, Wong MM, et al. Pseudohypoparathyroidism with osteitis fibrosa cystica: Direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone. J Bone Miner Res 1993;8:83-91. 104. Farfel Z. Pseudohypohyperparathyroidism-pseudohypoparathyroidism type Ib. JBone Miner Res 1999;14,1016. 105. Schipani E, Weinstein LS, Bergwitz C, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80:1611-1621. 106. Bettoun JD, Minagawa M, Kwan MY, et al. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1997;82:1031-1040.

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107. Suarez F, Lebrun JJ, Lecossier D, et al. Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 1995;80:965-970. 108. Fukumoto S, Suzawa M, Takeuchi Y, et al. Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1996;81: 2554-2558.

109. Jfippner H, Schipani E, Bastepe M, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci. USA 1998;95:11798-11803. 110. Thakker RV. Parathyroid disorders: Molecular genetics and physiology. In: Morris PJ, Wood WC, eds. Oxford:Oxford Textbook of Surgery, 2nd Edition, Oxford Univ. Press, 2000; 1121-1129. 111. Van Esch H, Groenen P, Nesbit MA, et al. GATA3 haplo-insufficiency causes human HDR syndrome. Nature 2000;406:419-422.

CHAPTF R 50

A u t o l•m m u n e

Hyp

oparat h y roldlsm " "

M I C H A E L P. W H Y T E Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, Missouri 63131; and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110

INTRODUCTION

a pathogenetic factor (4-10). In this chapter, the autoimmune aspects of hypoparathyroidism are reviewed.

Hypoparathyroidism may be transient or permanent, and the causes for either type are many. Mthough in most cases hypoparathyroidism is sporadic and acquired, in some cases hypoparathyroidism is lifelong and results from a genetic disorder that can exhibit autosomal recessive, X-linked recessive, or autosomal dominant transmission (see Chapter 49). Hypoparathyroidism occurs as a component of an autosomal recessive disorder that features hypofunction of one or more endocrine glands as well as a variety of additional problems due to autoimmune disease (1) (MIM #240300). Initially termed polyglandular autoimmune syndrome, type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) to reflect the diverse tissues that are affected, it is now often designated as autoimmune polyendocrinopathy syndrome, type 1 (APS1) (1). The major features of APS1 are hypoparathyroidism, adrenal insufficiency, and chronic mucocutaneous candidiasis (moniliasis), which have led to the acronym HAM. The pathogenesis of APS1, i.e., the autoimmune destruction of endocrine tissues, is now understood at a molecular level and the defective gene has been identified (1). Although immune dysfunction also occurs in the DiGeorge anomaly (III-1V brachial pouch dysembryogenesis), dysembryogenesis rather than autoimmunity causes the associated hypoparathyroidism (2,3). However, there are other less well-characterized syndromes with hypoparathyroidism in which autoimmunity may be

The Parathyroids, Second Edition

HISTORY

At the beginning of the twentieth century, pituitary failure was considered to be the basis for hypofunction of multiple endocrine glands in one person. In 1908, however, Claude and Gougerot (11) described a second possible cause when they reported infiltration of lymphocytes and fibrosis within several endocrine tissues of individuals, a condition they termed pluriglandular endocrine atrophy (11). Four years later, Falta (12) suggested that a mild form of this syndrome could lead to partial atrophy of endocrine glands. Indeed, this mechanism for endocrine disease was documented in 1926 when Schmidt described two patients with adrenal insufficiency together with thyroid hypofunction due to chronic lymphocytic thyroiditis (13). Reports of endocrine deficiencies with candidiasis then began to appear in the medical literature. In 1929, Thorpe and Handley (14) described a child with both hypoparathyroidism and oral moniliasis. In 1943, familial occurrences of idiopathic hypoparathyroidism with Addison's disease (15) and with candidiasis (16) were published, offering the possibility of a single heritable defect. The first description of hypoparathyroidism, candidiasis, and idiopathic adrenal insufficiency also appeared that year (17). By 1946, adrenal insufficiency had become the first endocrinopathy associated

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pathogenetically with idiopathic hypoparathyroidism (18). In 1955, Craig and co-workers (19) emphasized the familial occurrence of chronic candidiasis with hypoparathyroidism or nontuberculous Addison's disease. One year later, Whitaker and colleagues (20) called attention to the syndrome of "familial juvenile hypoadrenocorticism, hypoparathyroidism, and superficial moniliasis." Further insight concerning the potential pathogenesis for this syndrome came in 1956 when Roitt and co-workers discovered circulating thyroglobulin autoantibodies in patients with lymphocytic (Hashimoto's) thyroiditis (21). They proposed that organ-specific immunoglobulins might cause endocrine atrophy (21). In 1957 and 1962, Anderson et al. (22) and Blizzard et al. (23), respectively, hypothesized that autoantibodies could account for some cases of nontuberculous Addison's disease. During the early 1960s, the clinical spectrum of APSlminvolving hypoparathyroidism, Addison's disease, and mucocutaneous candidiasismbroadened. In 1962, Gass (24) reported the syndrome of keratoconjunctivitis, superficial moniliasis, idiopathic hypoparathyroidism, and Addison's disease. In 1963, Kunin and colleagues (25) noted an association of steatorrhea, macrocytic anemia, and posthepatitic cirrhosis with moniliasis, hypoparathyroidism, and Addison's disease. In 1966, recognizing that approximately onethird of patients with these combined endocrine deficiencies also had moniliasis, Taitz and co-workers (26) coined the acronym HAM (hypoparathyroidAddison's-monilia) to describe this syndrome. By 1972 (27), eight patients with this condition had been reported (15,16,19,20,28-30). Further progress in defining APS1 and elucidating its pathogenesis was made during the early 1980s. In a series of publications spanning 1980 and 1981, Neufeld

TABLE 1

and colleagues (31-33) characterized a group of disorders they called polyglandular autoimmune (PGA) disease. Three principal clinical forms, types I, II, and III, and possibly an additional type IV, were delineated. As summarized in Table 1, PGA disease, type I (APS1) includes hypoparathyroidism, adrenocortical insufficiency, a n d / o r chronic mucocutaneous candidiasis that appear during childhood. PGA disease, type I could also be complicated by insulin-dependent diabetes mellitus, primary hypogonadism, autoimmune thyroid disease, pernicious anemia, chronic active hepatitis, steatorrhea with malabsorption, alopecia, a n d / o r vitiligo (see below) (33). PGA disease, type II is not associated with hypoparathyroidism, but does include adrenocortical insufficiency as well as autoimmune thyroid disease a n d / o r insulin-requiring diabetes mellitus (33,34). Onset of PGA disease, type II is usually during adult life, and women are more commonly affected than men (32). PGA disease, type III features autoimmune thyroid disease with insulin-dependent diabetes mellitus, pernicious anemia, or vitiligo a n d / o r alopecia, or additional organ-specific autoimmune diseases (e.g., hepatic dysfunction) (33). Finally, Neufeld and Blizzard (33) defined PGA disease, type IV as the presence of two or more organ-specific autoimmune diseases that do not fall into types I, II, or III. Additionally, Neufeld and colleagues (32) reported a 20% incidence of antiparathyroid antibodies in patients with isolated Addison's disease. This and subsequent advances in our knowledge concerning the pathogenesis of PGA disease, type I (APS1) are discussed later. Several hundred patients with APS1 have been described, often reported using other labels (17). In 1956, APS1 was called familial juvenile hypoadrenocorticism, hypoparathyroidism, and superficial moniliasis (20) and, in 1966, as noted above, HAM syndrome (26). The terms multiple endocrine deficiency, autoimmune

Classification of Polyglandular Autoimmune Disease a

Type

Features

II III-A III-B III-C IV

Candidiasis, hypoparathyroidism, Addison's disease and two or three of the following: insulin-dependent diabetes mellitus, primary hypogonadism, autoimmune thyroid disease, pernicious anemia, chronic active hepatitis, steatorrhea (malabsorption), alopecia (totalis or areata), and vitiligo ~ Addison's disease + thyroid autoimmune disease and/or insulin-dependent diabetes mellitus Thyroid autoimmune disease + insulin-dependent diabetes mellitus Thyroid autoimmune disease + pernicious anemia Thyroid autoimmune disease + vitiligo and/or alopecia and/or other organ-specific autoimmune disease Two or more organ-specific autoimmune diseases not falling into types I, II, or III

aFrom Ref. 33, with permission. ~Adapted from Ref. 32.

AUTOIMMUNE HYPOPARATHYROIDISM / candidiasis (MEDAC) syndrome, type I polyendocrine autoimmune disease, and autoimmune polyendocrinecandidiasis syndrome (APECS) were introduced later (35). APS1 gained favor more recently (1). Idiopathic hypoparathyroidism associated with yet additional disorders has also been hypothesized to have an autoimmune basis (10). Hypoparathyroidism (and occasionally endocrine disturbances such as hypothyroidism and diabetes mellitus) can occur in patients with Kearns-Sayre syndrome (oculocraniosomatic neuromuscular disease with mitochondrial myopathy) (6). In one patient, hypoparathyroidism complicated Down syndrome and autoimmune hyperthyroidism (4). Primary hypoparathyroidism has accompanied acute interstitial nephritis and uveitis (5). An elderly man with hypoparathyroidism, and probably incidental Paget disease of bone, had parathyroid glands that showed severe lipomatosis and a diffuse lymphocytic infiltration with atrophy of the endocrine cells (8). An elderly woman with postsurgical hypoparathyroidism had spuriously elevated serum parathyroid hormone (PTH) levels in a radioimmunoassay that used antiserum specific for the carboxy-terminal region of human PTH. She appeared to have an immunoglobulin G (IgG) that was cross-reactive with the assay antiserum (7). Another elderly patient with acquired hypoparathyroidism had detectable serum levels of PTH owing to the presence of antiidiotypic PTH autoantibodies (9). Finally, in other disorders believed to have an autoimmune basis, hypoparathyroidism can apparently result from fibrosis of the parathyroid glands [e.g., progressive systemic sclerosis (36) and Riedel's thyroiditis (37) ]. Despite the existence of a seemingly growing number of autoimmune (or autoimmune-like syndromes) that manifest with hypoparathyroidism, distinctive clinical features can be recognized that distinguish APS1. These problems are detailed below.

C L I N I C A L FEATURES O F APS 1 The incidence of autoimmune disease increases significantly with age. Syndromes in which multiple tissues are affected are possibly more prevalent than disorders involving only a single tissue. In newly diagnosed children and adolescents with an autoimmune disorder, additional conditions will frequently be detected. In early infancy and childhood, the most common association of endocrine and nonendocrine autoimmune disease is APS1 (17). APS1 is an autosomal recessive disease characterized by (1) autoimmune polyendocrine deficiencies (hypoparathyroidism, adrenocortical failure, diabetes mellitus, gonadal failure, and hypothyroidism), (2) chronic mucocutaneous candidiasis, and (3) ectodermal dystrophies (vitiligo, alopecia, keratopathy and dys-

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trophy of dental enamel, nails, and tympanic membranes) (38,39). In addition, there can be pernicious anemia, hepatitis, intestinal malabsorption, splenic atrophy, or gallstones, and a large proportion of patients develop squamous-cell carcinoma of the oral mucosa (38,40). Two of the classical triad of manifestations (hypoparathyroidism, mucocutaneous candidiasis, and adrenocortical insufficiency) are required for the diagnosis of APSI (31-33). Most reported cases of APS1 have been familial. Genetic analysis of affected families indicates an autosomal recessive mode of inheritance (1,41,42). APS1 is reported worldwide, but is exceptionally prevalent among the Finnish population (incidence 1:25,000) and Iranian Jews (incidence 1:9000) (38). Consanguinity has been recorded (41,43). The age of onset does not differ between familial and sporadic cases (17,41 ). APS1 occurs with equal frequency in girls and boys (17,41). The average age of first symptoms is ---8 years. A few individuals have apparently developed the condition after 10 years of age (32,44). The sequence for presentation of the three major components is typically chronic mucocutaneous candidiasis, hypoparathyroidism, and then Addison's disease. In one review, these disorders manifested, on average, at 5, 9, and 14 years of age, respectively (35). Of interest, within a single family, one individual with APS1 may have hypoparathyroidism, candidiasis, and adrenal insufficiency, whereas other affected siblings may develop only one or two of these problems (20,41,45). Hypoparathyroidism or Addison's disease may seem to occur independently within a sibship (42). Because hypoparathyroidism almost invariably precedes Addison's disease, very few patients who have isolated Addison's disease will later develop hypoparathyroidism (32,41). As discussed below, many patients with two or all three of these major components of APS1 manifest additional "atrophic" problems, including pernicious anemia, alopecia, premature ovarian failure, diabetes mellitus, vitiligo, and autoimmune thyroid disease (32,35). The autoimmune thyroiditis is indistinguishable from the isolated disorder (46). In 1966, Blizzard and co-workers (47) described 32 patients with idiopathic hypoparathyroidism who had one or more of the following associated conditions: moniliasis (66%), Addison's disease (56%), pernicious anemia (22%), thyroid disease (19%), alopecia totalis (13%), premature menopause before the age of 25 (6%), and juvenile cirrhosis (6%). Review of a large series of patients in 1969 by Fanconi (48) disclosed that moniliasis occurred in 72% (mean age 3 years), Addison's disease in 58% (mean age 11 years), steatorrhea in 26% (mean age 8 years), and pernicious anemia in 9% (mean age

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17 years). The complete APS1 syndrome affected 32%, whereas hypoparathyroidism occurred with moniliasis alone in 40% and with Addison's disease alone in 28%. In 1990, A h o n e n and co-workers reported their followup of 68 patients (48a). Clinical manifestations varied greatly, but all had candidiasis at some time. The majority of patients had three to five manifestations; all required lifelong follow-up (48a). The major and less c o m m o n clinical features of APS1 are discussed below.

Idiopathic Hypoparathyroidism Patients with APS1 have an especially interesting and challenging form of "idiopathic" hypoparathyroidism. The considerable n u m b e r of associated disorders diversifies its clinical presentation and offers important diagnostic and therapeutic hurdles (27,35). Idiopathic hypoparathyroidism with or without Addison's disease a n d / o r mucocutaneous candidiasis manifests between ages 6 months and 20 years (24). In APS1, symptoms of hypoparathyroidism begin, on average, 4 years after candidiasis is noted, with a mean age of onset from 6 to 9 years (35), d e p e n d i n g on the reported population. Importantly, hypoparathyroidism almost invariably precedes Addison's disease (32,41). Hypoparathyroidism in patients with APS1 seems not to differ clinically or biochemically from isolated idiopathic hypoparathyroidism. As with other forms of PTH deficiency, high serum levels of creatine kinase can accompany episodes of hypocalcemia (49).

Addison's Disease By 1974, idiopathic and tuberculosis-related Addison's disease were recognized to be distinct entities (50). In contradistinction to Addison's disease due to tuberculosis, --~14% of patients with idiopathic Addison's disease have additional problemsm hypoparathyroidism, diabetes mellitus, thyroid disease, pernicious anemia, or gonadal insufficiency (50). Addison's disease, on an a u t o i m m u n e basis, is a comp o n e n t of both PGA disease, types I (APS1) and II (Table 1). However, each disorder has a different mean age of onset, apparent genetic basis, and pathogenesis (32). In APS1, Addison's disease may rarely occur before hypoparathyroidism (32,45,51), but it never appears prior to moniliasis. Addison's disease is noted, on average, 5 years after hypoparathyroidism, at a mean age of 14 years (35). If unrecognized and untreated (see below), Addison's disease can be fatal (18). Rarely, selective involvement of the adrenal cortex may limit destruction to the zona glomerulosa, with development of isolated hypoaldosteronism (52). The unusually high mortality rate of adrenal insufficiency relates to the early age of onset, difficulty in managing hypoadrenalism,

and additional clinical problems, including hypocalcemia (29,53). It is critical to appreciate that untreated Addison's disease can conceal the presence of coexisting hypoparathyroidism (see below).

Candidiasis Chronic mucocutaneous candidiasis is a c o m m o n complication of immune deficiency conditions (54). Included are disorders with morphologic abnormalities of the thymus and thymus-dependent tissues that lead to profound deficiencies of cell-mediated immunity (e.g., DiGeorge anomaly), and those associated with both defective cellular and humoral immunity (e.g., Swiss-type agammaglobulinemia and thymic dysplasia) (54). In fact, clinicians should consider chronic mucocutaneous candidiasis to be a manifestation of an underlying disorder, rather than a primary disease (54). Chronic mucocutaneous candidiasis is usually accompanied by hypoparathyroidism, but Addison's disease etc. may also occur (55). Infection with Candida albicans occurs in --~14% of patients with idiopathic hypoparathyroidism. Candidiasis is usually the initial manifestation of APS1 and generally appears in early childhood. It develops 1-4 years before there is overt evidence of hypoparathyroidism or other endocrine failure (17,29). It is more apt to occur in patients who subsequently develop adrenocorticoid insufficiency or pernicious anemia. Candidiasis may be limited in distribution, but occasionally can involve almost the entire body (Fig. 1). Mucocutaneous candidiasis invariably affects the oral cavity. Oral lesions are diffuse and commonly cause per16che (cracks at the corners of the mouth) and fissures of the lip. The next most frequently involved site is the fingernails. The toenails are only sometimes infected. The entire width of the nail plate is affected, and the nails become thickened and dystrophic. There may be associated paronychia. Affected nails are typically pitted and friable m features that may mistakenly be attributed to hypocalcemia from associated hypoparathyroidism or hepatobiliary or gastrointestinal disease (16). The vagina can also be infected. Rarely, the skin of the hands and feet becomes hyperkeratotic and disfigured (51). Patients with chronic mucocutaneous candidiasis are predisposed to other skin infections (54). By contrast, systemic candidiasis is rare and occurs only if there is another predisposing factor that impairs immune defense mechanisms (e.g., diabetes mellitus). It is important to recognize the association between hypoparathyroidism and candidiasis, although the basis of the relationship is unknown (see below). Many individuals who suffer from chronic mucocutaneous candidiasis will later develop endocrine dysfunction, but such patients are clinically indistinguishable from those who will not (54,55).

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FI6. 1 A young boy with APS1 and severe mucocutaneous candidiasis. There is striking hyperkeratosis from infection of the skin of the hands and face in addition to involvement of the oropharynx and fingernails. (See color plates.)

Other Endocrine Disease Other endocrinopathies that may occur with hypoparathyroidism in APS1 include insulin-dependent diabetes mellitus, Hashimoto's thyroiditis (43,46,56), hypothyroidism (57), and ovarian failure (58-62). Several APS1 patients have developed severe, multiple

endocrine gland hypofunction after moniliasis, in some cases involving the parathyroid, adrenal, and thyroid glands as well as ovaries in succession (43,58). The pituitary gland has not been directly affected, although hypopituitarism due to a pituitary tumor has been reported and growth hormone deficiency has been described.

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Ovarian failure associated with antiovarian antibodies has been well documented in APS1 (58). In cases of primary amenorrhea, ovarian histopathology may resemble gonadal dysgenesis (43). Otherwise, menses are typically regular until the onset of secondary amenorrhea and premature menopause. Affected young women can, however, be fertile. As in other forms of primary gonadal failure, serum levels of follicle-stimulating h o r m o n e are elevated to a greater extent than those of luteinizing hormone.

Nonendocrine Diseases Nonendocrine problems, in addition to candidiasis, occur frequently in patients with APS1. Pernicious anemia is the most common (27,28,43,44,53,61,63,64). It usually manifests 5-10 years after hypoparathyroidism (27), and occasionally presents during adult life (44). Despite its early onset, the pernicious anemia is the "adult type," which is otherwise rare before age 20 years (27). There is acquired gastric atrophy, with loss of all secretory components. Typically, antibodies to parietal cells and intrinsic factor can be detected in the serum. This contrasts with '~juvenile type" pernicious anemia in which there is selective deficiency of intrinsic factor and absence of antibodies. Distinction should be made between cases of true pernicious anemia, in which intrinsic factor is deficient, and those cases in which absorption of vitamin B12 is impaired by steatorrhea or nonspecific gastrointestinal defects. Ikkala and colleagues (64) showed in 1964 that these latter changes may also occur in cases of postoperative hypoparathyroidism. Indeed, in other types of hypoparathyroidism, successful treatment of the hypocalcemia occasionally leads to improved absorption of fat and vitamin B12. Corneal abnormalities in a patient with idiopathic hypoparathyroidism were first described in 1929 (65), but initially, phlyctenular keratoconjunctivitis was thought to be an infrequent complication of APS1 (16,18,66). However, in 1962, a review by Pohjola indicated the prevalence was 10% (67). That same year, Gass (24) reported 12 cases of keratoconjunctivitis, all with moniliasis a n d / o r endocrinopathies. Keratoconjunctivitis is now recognized to occur in as many as 50% of patients with APS1 (24). In fact, it is one of the earliest presenting manifestations (24). In most patients, the symptoms are often chronic and disabling. However, they can also be minimal. There may also be long periods of remission (24). In mild cases, slight redness of the eyes, photophobia, and blepharospasm can occur. Intense photophobia and excessive lacrimation manifest during the acute phase of the keratitis (18,45,66). In severe cases, there is ulceration, scarring, and vascularization that can opacify the cornea and impair vision (24). Conjunctival biopsy specimens

may disclose heavy subepithelial infiltration of lymphocytes and plasma cells (68). Bilateral superficial keratitis accompanies corneal vascularization (66,69,70). Keratoconjunctivitis has been postulated to be a hypersensitivity response to the candidiasis, rather than an independent component of the syndrome. However, this complication has occurred in the absence of Candida infection (24). In 1987, Wagman and coworkers (68) studied 16 cases of APS1 and identified four children with bilateral, self-limited keratitis that began between 2 to 9 years of age (68). It preceded endocrinopathy in two of these patients and caused some impairment of vision. The authors concluded that the keratitis was not caused by the hypoparathyroidism or candidiasis (68). Nevertheless, others have noted that keratitis may improve when serum calcium levels are well controlled (66). Other reported ocular abnormalities of APS1 include strabismus, loss of eyebrows and eyelashes, recurrent blepharitis, keratic precipitates, retinitis pigmentosa, exotropia, pseudoptosis, cataracts, and papilledema (66,68). The cataracts and papilledema are probably related to the hypocalcemia and hyperphosphatemia that occur in untreated hypoparathyroidism (68). In 1999, a patient was reported with an extremely rapid evolution of typical hypocalcemic cataracts during acute idiopathic hepatic and renal failure, and serum calcium and phosphorus were unbalanced (71). Patients with APS1 and idiopathic hypoparathyroidism also have ectodermal problems that include dry, rough skin (18), coarse and brittle hair, and lusterless, somewhat hypoplastic, distally split nails (18,56,65,72). Alopecia totalis is another frequently mentioned complication (18,47,55,56,62). There can also be alopecia areata, (16,18,30,56,62,65), piebaldism (65), or vitiligo (56,63). Alopecia occurs in about 30% of affected individuals and may include loss of eyebrows and eyelashes (66). There can be dental abnormalities in APS1 (18,30), including partial anodontia, enamel hypoplasia, and delayed eruption of teeth (44,53,66,73). Dental dysplasia may occur before or in the absence of hypocalcemia. In a small series of patients, enamel hypoplasia affected canines most severely among maxillary and mandibular teeth, but all tooth types were involved (74). There may also be darkly pigmented skin (18,30) with hyperkeratosis (16). Pigmented nevi have been described (20). One patient with prominent ectodermal dysplasia has been reported (72). Occasionally, there is cutaneous infection with other fungi, such as Trichophytonrubrum (62,65). Recurrent staphylococcal infections of the skin are also common (54). Intracranial calcification (18,53,72) may occur with longstanding hypoparathyroidism, and some affected individuals can be mentally deficient (53). Papilledema

AUTOIMMUNE HYPOPARATHYROIDISM / can occur from raised intracranial pressure (16,18). Patients with APS1 may also develop a variety of intraabdominal disorders. Hepatitis that can progress to cirrhosis is well established (19,20). Indeed, it has been speculated that the hypoparathyroidism and Addison's disease could be sequellae (25). Chronic active hepatitis (45), '~juvenile cirrhosis" (47), posthepatitic cirrhosis (20,24,25,73), pancreatic cystic fibrosis or mucoviscidosis (53,63), and steatorrhea (45,61,63,75,76) have been described. Steatorrhea can lead to overt malabsorption (60,61). The steatorrhea of APS1 patients with idiopathic hypoparathyroidism has been explained as an independent feature, a direct effect of the hypoparathyroidism and hypocalcemia, a complication of intestinal candidiasis (77-81), or a result of abnormal liver function (25,82). There may be predisposition to severe bouts of viral hepatitis, measles, and encephalomyelitis (65).

PATHOGENESIS DiGeorge syndrome shares several clinical problems with APS1, specifically hypoparathyroidism and candidiasis. In DiGeorge syndrome these features result from gene defects that affect parathyroid and thymic development (3,4). Absence of the thymus leads to diminished T lymphocyte function, which seems to explain the predisposition to candidiasis (3,4). The pathogenesis of immune dysfunction in APS1, is less clear however. APS1 is due to defects in a gene that regulates immune function (83) (see below). The hypothesis that the endocrine dysfunction of APS1 is pathogenetically related to the associated candidiasis led to early (and correct) proposals of an autoimmune pathogenesis for the disorder. Subsequently, an autoimmune basis was considered as the cause for idiopathic hypoparathyroidism in several additional syndromes (4-9). By contrast, in 1961, Morse and co-workers (77) postulated inheritance of a gastrointestinal defect that impaired absorption of a factor necessary for parathyroid and adrenal function. In 1963, Kunin and colleagues (25) reviewed cases of APS1 with posthepatitic cirrhosis, and considered the possibility of a viral etiology that could present with hepatitis. In 1966, Sj6berg (61) found high serum titers of antibodies against C. albicans in three patients. Earlier reports had identified a potent, heat-stable, inhibitory factor(s) in serum that prevented the growth of C. albicans (84) that was deficient in patients with chronic moniliasis (85). However, in 1967, Esterly and coworkers (86) showed that lack of this inhibitory substance was not an explanation for the fungal disease. Until the 1970s, it was hypothesized that candidiasis could explain the hypoparathyroidism and other

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endocrine deficiencies of APS1 (77,87,88). However, autopsy studies have never shown invasion of the viscera or parathyroid glands by C. albicans (20,43,63). As an alternative to direct infection by Candida, in 1966 Sj6berg (61) and in 1970 Windorfer (89) postulated that this fungus elaborated a toxin that impairs endocrine gland function. In fact, in 1971, it was proposed that should a causal relationship between candidiasis and endocrinopathy be documented, it would be rationale to amputate fingertips because of the high mortality rate from Addison's disease (90). However, such a toxin has not been demonstrated, and candidiasis does not cause the endocrinopathies of APSl (55). In 1956, Whitaker and colleagues (20) had proposed that the candidiasis emanated from the other dystrophic lesions of the skin, mucosa, and nails due to the hypoparathyroidism. This hypothesis seems incorrect, however, because candidiasis precedes idiopathic hypoparathyroidism by an average of 5 years. Additionally, the dystrophic ectodermal changes improve when hypocalcemia is corrected, whereas the fungal disease usually does not (16). An autoimmune pathogenesis for APS1 was suspected from histopathologic and serologic studies of patients (50). Early on, postmortem investigations disclosed fibrous and lymphocytic infiltration of endocrine glands that replaced healthy tissue and presumably caused gradual destruction of epithelial cells (65). In the parathyroids, fatty replacement, atrophy with atypical cells, and various degrees of lymphocytic accumulation were documented at autopsy (24). Remnants (19,43) or complete absence of parathyroid tissue (18,20,91) were observed, suggesting that the parathyroid lesion was an acquired atrophy, rather than developmental aplasia or hypoplasia (20). The adrenal cortex also appeared atrophic and was replaced by dense fibrous connective tissue infiltrated by lymphocytes (18,24). The role of autoimmunity in APS1 became established in the 1960s. In 1966, the notion that autoantibodies could cause disease of the parathyroid glands was given considerable support by Blizzard, Chee, and Davis (47), who used both normal and pathologic human parathyroid tissue to demonstrate antiparathyroid antibodies in the sera of 38% of patients with idiopathic hypoparathyroidism, 26% with Addison's disease, 12% with Hashimoto's thyroiditis, and 6% of healthy controls. These autoantibodies seemed to be parathyroid specific. The antigen was detected in some, but not all, parathyroid adenomas. However, these investigators were hindered by limited availability of normal parathyroid tissue. Therefore, it was not possible to learn as much about the incidence, characteristics, and significance of parathyroid autoantibodies as was known about antibodies against other endocrine

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tissues (47). These researchers also found that many children and some adults with idiopathic hypoparathyroidism had antiadrenal antibodies (10%) and antigastric parietal cells antibodies (7%) (47). The antibodies did not react with C. albicans. Later, in 1981, a 20% incidence of antiparathyroid antibodies was reported in patients with isolated idiopathic adrenocortical insufficiency (32). In 1967, Wuepper and Fundenberg (45) identified immunologic abnormalities in family members of patients with hypoparathyroidism and speculated that the autoimmune pathogenesis probably had a genetic basis. Their patients showed delayed hypersensitivity to Candida (positive skin test), but lacked an anti-Candida factor detected in normal serum (45). The following year, Blizzard and Gibbs (55) reported that 40% of 44 patients with mucocutaneous moniliasis had an associated disease with a presumed autoimmune basis, and that most had antibodies that were reactive with endocrine glands or stomach. The presence of such antibodies was believed to reflect focal or generalized lymphocytic infiltration and at least subclinical involvement of the various tissues. A direct relationship between the candidiasis and the endocrinopathies could not be established, however (55). There was no cross-antigenicity between C. albicans and adrenal or thyroid tissue (92). In 1969, Irvine and Scarth found IgG antibodies against parathyroid oxyphil cells in only one of nine patients with idiopathic hypoparathyroidism (93), but those with "autoimmune" hypoparathyroidism were not tested. That same year, Spinner and co-workers (94) studied parents and siblings for antibodies to endocrine and gastric tissue and provided additional evidence to support their genetic/clinical classification of individuals with hypoparathyroidism a n d / o r Addison's disease (41). Beginning in the 1960s, several groups of investigators were able to show lymphocytic infiltration and even atrophy of parathyroid glands in animals that were immunized with parathyroid tissue (95,96). In 1968, Lupulescu and co-workers (97) produced lymphocytic and plasma cell infiltration and atrophy of the parathyroid glands in dogs that had been immunized with extracts of allogeneic parathyroid tissue. Moreover, similar changes were noted in adrenal cortex as well. In 1974, passive immunization of rats with antiserum against their parathyroid tissue caused a marked immunoparathyroiditis, but hypoparathyroidism did not ensue (78). Actually, there is little correlation between the presence of antibodies and clinical manifestations of APS1 even among members of a single kindred. The occurrence of antibodies in affected individuals, however, may be a harbinger of eventual endocrine deficiency, but the significance of antibodies found in apparently healthy family members is unclear.

In 1969, Hermans and co-workers (65) suggested that viral infection, in a genetically predisposed individual, might play a pathogenetic role in APS1 and that "immunologic deficiencies, especially those related to delayed hypersensitivity, appear to be of importance in the development of chronic mucocutaneous candidiasis." In 1971, Kirkpatrick and co-workers (54) and Block and colleagues (98) summarized evidence for various abnormalities in cell-mediated immunity in APS1. A woman with hypoparathyroidism, moniliasis, Addison's disease, and primary ovarian failure was described as lacking delayed hypersensitivity in vivo to C. albicans, although there was evidence of immune cellular activity in vitro (98). That same year, Levy et al. (99) reported impaired cellular immunity in a 17-year-old man with chronic mucocutaneous moniliasis, Addison's disease, pernicious anemia, and gastrointestinal malabsorption in whom thrush had cleared after transplantation of fetal thymus tissue. In a preliminary report in 1974, Rao and co-workers (100) noted that cell-mediated immunity to C. albicans could be impaired in idiopathic hypoparathyroidism. In 1975, patients with APS1 and hypoparathyroidism were shown to have abnormalities of T cell function (101). APS1 was postulated to be caused by a defect in T lymphocyte action. In 1979, abnormal suppressor T lymphocyte function, low circulating levels of IgA, and deficient cell-mediated immunity to C. albicans were reported (102). By that time, however, autoantibodies to endocrine glands, intrinsic factor, and the parietal cells of the stomach had been described. In the late 1970s, some evidence suggested that the disorders deemed to be polyglandular failure syndromes were HLA-B8 associated (103,104). Thus, immunologic dysfunction involving genes or chromosome 6 could be a factor in their pathogenesis (104,105). However, other studies of HLA type showed no consistent haplotypes in APS1 (32,102,105), although PGA disease, type II is indeed associated with HLA-B8 (DW3) (32,104,105). Discovery of the molecular basis for APS1 clarified this issue (see below). In 1977, Swana and co-workers (106) reported that reactivity against parathyroid tissue in patients with hypoparathyroidism was due to an antimitochondrial autoantibody. Yet, in 1981, Doniach and Bottazzo (107) noted that organ-specific parathyroid autoantibodies occurred rarely in patients with autoimmune polyendocrinopathy a n d / o r hypoparathyroidism. In 1983, Haspel and co-workers (108) discovered that mice infected with retrovirus type I can develop an autoimmune polyendocrine disease in which autoantibodies are organ specific. Subsequently, in 1985, Betterle and co-workers (109) described an IgG-class mitochondrial antibody that reacted with a 46-kDa mitochondrial membrane pro-

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tein in 31% of patients with APS1, but this antibody was not specific for parathyroid tissue. Furthermore, antibodies could appear before endocrine dysfunction (110). In 1986, Posillico and co-workers (111) described three patients with idiopathic hypoparathyroidism in whom sera contained autoantibodies that inhibited secretion of PTH from dispersed h u m a n parathyroid cells. These antibodies were reactive with epitopes on the extracellular surface of the parathyroid cell and possibly interfered with signal recognition/transduction mechanisms for calciumregulated PTH secretion (111). In 1986, Brandi and co-workers (112) developed a 51Cr release assay using bovine parathyroid cells in culture and showed that complement-dependent cytotoxic antibodies were present in sera from seven patients with autoimmune hypoparathyroidism, but not in 15 healthy individuals or 41 patients with other diverse conditions associated with immune dysfunction (112). Absorption of the sera with parathyroid or adrenal tissue caused a marked decrease in this effect. In 1988, Fattorossi and co-workers (113) extended this work and used fluorescence flow cytometry and tissue immunohistology to show IgM in patient sera that reacted with cultured endothelial cell membranes and tissue sections from bovine parathyroid glands. The antibody was not, however, completely species or organ specific. Two major bands (130 and 200 kDa) were associated with the parathyroid endothelial membranes and perhaps the principal targets of the antibody. An intriguing postulate is that these immunoglobulins disturb an important physiologic relationship between endocrine and epithelial cells (113). Mice with autoimmune polyendocrine disease also have antibodies of the IgM class. In 1992, Wortsman and co-workers (114) found generalized T cell activation to be a novel feature of adultonset idiopathic hypoparathyroidism. This observation suggested that an immune disturbance, possibly related to autoimmunity, could cause this type of hypoparathyroidism as well (114). Although the pathogenesis of the chronic bilateral keratoconjunctivitis in APS1 is unclear, hypoparathyroidism appears not to be the cause. This ophthalmic disorder is not a feature of other types of hypoparathyroidism and can occur without hypoparathyroidism (68). Although hypothesized to be an allergic reaction to C. albicans protein (24), for similar reasons phlyctenular keratitis is unlikely to reflect a hypersensitivity to candidiasis (68). Laboratory studies have not confirmed an autoimmune basis for the keratitis (68). In 1996, to characterize the antibody responses to C. albicans, Peterson and co-workers screened a candidal cDNA expression library with patient sera (115). The highest antibody reactivity was found with enolase (80% of patients reactive), but significant serological responses were also found with heat-shock protein 90

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(67%), pyruvate kinase (63%), and alcohol dehydrogenase (64%) (115). Overall, 96% of APS1 patients had detectable antibodies to at least one of these proteins. Hence, the four abundant candidal proteins are the major antigens and can be used as accurate markers of candidiasis in APS1 (115). Evidence reported in 1996 suggested that acquired hypoparathyroidism is associated with autoantibodies to the extracellular domain of the calcium-sensing receptor (116). Patients with APS1 have autoantibodies against several endocrine and nonendocrine organs (117,118). A new autoantigen related to this syndrome, tyrosine hydroxylase, was identified in sera from patients with alopecia areata through immunoscreening of a scalp cDNA library (117). Immunoreactivity against in vitro-expressed tyrosine hydroxylase was found in 41 (44%) of 94 APS1 patients studied (117). These findings stress the importance of enzymes involved in neurotransmitter biosynthesis as important targets in APS1 (117). Antibodies to glutamic acid decarboxylase occur frequently (119). Alopecia areata occurs in about one-third of APS1 patients, usually in the more severely affected individuals (118). Many patients have high-titer autoantibodies directed against the anagen matrix, cuticle, and cortex keratinocytes and a melanocyte nuclear antigen (118). Also, hair follicle keratinocyte immunostaining accompanies alopecia, especially alopecia totalis. Alopecia is a common feature in APS1, affecting 29-37% of the patients and occurring between ages 3 and 30 years (17). Tryptophan hydroxylase is an intestinal autoantigen in APS1 (120). Antibodies associated with gastrointestinal dysfunction in APS1 are specific for this disease and not present in patients with other bowel disorders or healthy controls (120). Intermittent gastrointestinal dysfunction occurs in 25-30% of APS1 patients, consisting of therapy-resistant steatorrhea, diarrhea, or constipation (120). Measurement of tryptophan hydroxylase antibodies is a valuable diagnostic tool to identify these patients, often young children presenting with atypical gastrointestinal disease, as having APS1 (120). There is as yet no animal model for APS1.

ETIOLOGY APS1 is the only systemic autoimmune disease with an established monogenic background, and the first autoimmune disorder localized outside the major histocompatibility complex region on chromosome 6 (1). In 1994, the founder effect in Finnish APS1 kindreds enabled the genetic mapping of APS1 to chromosome 21q22.3 (121). In 1997, the defective gene in APS1 was isolated and characterized using positional cloning, and was named AIRE-1, denoting an autoimmune regulator (83).

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AIRE-1 encodes a 545-amino acid, proline-rich protein with a molecular mass of 58 kDa. The amino acid sequence includes a putative nuclear targeting signal between amino acids 113 and 133 (122). APS1 protein is mainly localized, both in vitro and in vivo, to the cell nucleus, (122). The protein contains two zinc fingers of plant homeodomain (PHD) type-motifs that are found in many nuclear proteins (122), which implies a role in the regulation of gene expression (122,123). Mutations in the AIRE-1 gene cause APS1 (123). A total of 16 different mutations have been identified spread throughout the coding region (122). A common haplotype spanning the locus carrying a 964de113 mutation suggests a founder effect in the British population (40). Genotype-phenotype correlations for APS1 remain difficult, suggesting that other genetic or environmental factors, or both, influence the clinical presentation and disease progression in individual patients (124).

TREATMENT One must consider that children with hypoparathyroidism or Addison's disease may develop fullblown APS1. Furthermore, clinicians need to know that Addison's disease can conceal coexisting hypoparathyroidism (91), and that glucocorticoid therapy alone can lead to a fatal outcome (18-20,30,82,124a). Serum calcium concentrations rise in adrenocortical insufficiency, but may suddenly decrease after corticosteroid therapy is begun, because of both diminished gastrointestinal absorption and increased renal excretion of calcium (20). Similarly, estrogen replacement therapy for ovarian failure can diminish serum calcium levels (61). Patients with hypoparathyroidism and such additional disorders are, therefore, more likely to show fluctuations of serum calcium concentrations, and require especially careful regulation of doses of vitamin D sterols, etc. (30). This is discussed further below.

Hypoparathyroidism Management of hypoparathyroidism in patients with APS1 is essentially as described in Chapter 52 for patients with isolated idiopathic hypoparathyroidism. However, APS1 is a far more complex disorder than idiopathic hypoparathyroidism, and many potentially interacting medical problems will likely complicate therapy. A major factor, noted above, is Addison's disease. Another is exemplified by a case of a child with candidiasis and hypoparathyroidism, who seemed to have defective 25-hydroxylation of vitamin D due to concomitant giant cell hepatitis and severe cirrhosis (125). Here, 1,25-dihydroxyvitamin D 3 was considered

to be the best form of vitamin D therapy (125). Steatorrhea could also determine the type of vitamin D that would be most efficacious. As discussed below, the presence or absence of Addison's disease is an especially important consideration.

Addison's Disease Among the major confounding factors to consider when treating hypoparathyroidism in APS1 is the likelihood of Addison's disease. Prior to the 1950s, adrenocortical insufficiency was a fatal disorder, and more than 80% of patients were dead 2 years after receiving this diagnosis (65). In 1969, one-third of patients succumbed (48). Now, conventional replacement therapy using glucocorticoids and mineralocorticoids enables long-term survival. Addison's disease and its treatment both impact significantly on the manifestations of hypoparathyroidism. When hypoadrenalism supervenes in APS1, the clinical and biochemical features of hypoparathyroidism can diminish considerably, but will rapidly return when glucocorticoid replacement therapy is begun (30). Glucocorticoids can lower blood calcium levels by decreasing dietary absorption of calcium, and by enhancing renal excretion of calcium because glomerular filtration rate is increased. Accordingly, hypoparathyroidism must be excluded before glucocorticoid therapy is started for Addison's disease. If hypocalcemia is present, it may be rapidly and severely exacerbated by glucocorticoid treatment. An example is case 2 described in 1961 by Morse and colleagues (53). This child with hypoparathyroidism and a serum calcium concentration of 5.5 m g / d l experienced a spontaneous "remission" with a serum calcium level of 10.3 m g / d l as Addison's disease developed. When glucocorticoid replacement treatment (without additional vitamin D therapy) was initiated, the serum calcium level abruptly fell to 4.7 mg/dl. Similar clinical scenarios have been described by Leonard (18), Leifer and Hollander (126), Papadatos and Klein (30), and Quichaud and colleagues (127). Conversely, in 1964, Kenny and Holliday (46) reported that the onset of Addison's disease in a patient with well-controlled hypoparathyroidism was followed by marked hypercalcemia of 14.5 mg/dl.

Candidiasis The ectodermal changes that result from hypoparathyroidism per se, including brittleness of nails, respond to successful treatment of the mineral disturbances. However, the candidiasis often will not (16). Intractable superficial candidiasis involving mucous membranes, skin, and nails is one of the most perplexing and frustrating problems associated with

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autoimmune hypoparathyroidism. Candidiasis, once established, may be difficult to eradicate even when serum calcium concentrations are maintained in the normal range. Although treatment for mucocutaneous candidiasis had been disappointing generally, success was reported for a significant n u m b e r of patients who received transfer factor and amphotericin B. Transfer factor was prepared from lymphocytes of healthy individuals immunized against C. albicans. Kirkpatrick and Greenberg (128), however, studied 19 patients with chronic mucocutaneous candidiasis, the majority of whom had abnormalities in cell-mediated immunity. All had normal numbers of circulating T and B lymphocytes and normal lymphocyte responses to phytohemagglutinin and concanavalin A. Nevertheless, most had negative skin tests for C. albicans. Transfer factor administered for several months (with only local treatment with antifungal agents) was ineffective. Amphotericin B alone given intravenously, however, induced remissions in most of the patients (54). Nevertheless, complete and lasting eradication of all clinical evidence of the disease proved to be an elusive goal (88). Face and scalp lesions cleared most readily and relapsed least frequently. Thrush often reoccurred and remained the most symptomatic lesion (54). Local hypochlorite treatment has been suggested, because candidiasis of skin and nails improves in patients who swim frequently in chlorinated pools. Infected nails have been avulsed, but recurrence during nail regrowth ensues unless the candidiasis is controlled elsewhere (54). Long-term therapy with oral ketoconazole is now considered the treatment of choice for mucocutaneous candidiasis (129). Ketoconazole, 200 to 400 m g / d a y orally, is very effective for extensive and resistant disease (130,131). Nystatin applied topically as well as administered orally (to reduce intestinal colonization) may prevent the spread of the fungal lesions, but will rarely eradicate the infection.

Additional Disorders Absence of intrinsic factor production by gastric mucosa can lead to pernicious anemia in patients with APS1. The treatment is vitamin B12, as in any other form of intrinsic factor deficiency. However, steatorrhea is also a complication of autoimmune hypoparathyroidism. Patients with steatorrhea, hypocalcemia, and megaloblastic anemia have, on occasion, mistakenly been thought to have folate deficiency. When steatorrhea is severe, with its potential for vitamin D malabsorption, etc., hypoparathyroidism can be especially difficult to treat. Calcitriol is more water soluble than

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other forms of vitamin D, and thus may be the treatment of choice. It may be necessary to give calcium intravenously for a period of time (60). A diet enriched with medium-chain triglycerides has been reported to be helpful, perhaps by decreasing calcium loss due to saponification (60). One patient with APS1 who suffered recurrent episodes of severe intractable diarrhea, steatorrhea, and hypocalcemia was successfully treated only when given immunosuppression using intravenous high-dose methylprednisolone and oral methotrexate maintenance therapy (132). Of interest, a 10-year-old girl with APS1 who developed pure red cell aplasia resistant to conventional therapy showed hematologic remission after intramuscular injections of gamma globulin (13). The investigators postulated an idiotype-antiidiotype interaction in which specific suppression by antiidiotype antibodies corrected the hematologic disease (133). Patients with features of APS1 must be screened regularly for the associated abnormalities (32). Genetic testing for mutations in the AIRE-1 gene may confirm the diagnosis of APS1 and support this process, and can facilitate screening of healthy siblings. In the absence of genetic testing, these children should be examined for endocrine defects during at least the first decade of life (134). Because ---13% of individuals with APS1 will develop chronic active hepatitis, liver function tests and assays for smooth muscle and mitochondrial antibodies should be included (32). Medical therapy for keratoconjunctivitis can include corticosteroid eye drops. Surgical treatment involves keratectomy or corneal transplantation (66,68). Wagman and colleagues (68) r e c o m m e n d medical m a n a g e m e n t of the corneal disease without surgical intervention (68). The active phase is helped by topical antibiotic/corticosteroid medication; systemic corticosteroids or immunosuppression were not required (68). Of importance, these authors noted an apparent transition from an active to quiescent phase --~10 years after the onset. Interestingly, cimetidine has also been reported to have some efficacy, perhaps acting as an immunomodulator (135). Ward and colleagues reported complete resolution of photophobia and considerable hair regrowth, with minimal side effects, in a girl who received oral cyclosporine for severe APSl-associated exocrine pancreatic failure and keratoconjunctivitis (136).

ACKNOWLEDGMENTS This work was supported by Grant 8480 from Shriners Hospitals for Children. The author is grateful to Becky Whitener, CPS, for expert secretarial help.

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97. Lupulescu A, Potorac E, Pop A, et al. Experimental investigations on immunology of the parathyroid gland. Immunology 1968;14:475-482. 98. Block MB, Pachman LM, Windhorst D, Goldfine ID. Immunological findings in familial juvenile endocrine deficiency syndrome associated with mucocutaneous candidiasis. A m J M e d Sci 1971;261:213-218. 99. Levy RL, Huang S-W, Bach ML, et al. Thymic transplantation in a case of chronic mucocutaneous candidiasis. Lancet 1971; 2:898-900. 100. Rao KJ, Tomar RH, Moses AM. Cell mediated immunity in hypoparathyroidism Clin Res 1974;22:704A (abstract). 101. Irvine WJ, Barnes EW. Addison's disease, ovarian failure, and hypoparathyroidism. Clin Endocrinol Metab 1975;4:379-434. 102. Arulanantham K, Dwyer JM, Genel MD. Evidence for defective immunoregulation in the syndrome of familial candidiasis endocrinopathy. N EnglJ Med 1979;300:164-168. 103. Wirfalt A. Genetic heterogeneity in autoimmune polyglandular failure. Acta Med Scand 1981;210:7-13. 104. Eisenbarth G, Wilson P, Ward E Lebovitz HE. HLA type and occurrence of disease in familial polyglandular failure. N E n g l J Med 1978;298:92-94. 105. Eisenbarth GS, Wilson PW, Ward F, Buckley C, Lebovitz H. The polyglandular failure syndrome: Disease inheritance, HLA type, and immune function: Studies in patients and families. Ann Intern Med 1979;91:528-533. 106. Swana GT, Swana MR, Bottazzo CE Doniach D. A human specific mitochondrial antibody: Its importance in the identification of organ-specific reactions. Clin Exp Immunol 1977;28:517-525. 107. Doniach D, Bottazzo GE Polyendocrine autoimmunity. In: Franklin EC, ed. Clinical immunology update, Vol 2. New York: Elsevier, 1981:95-121. 108. Haspel MV, Onodera T, Prabhakar BS, Horita M, Suzuki H, Notkins AL. Viral-induced autoimmunity: Monoclonal antibodies that react with endocrine tissues. Science 1983;220: 304-306.

109. Betterle C, Caretto A, Zeviani M, Pedini B, Salviati C. Demonstration and characterization of anti-human mitochondria autoantibodies in idiopathic hypoparathyroidism and in other conditions. JExp Immunol 1985;62:353-360. 110. Burckhardt P. Idiopathic hypoparathyroidism and autoimmunity. Horm Res 1982;16:304-307. 111. Posillico JT, WortsmanJ, Srikanta S, Eisenbarth GS, Mallette LE, Brown EM. Parathyroid cell surface autoantibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells.JBone Miner Res 1986;1:475-483. 112. Brandi M-L, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA. Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci USA

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113. Fattorossi A, Aurbach GD, Sakaguchi K, et al. Anti-endothelial cell antibodies: Detection and characterization in sera from patients with autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 1988;85:4015-4019. 114. Wortsman J, McConnachie P, Baker JR, Jr, Mallette LE. TLymphocyte activation in adult-onset idiopathic hypoparathyroidism. Am J Med 1992;92:352-356. 115. Peterson P, Perheentupa J, Krohn KJE. Detection of candidal antigens in autoimmune polyglandular syndrome type 1. Clin Diag Lab Immun 1996;3:290-294. 116. Rose NR. Is idiopathic hypoparathyroidism an autoimmune disease? J Clin Invest 1996;97:899-900. 117. Hedstrand H, Ekwall O, Haavik J, Landgren E, Betterle C, PerheentupaJ, GustafssonJ, Husebye E, Rorsmann E Kfimpe O. Identification of tyrosine hydroxylase as an autoantigen in autoimmune polyendocrine syndrome type 1. Biochem Biophys Res Commun 2000;265:456-461. 118. Hedstrand H, Perheentupa J, Ekwall O, Gustafsson J, Micha61sson G, Husebye E, Rorsman E Kfimpe O. Antibodies against hair follicles are associated with alopecia totalis in autoimmune polyendocrine syndrome type I. Soc Invest Dermatol 1999;113:1054-1058. 119. Kelmetti P, Bj6rses P, Tuomi T, Perheentupa J, Partanen J, Rautonen N, Hinkkanen A, Ilonen J, Vaarala O. Autoimmunity to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Clin Exp Immuno12000; 119:419--425. 120. Ekwall O, Sj6berg K, Mirakian R, Rorsman E K/impe O. Tryptophan hydroxylase autoantibodies and intestinal disease in autoimmune polyendocrine syndrome type 1. Lancet 1999;354:568. 121. Aaltonen J, Bj6rses P, Sandkuijl L, Perheentupa J, Peltonen L. An autosomal locus causing autoimmune disease: Autoimmune polyglandular disease type I assigned to chromosome 21. Nat Genet 1994;8:83-87. 122. Bj6rses P, Halonen M, PalvimoJJ, Kolmer M, AaltonenJ, Ellonen P, PerheentupaJ, Ulmanen I, Peltonen L. Mutations in the AIRE gene: Effects on subcellular location and transactivation function of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy protein. A m J H u m Genet 2000;66:378-392. 123. Gibson TJ, Ramu C, Gemfind, Aasland R. The APECED polyglandular autoimmune syndrome protein, AIRE-1, contains the SAND domain and is probably a transcription factor. Elsevier Science. Trends Biochem Sci 1998;23:242-244. 124. Heino M, Scott HS, Chen 02, Peterson P, M/ienpfifi U, Papasavvas M-P, Mittaz L, Barras C, Rossier C, Chrousos GP, Stratakis CA, Nagamine K, Kudoh J, Shimizu N, Maclaren N, Antonarakis SE, Krohn K. Mutation analyses of North American APS-1 patients. Hum Mutat 1999;13:69-74. 124a.Jeffcoate wJ, Hosking DJ, Jones RM. Hypoparathyroidism and Addison's disease: A potentially lethal combination. J Royal Soc Med 1987;80:709-710.

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131. Horsburg CR, Jr, Kirkpatrick CH. Long-term therapy of mucocutaneous candidiasis with ketoconazole: Experience with twenty-one patients. A m J M e d 1983;74:23-29. 132. Padeh S, Theodor R, Jonas A, PasswellJH. Severe malabsorption in autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy syndrome successfully treated with immunosuppression. Arch Dis Child 1997;76:532-534. 133. Etzioni A, Atias D, Pollack S, et al. Complete recovery of pure red cell aplasia by intramuscular gammaglobulin therapy in a child with hypoparathyroidism. Am J Hematol 1986;22:409-414. 134. Daneman D, Kooh SW, Fraser D. Hypoparathyroidism and pseudohypoparathyroidism in childhood. Clin Endocrinol Metab 1982;11:211-231. 135. Jorizzo JL, Sams WM, Jr, Jegasothy BV, Olansky AJ. Cimetidine as an immunomodulator: Chronic mucocutaneous candidiasis as a model. A n n Intern Med 1980;92:192-195. 136. Ward L, PaquetteJ, Seidman E, Huot C, Alvarez F, Crock P, Delvin E, Kfimpe O, Deal C. Severe autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy in an adolescent girl with a novel AIRE mutation: Response to immunosuppressive therapy. J Clin Endocrinol Metab 1999;84:844-852.

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CHAPTER 51

P se u d o h y p o p ara thyr o i dis m Clinical, Biochemical, and Molecular Features

SUZANNE

M.

JAN

D E B E U R Division of Endocrinology, Department of Medicine and Metabolism, The Johns Hopkins University School of Medicine,

Baltimore, Maryland 21287

MICHAEL A. LEVINE Departments of Pediatrics, Medicine, and Pathology, TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

PATHOPHYSIOLOGY

Pseudohypoparathyroidism (PHP) is an u n c o m m o n metabolic disorder characterized by biochemical hypoparathyroidism (i.e., hypocalcemia and hyperphosphatemia), increased secretion of parathyroid hormone (PTH), and target tissue unresponsiveness to the biologic actions of PTH. Thus the pathophysiology of PHP differs fundamentally from true hypoparathyroidism, in which PTH secretion rather than PTH responsiveness is defective (1). In addition to functional hypoparathyroidism, many patients with PHP exhibit a distinctive constellation of developmental and skeletal defects, collectively termed Albright's hereditary osteodystrophy (AHO) (2), which includes a round face, short stature, obesity, brachydactyly, heterotopic ossification, and mental retardation (Fig. 1). The relationship between the metabolic abnormalities of PHP and the various features of AHO remains unknown. Indeed, in certain families some affected members manifest both AHO and PTH resistance (i.e., PHP) whereas other family members have AHO without evidence of any endocrine dysfunction, a variation termed "pseudopseudohypoparathyroidism" (pseudo-PHP) (3) Several forms of PHP have been described, which has led to the development of a diagnostic classification that is based on clinical, biochemical, and genetic characteristics (Table 1)

The first insights into the molecular basis for PHP emerged from the parallel observations that cAMP mediates many of the actions of PTH on kidney and bone, and that administration of PTH to normal subjects markedly increases the urinary excretion of nephrogenous cAMP and phosphate (4). Although the PTH infusion test remains a reliable test for the diagnosis of PHP, more important perhaps is the ability of the test to distinguish between several variants of the syndrome (Fig. 2). Patients with PHP type 1 fail to show an appropriate increase in urinary excretion of both nephrogenous cAMP and phosphate (4), suggesting that an abnormality in the renal PTH receptoradenylyl cyclase complex that produces cAMP is the basis for impaired PTH responsiveness (Fig. 3). This early hypothesis was subsequently confirmed by administration of dibutyryl cAMP to patients with PHP type 1. These patients showed a normal phosphaturic response to the infused cAMP analog, indicating that the renal response mechanism to cAMP was intact (5). These studies have led to the conclusion that proximal renal tubule cells are unresponsive to PTH. By contrast, cells in other regions of the nephron appear to respond normally to PTH, as evidenced by reduced urinary calcium excretion in PHP type 1 patients compared to those with hormonopenic hypoparathyroidism (6,7). Furthermore, renal handling of calcium (and sodium)

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A

B

D i

C

FIG. 1 Typical features of Albright hereditary osteodystrophy. (A) A young woman with characteristic features of AHO; note the short stature, disproportionate shortening of the limbs, obesity, and round face. (B) Radiograph of the AHO patient showing marked shortening of fourth and fifth metacarpals. (C) Archibald sign, the replacement of "knuckles" with "dimples" due to the marked shortening of the metacarpal bones. (D) Brachydactyly of the hand; note thumb sign (Murderer's thumb or potter's thumb) and shortening of the fourth and fifth digits.

TABLE 1 Feature Physical appearance Response to PTH Urine cAMP Urine phosphorous Serum calcium level Hormone resistance GsoL activity Inheritance Molecular defect

in response to exogenous PTH is normal in PHP type 1 (8). These observations suggest that the distal renal tubule is responsive to PTH in subjects with PHP type 1, and imply that adequate amounts of cAMP can be produced in these cells or that other second messengers (e.g., cytosolic calcium or diacylglycerol) may mediate these PTH responses. Administration of PTH to subjects with the less comm o n form of the disorder, PHP type 2, produces a normal increase in urinary cAMP but fails to elicit an appropriate phosphaturic response (9). This pattern of response has suggested that PTH resistance in PHP type 2 results from a biochemical defect that is either distal or unrelated to the PTH-stimulated generation of cAME It has b e e n generally assumed that bone cells of patients with PHP type 1 are innately resistant to PTH, based primarily on the observation that patients with PHP type 1 are hypocalcemic and that administration of PTH does not increase the plasma calcium level. In fact, cultured bone cells from a patient with PHP type 1 have been shown to increase intracellular cAMP normally in response to PTH treatment in vitro (10). Moreover, many patients with PHP type 1 develop radiologic (Fig. 4) or histologic evidence of significant bone resorption and hyperparathyroid bone disease (11). Patients with PHP may develop additional abnormalities in bone metabolism resulting from excessive PTH or deficient 1,25-dihydroxyvitamin D~ [1,25(OH)zD3], including osteomalacia (11), rickets (12), renal osteodystrophy (13), and osteopenia (14). One possible explanation for the variable responsiveness of bone to PTH is the existence of two distinct cellular systems in bone on which PTH exerts action: the remodeling system and mineral homeostasis. In

Classification of the Various Forms of Pseudohypoparathyroidism Based on Clinical, Biochemical, and Genetic Features

PHP type la

PseudoPHP

PHP type 1b

PHP type lc

PHP type 2

Albright hereditary osteodystrophy typical, but may be subtle or absent

Normal

Albright hereditary osteodystrophy

Normal

Defective Defective Low or (rarely) normal Generalized

Defective Defective Low or (rarely) normal Limited to PTH target tissues Normal Autosomal dominant (most cases) 20q13.3

Defective Defective Low

Normal Defective Low

Generalized Normal Unknown

Limited to PTH target tissues Normal Unknown

Unknown

Unknown

Reduced

Normal Normal Normal Absent

Reduced Autosomal dominant

Heterozygous mutations in the GNAS 1 gene

PSEUDOHYPOPARATHYROIDISM /

809

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300 A

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FIG. 2 Urinary cAMP excretion in response to an infusion of bovine parathyroid extract (300 USP units). The peak response in normal subjects (A) as well as those with pseudoPHP (not shown) is 50- to 100-fold times basal. Subjects with PHP type la (©) or PHP type 1b (O) show only a 2- to 5-fold increase. Urinary cAMP is expressed as nanomoles per 100 ml of GF, UoAMP(nanomoles per 100 ml GF) = UoAMP (nanomoles/dl) x Scr e ( m g / d l ) / U c r e (mg/dl). Reprinted with permission from Ref 23; MA Levine, TS Jap, RS Mauseth, RW Downs, AM Spiegel. Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 1986;62:497-502. © The Endocrine Society.

Ri

PTH/PTHrP Rc

l i III1\1 t

{ IIii//

I

cAMP

1

"

IP3

+

DAG

c

PKA

PKC

FIG. 3 Cell surface receptors for PTH are coupled to two classes of G proteins. G s mediates stimulation of adenylyl cyclase (AC) and the production of cAME which in turn activates protein kinase A (PKA). Gq stimulates phospholipase C (PLC) to form the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from membrane-bound phosphatidylinositol 4,5-bisphosphate. IP3 increases intracellular calcium (Ca2+) and DAG stimulates protein kinase C (PKC) activity. Each G protein consists of a unique oL chain and a 13",/dimer.

FIG. 4 Photograph and radiograph of hands of a patient with marked hyperparathyroid bone disease. Marked periosteal bone erosion in terminal phalanges has resulted in "pseudoclubbing" From Levine MA, Parfrey NA, Feinstein RS. Pseudohypoparathyroidism. Johns Hopkins Medical Journal (1982), 151:137-146. © The John Hopkins University Press. Reprinted with permission of The John Hopkins University Press.

PHP type 1, the bone remodeling system appears to be more responsive to PTH than the homeostatic system. This variability may reflect that the remodeling system is less d e p e n d e n t on normal plasma levels of 1,25(OH)zD 3. Plasma levels of 1,25(OH)zD s are low in hypocalcemic patients with PHP type 1 (15), a finding that could explain the concurrence of hypocalcemia and increased skeletal remodeling in many of these patients. Hypocalcemia leads to a compensatory overproduction of PTH, which could eventually overcome the 1,25(OH)2D s dependency for remodeling but not for PTH-stimulated calcium mobilization. A role for 1,25(OH)zD ~ in modulating the responsiveness of the calcium homeostatic system to PTH is suggested by several observations. First, the calcemic response to PTH is deficient not only in patients with PHP type 1, but also in patients with other hypocalcemic disorders in which plasma levels of 1,25(OH)zD 3 are low. Moreover, normalization of the plasma calcium level in patients with PHP type 1 by administration of physiologic amounts of 1,25(OH)zD s or pharmacologic amounts of vitamin D restores calcemic responsiveness

810

/

CIJ~TwR51

to PTH (16). Second, patients with PHP type 1 who have normal serum levels of calcium and 1,25(OH)2D a without vitamin D treatment (so-called normocalcemic PHP) show a normal calcemic response to administered PTH (16). These findings suggest that 1,25(OH)zD S deficiency is the basis for the lack of a calcemic response to PTH in hypocalcemic patients with PHP type 1, and challenge the premise that bone cells are intrinsically resistant to the actions of PTH. Subjects with PHP type 1 have increased serum levels of phosphate owing to an inability of PTH to decrease phosphate reabsorption in the proximal renal tubule. Hypocalcemia per se may also contribute to the develo p m e n t of hyperphosphatemia, secondary to impaired renal phosphate clearance by very low levels of intracellular calcium. Accordingly, restoration of plasma calcium levels to normal by chronic treatment with calcium and vitamin D can reduce elevated levels of serum phosphorous. Similar therapy has been shown to reverse the defective phosphaturic response to administered PTH in certain patients with PHP type 1, although the urinary cAMP response remains markedly deficient (17). Therefore, persistence of a blunted urinary cAMP response to PTH in PHP type 1 patients in whom chronic vitamin D therapy has led to normalization of plasma calcium levels and restoration of a phosphaturic response need not imply, as has been at least suggested (17), that there is no relationship between cAMP production and phosphate clearance. A circulating inhibitor of PTH action has been proposed as a cause of PTH resistance on the basis of studies showing an apparent dissociation between plasma levels of e n d o g e n o u s immunoreactive and bioactive PTH in subjects with PHP type 1. Despite high circulating levels of immunoreactive PTH, the levels of bioactive PTH in many patients with PHP type 1 have been found to be within the normal range when measured with highly sensitive renal (18) and metatarsal (19) cytochemical bioassay systems. Furthermore, plasma from many of these patients has been shown to diminish the biologic activity of exogenous PTH in these in vitro bioassays (20). Currently, the nature of this putative inhibitor or antagonist remains unknown. The observation that prolonged hypercalcemia can remove or reduce significantly the level of inhibitory activity in the plasma of patients with PHP has suggested that the parathyroid gland may be the source of the inhibitor. In addition, analysis of circulating PTH immunoactivity after fractionation of patient plasma by reversed-phase high-performance liquid chromatography has disclosed the presence of aberrant forms of immunoreactive PTH in many of these patients (21). Although it is conceivable that a PTH inhibitor may cause PTH resistance in some patients with PHP, it is more likely that circulating antagonists of PTH action arise as a

consequence of the sustained secondary hyperparathyroidism that results from the primary biochemical defect. The identification of circulating fragments of PTH that may act as competitive antagonists of intact PTH in uremic patients with secondary hyperparathyroidism now provide at least a theoretical basis for this hypothesis (22). The overall evidence indicates that the disturbances in calcium, phosphorous, and vitamin D metabolism in patients with PHP type 1 result directly or indirectly from reduced responsiveness of both bone and kidney to PTH. Hypocalcemia results from impaired mobilization of calcium from bone, reduced intestinal absorption of calcium [via deficient generation of 1,25(OH)2D3], and urinary calcium loss. Of these defects, the diminished movement of calcium out of bone stores into the extracellular fluid probably has the greatest role in producing hypocalcemia. Intensive treatment with calcitriol [1,25 (OH) zD3] or other vitamin D analogs improves intestinal calcium absorption and bone calcium mobilization, restores plasma calcium to normal, and reduces circulating PTH levels. Although resistance of the proximal renal tubule to PTH appears to be the primary biochemical defect, the major abnormalities in mineral metabolism found in patients with PHP type 1 can be largely explained on the basis of deficiency of circulating 1,25(OH)zD s.

PSEUDOHYPOPARATHYROIDISM

TYPE la

PHP type 1 can be subclassified into two apparently distinct disorders based on several important clinical and biochemical characteristics: (1) the absence or presence of AHO, (2) h o r m o n e resistance that is specific (PTH alone) or that is more generalized, and (3) normal or reduced tissue activity of the G protein (G~) that couples heptahelical receptors to stimulation of adenylyl cyclase (see Chapters 5 and 7). Subjects with the type la variant have an approximately 50% reduction in expression or activity of the ot chain of G s (Gs0t) in membranes from a wide variety of cells and tissues (23) (Fig. 5). The generalized deficiency of Gsot may impair the ability of PTH, as well as other hormones and neurotransmitters, to activate adenylyl cyclase, thereby accounting for the multihormone resistance that occurs in these patients. In addition to h o r m o n e resistance, patients with PHP type la also manifest AHO (Fig. 1) (1). By contrast, patients with PHP type l b lack features of AHO, have h o r m o n e resistance that is limited to PTH, and have normal levels of Gsot protein in cell membranes (Fig. 5). Early studies of PHP type la led to the identification of families in which some individuals had signs of AHO but lacked apparent h o r m o n e resistance (i.e.,

PSEUDOHYPOPARATHYROIDISM

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FIG. 5 Gso~activity of erythrocyte membranes. GsoLis quantified in complementation assays with $49 cyc- membranes, which genetically lack Gse~ but retain all other components necessary for hormone-response adenylyl cyclase activity. Activity is reduced approximately 50% in patients with AHO subjects with either PHP type l a or pseudoPHP, but is normal in patients with PHP type lb.

pseudoPHP). The observation that PHP type la and pseudoPHP can occur in the same family first suggested that these two disorders might reflect variability in expression of a single genetic lesion. Support for this view comes from studies indicating that within a given kindred, subjects with either pseudoPHP or PHP type la have equivalent functional Gse~ deficiency (Fig. 5) (23,24), and that a transition from h o r m o n e responsiveness to h o r m o n e resistance may occur (25). It therefore seems appropriate to apply the term AHO to both of these variants in acknowledgment of the clinical and biochemical characteristics that patients with PHP type l a and pseudoPHP share (2). M o l e c u l a r D e f e c t in A l b r i g h t

Hereditary Osteodystrophy

The discovery that Gsot deficiency results from inactivating mutations in the GNAS1 gene, located at 20q13.2 --+ 13.3 (26), provided confirmation of autosomal d o m i n a n t transmission in AHO and resolved longstanding controversies regarding the inheritance of this disorder (2,27-29). GNAS1 is a complex gene (30) composed of at least 16 exons, including 3 alternative first exons (Figure 6) (31-33). Alternative splicing of nascent transcripts derived from exons 1-13 generates four mRNAs that encode GsOt. Deletion of exon 3 results in the loss of 15 codons from the mRNA, whereas use of an alternative splice site in exon 4 results in the insertion of a single additional codon into the mRNA. This produces two Gse~ proteins with apparent molecular masses of 45 kDa and two isoforms with apparent molecular masses of 52 kDa (30) that exhibit specific patterns of tissue expression (34). Both long and short forms of Gs0t can stimulate adenylyl cyclase

Nesp551NESP

2-13

Maternal

Gnasxl [ XL~s

2-13

Paternal

Gsot-t

2-13

Paternal

2-13

Biallelic

Gsot

L1A I 1

FIG. 6 The structural organization of the GNAS1 gene in schematic representation. The 13 exons that comprise transcripts for the 52-kDa forms of Gso~protein are labeled 1-13; exon 3 is not present in transcripts that encode the smaller, 45-kDa forms of GsoL protein (see text). The three alternative first exons that are present in transcripts encoding NESP55, Gnasxl, and the truncated (GsoL-t) proteins are also indicated. The transcripts that are derived from maternal, paternal, or both (biallelic) parental alleles are noted.

and open calcium channels (35), but biochemical characterization of these isoforms has revealed subtle differences in the binding constant for GDP, the rate at which the forms are activated by agonist binding, efficiency of adenylyl cyclase stimulation, and the rate of GTP hydrolysis. The significance of these differences remains unknown (35-37), but these distinctions imply the existence of as-yet undefined roles for these G proteins (38). Additional complexity in the processing of the GNAS1 gene arises from the use of alternative first exons that generate novel transcripts (Fig. 6). Because these proteins lack amino acid sequences encoded by exon 1, which are required for interaction of Gscx with G[3~/ and attachment to the plasma membrane, it is unlikely that these proteins can function as transmembrane signal transducers. In one case, a Gsc~ transcript is p r o d u c e d with an alternative first exon (exon 1A) that lacks an initiator ATG; thus, a truncated, nonfunctional Gsot protein is conceivably translated from an inframe ATG in exon 2 (33,39). These transcripts encode a protein of 45 or 42 kDa, d e p e n d i n g on the presence or absence of exon 3, and are most highly expressed in the retina and brain. The function of this Gsc~ chain is unknown. In three other instances unique transcripts are generated using additional coding exons that are present upstream of the exon 1 used to generate functional Gse~ protein. The more 5' of these exons encodes the n e u r o e n d o c r i n e secretory protein NESP55, a chromogranin-like protein, and is generated from a transcript that contains sequences derived from exons 2-13 of GNAS1 in the 3' nontranslated region (40,41). Accordingly, NESP55 shares no protein homology with Gsot. Eleven kilobases further downstream, a second

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CHAPTER51

alternative exon, XLots, when spliced in-frame to exons 2-13, results in a transcript of a larger, 51-kDa Gse~ isoform (42). Both NESP55 and XLots have been implicated in regulated secretion in neuroendocrine tissues. Molecular studies of DNA from subjects with AHO have disclosed inactivating mutations in the GNAS1 gene (43-57) that account for a 50% reduction in expression or function of Gse~ protein (Table 2) (181-185). All patients are heterozygous and have one normal GNAS1 allele and one defective allele. Mutations in the GNASI gene are heterogeneous, including missense mutations (46,48-50,55), point mutations that disrupt efficient splicing (47) or terminate translation prematurely (53), and small deletions (47,48,51,52,57,58). Although novel mutations have

TABLE 2 Mutation M1V Q12X Y37X Q31X GA/gtaagt L99P (AAA___GGAG)--> (AAAGG) AA/gtacgt -> AA/gtacat P115(CCC) ~ (_CC) P115S L153(CTGT) --> ( C T ) R165C ATT GAC TGT --->ATTGT CAG GCT GAC --->CAGGC Y190N AG/gtgt --->AG/gcgt CAG GTG GAC --> CAG AC G206 (GGA) --> ( G ) R231H $250R R258W E259V E267(CAG) --> (CCAG) L271(CTC) --> (_TC) AG/gt ~ AG/ct R385H A366S z~1382 m

been found in nearly all of the kindreds studied, a fourbase deletion in exon 7 has been detected in multiple families (56,58-60) (54,61) and an unusual missense mutation in exon 13 (A366S; see below) has been identified in two unrelated young boys (62), suggesting that these two regions may be genetic "hot spots." Most gene mutations lead to reduced expression of GsoL mRNA (24,63), but in some subjects the mutant allele produces normal levels of Gse~ mRNA (24,63,64) that encodes dysfunctional GsOt proteins (49,50,55,65). The replacement of arginine by histidine at codon 385 in the carboxyl-terminal tail of Gsot selectively "uncouples" G s from receptors and prevents receptor activation (49). Substitution of arginine by histidine at position 231 also prevents receptor activation of Gs,

Mutations in the GNAS1 Gene

Location

Type

Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Intron 2 Exon 4 Exon 4 Exon 4 Intron 4 Exon 5 Exon 5 Exon 6 Exon 6 Exon 6 Exon 7 Exon 7 Intron 7 Exon 8 Exon 8 Exon 9 Exon 10 Exon 10 Exon 10 Exon 10 Exon 10 Intron 10 Exon 13 Exon 13 Exon 13

Missense Nonsense Nonsense Nonsense Deletion 38 bp Deletion 2 bp Missense Deletion 2 bp Deletion 43 bp Substitution Deletion 1 bp Missense Deletion 2 bp Missense Deletion 4 bp Deletion 4 bp Missense Substitution Deletion 4 bp Deletion 2 bp Missense Missense Missense Missense Insertion 1 bp Deletion 1 bp Substitution Missense Missense Deletion 3 bp

Effect

Ref.

Initiator codon Truncated protein Truncated protein Truncated protein Frameshift Donor splice site

(46) (181 ) (181 ) (181 ) (52) (182)

--

(48)

Frameshift Frameshift Donor splice site Frameshift

(57) (45) (182) (51 )

Frameshift

(182)

Frameshift Frameshift

(182) (54,56,58-61,183) (182) (182) (48) (57) (50) (184) (184) (56) (51 ) (47) (47) (49) (44) (185)

Donor splice site Frameshift Frameshift 13"yinteraction Stability Stability Stability Frameshift Frameshift Donor splice site Receptor coupling GDP release Defective coupling to PTH 1R

(56)

(48)

PSEUDOHYPOPARATHYROIDISM

but by an entirely different mechanism (50). The replacement of Arg-231 hinders binding of GTP to the e~ chain, and thereby inhibits receptor-induced dissociation of Gse~ from G~7. Likewise, substitution of arginine at position 258 with tryptophan leads to increased GDP release and impaired receptor-mediated activation (55).

Multiple Hormone Resistance in Pseudohypoparathyroidism Type la Although biochemical hypoparathyroidism is the most commonly recognized endocrine deficiency in PHP type l a, early clinical studies described additional hormonal abnormalities, such as hypothyroidism (66,67) and hypogonadism (68). Because available evidence suggests that Gse~ is present in all tissues, generalized deficiency of this protein could be the basis for not only PTH resistance, the hallmark of PHP type la, but could also explain the decreased responsiveness of diverse tissues (e.g., kidney, thyroid gland, gonads, and liver) to hormones that act via activation of adenylyl cyclase (e.g., PTH, TSH, gonadotropins, and glucagon) (7,69,70). Primary hypothyroidism occurs in most patients with PHP Type la (69). Typically, patients lack a goiter or antithyroid antibodies and have an elevated serum TSH with an exaggerated response to TRH. Serum levels of Y 4 may be low or low normal. Hypothyroidism may occur early in life prior to the development of hypocalcemia, and elevated serum levels of TSH are not uncommonly detected during neonatal screening (71-73). Unfortunately, early institution of thyroid hormone replacement does not seem to prevent the development of mental retardation (72). Reproductive dysfunction occurs commonly in subjects with PHP type la. Women may have delayed puberty, oligomenorrhea, and infertility (69). Plasma gonadotropins may be elevated but are more commonly normal (74). Some patients show an exaggerated serum gonadotropin response to GnRH (68,75). Features of hypogonadism may be less obvious in men with variable serum testosterone levels ranging from normal to frankly reduced. Testes may show evidence of a maturation arrest or may fail to descend normally. Fertility appears to be decreased in men with PHP type l a. Deficiency of prolactin secretion (basal and in response to secretagogues such as TRH) had been reported in some patients with PHP type 1 (76), but later studies have not confirmed these early findings (69). Obesity is common in subjects with PHP type la and pseudohypoparathyroidism, and may reflect a defective lipolytic response to hormonal stimulation due to Gsc~ deficiency (77,78). Others have hypothesized that Gsc~ deficiency results in reduced signaling through the

/

813

melanocortin 4 receptor, which might account for the disinhibition of satiety and hyperphagia observed in many patients with PHP la (79). Abnormal h o r m o n e responsiveness may occur in some tissues without obvious clinical sequellae. For example, the hepatic glucose response to glucagon is normal although plasma cAMP concentrations fail to increase normally (69,80). In other tissues significant hormone resistance does not occur despite the apparent reduction in GsoL. Diabetes insipidus is not a feature of AHO, and urine is concentrated normally in response to vasopressin in patients with PHP type la (81). Although there is a report of adrenal insufficiency in a single individual with PHP type la (82), hypoadrenalism is not a typical feature of PHP type la and adrenocortical responsiveness to ACTH is normal (69).

Neurosensory Defects in Pseudohypoparathyroidism Type la Patients with PHP type la frequently manifest distinctive olfactory (83), gustatory (84), and auditory (85) abnormalities that are apparently unrelated to endocrine dysfunction. The molecular basis of these neurosensory deficits has become more obscure with the discovery of unique G proteins that regulate signal transduction pathways related to vision (86,87), olfaction (88), and taste (89). Mild to moderate mental retardation is common in patients with PHP type la. Farfel and Friedman assessed intelligence in 25 patients with PHP type 1 whose GsOt activity had been determined (90). The authors found an association between mental deficiency and GsOt deficiency, and speculated that reduced cAMP levels in cortical tissue may lead to mental retardation. Other factors that might contribute to mental retardation in patients with PHP type la include hypothyroidism and hypocalcemia; however, early detection and treatment have not prevented cognitive dysfunction in all patients, suggesting that Gs0t deficiency may cause a primary abnormality of neurotransmitter signaling.

The Somatic Phenotype of Albright Hereditary Osteodystrophy AHO is characterized by a unique constellation of developmental defects, including short stature, obesity, a round face, shortening of the digits (brachydactyly), subcutaneous ossification, and dental hypoplasia (Fig. 1) (1,3). Considerable variability occurs in the expression of these features even among affected members of a single family, and all of these features may not be present in every individual (91). Sometimes it may

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CHAPTER51

not be possible to detect any features of AHO in an individual with Gs~ deficiency (47,48). Although patients with AHO may be of normal height and weight, approximately 66% of children and 80% of adults are below the tenth percentile for height. This reflects a disproportionate shortening of the limbs, and arm span is less than height in the majority of patients. Obesity is a c o m m o n feature of AHO and about one-third of all patients with AHO are above the ninetieth percentile of weight for their age, despite their short stature (92) (Fig. 1A). Patients with AHO typically have a r o u n d face, a short neck, and a flattened bridge of the nose. Numerous other abnormalities of the head and neck have also been noted. Ocular findings include hypertelorism, strabismus, nystagmus, unequal pupils, diplopia, microphthalmia, and a variety of abnormal findings on funduscopic exam that range from irregular pigmentation to optic atrophy and macular degeneration. Head circumference is above the ninetieth percentile in a significant minority of children (92). AHO subjects with long-standing hypocalcemia (i.e., PHP type la) frequently develop dental abnormalities that include dentin and enamel hypoplasia, short and blunted roots, and delayed or absent tooth eruption (93). Brachydactyly is often the most obvious clinical sign of AHO, and may be a useful criterion to distinguish AHO from other causes of obesity or short stature. Brachydactyly can be symmetrical or asymmetrical and involve one or both hands or feet (Fig. 1B). Shortening of the distal phalanx of the thumb is the most c o m m o n abnormality; typically, the ratio of the width of the nail to its length is increased (so-called Murder's thumb or potter's thumb; Fig. 1D). Shortening of the metacarpals causes shortening of the digits, particularly the fourth and fifth, frequently recognized on physical exam as dimpling over the knuckles of a clenched fist (Archibald sign; Fig. 1C). A definitive diagnosis requires careful examination of radiographs of the hands and feet (Fig. 1B). Severe shortening of the distal phalanx of the thumb and third through fifth metacarpals is the most specific pattern of brachydactyly in AHO (94,95), distinguishing it from other syndromes with brachydactyly, such as familial brachydactyly, Turner syndrome, and Klinefelter syndrome (94). In addition to brachydactyly, several other skeletal abnormalities occur in subjects with AHO. Numerous deformities of the long bones have been observed, including a short ulna, bowed radius, deformed elbow or cubitus valgus, coxa vara, coxa valga, genu varum, and genu valgus deformities (92). The most c o m m o n abnormalities of the skull are hyperostosis frontalis interna and a thickened calvarium. Rarely, spinal cord compression has been reported in AHO (96). The bone age is advanced by 2-3 years in the majority of

patients (92). It should be noted that the skeletal abnormalities of AHO may not be apparent until a child is 5 years old (97). Patients with A H O develop heterotopic ossifications of the soft tissues or skin (osteoma curls) that are frequently mistaken for subcutaneous calcifications. However, these lesions are unrelated to abnormalities in serum calcium or phosphorous levels and represent true, ectopic bone consisting of spicules of mineralizing osteoid and calcified cartilage. Osteoma cutis is present in 25 to 50% of patients and is typically noted in infancy or early childhood. Occasionally, ossification of the skin is the presenting feature of AHO (98,99). Blue-tinged, stony hard papular or nodular lesions that range in size from pinpoint up to 5 cm in diameter often occur at sites of minor trauma and may appear to be migratory on serial examinations (98). More extensive and progressive ossification that affects the deep connective tissues occurs in subjects with progressive osseous heteroplasia (POH), a rare genetic disorder with apparent autosomal d o m i n a n t inheritance (100). POH patients do not share any other phenotypic features with AHO or display hormonal resistance. A mutation in GNAS1 has been identified in a girl with POH-like heterotopic ossification, no h o r m o n e resistance, and no features of AHO except mild brachydactyly of the fourth and fifth metacarpals (101). The discovery of GNAS1 mutations in an overlap syndrome of AHO and POH lead to the analysis of GNAS1 in several POH kindreds. Heterozygous inactivating mutations in GNAS1, including the 4-bp deletion in exon 7 that is a mutational hot spot in AHO, were identified (102). The mechanism by which heterozygous loss-of-function mutations in GNAS1 lead to heterotopic bone formation is unclear. The association of POH with mutations in GNAS1 further widens the phenotypic expression of GsCX deficiency, and the molecular modifiers that modulate the phenotypic expression of identical gene defects are a subject of active investigation One or more of the developmental and skeletal abnormalities of AHO can occur in subjects who do not have AHO or a defect in the GNAS1 gene, and thus need not imply that the subject has PHP type l a or pseudoPHP. Features of AHO, particularly shortened metacarpals or metatarsals, may be present in normal subjects, and have been described in patients with h°rm°ne-deficient hypoparathyroidism (103-106), renal hypercalciuria (107), and primary hyperparathyroidism (108). Furthermore, obesity, round face, brachydactyly, and mental retardation are also present in several other genetic disorders (e.g., Prader-Willi syndrome, acrodysostosis, Ullrich-Turner syndrome, Gardener syndrome). In some instances overlapping clinical features between AHO and other syndromes may cloud the diagnosis. For example, an AHO-like

PSEUDOHYPOPARATHYROIDISM / syndrome has been described in a mother and her daughter who have a proximal 15q chromosomal deletion that resembles that found in Prader-Willi syndrome (109). Terminal deletion of 2q37 [del(2) (q37.3)] is a consistent karyotypic abnormality that has been documented in patients with an AHO-like syndrome (110,111). In contrast to AHO, these patients have normal endocrine function and normal GsOLactivity (111). Thus, high-resolution chromosome analysis, biochemical/molecular analysis, and careful physical and radiologic examination are essential in discriminating between these phenocopies and AHO.

Phenotypic Variability in AHO: The Paradox of Pseudohypoparathyroidism Type la and Pseudopseudohypoparathyroidism Molecular studies have provided a basis for Gs0Ldeficiency, but they do not explain the striking variability in biochemical and clinical phenotype. Why do some GsoLcoupled pathways show reduced hormone responsiveness (e.g., PTH, TSH, gonadotropins) whereas other pathways are clinically unaffected (ACTH in the adrenal gland and vasopressin in the renal medulla)? Perhaps even more intriguing is the paradox that GsoL deficiency can be associated with hormone resistance and AHO (PHP type la), AHO only (pseudoPHP), or no apparent consequences at all (48). These observations, when considered in the context of studies showing that the n u m b e r of G s molecules in cell membranes greatly exceeds the number of either receptor or adenylyl cyclase molecules (112), raise issue with the hypothesis that a 50% deficiency of GsoL can impair h o r m o n e responsiveness. Indeed, in vitro studies of tissues and cells from subjects with PHP type la have often demonstrated normal hormonal responsiveness despite a 50% reduction in GsoLexpression (113). Although the basis for the variable expression of GsOL deficiency remains unknown, several observations provide important insights. First, clinical genetic studies have documented that PHP type la and pseudoPHP frequently occur in the same family, but are not present in the same generation. Second, analysis of published pedigrees has indicated that in most cases maternal transmission of GsoL deficiency leads to PHP type l a whereas paternal transmission of the defect leads to pseudoPHP (23,54,114,115). These findings are inconsistent with models in which chance determines phenotype or in which a second gene is interactive with the defective GNAS1 gene, because both PHP type la and pseudoPHP would be expected to occur with equal frequency and in the same sibship. By contrast, these observations first suggested the possibility of genomic imprinting of the GNAS1 gene locus as an explanation for the variable phenotypic expression of a single

815

genetic defect (115). Genomic imprinting is an unusual mode of regulation of gene expression that results in the expression of only one parental allele in somatic tissues. Thus, genomic imprinting can account for functional differences that arise as a consequence of the parental origin of a gene allele. Studies have indeed confirmed that the GNAS1 gene is imprinted, but in a far more complex m a n n e r than had been anticipated. Two upstream promoters, each associated with a large coding exon, lie 35 kb upstream of GNAS1 exon 1 (Fig. 6). These promoters are only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5' of these exons encodes NESP55, which is expressed exclusively from the maternal allele. By contrast, the XLoLs exon is paternally expressed (31,32). Exon 1A is a third upstream exon that apparently consists only of untranslated sequence that is spliced to exons 2-13 (33). This exon is also imprinted and is derived exclusively from the paternal allele (116). Despite the simultaneous imprinting in both the paternal and maternal directions of the GNAS1 gene, expression of GsOLappears to be biallelic in all h u m a n tissues that have been examined thus far (31,32,117). Moreover, the lack of access to relevant tissues (i.e., kidney) from subjects with PHP type la has prevented direct analysis of GsoL expression in patients with this disorder. To overcome these difficulties, murine models of PHP type la have been developed through disruption of a single Gnas gene in embryonic stem cells (118,119) Although these mice have reduced levels of GsoLprotein, they lack many of the features of the h u m a n disorder. Biochemical analyses of these heterozygous Gnas knockout mice suggest that Gs0Lexpression may derive from only the maternal allele in some tissues (e.g., renal cortex) and from both alleles in other tissues (e.g., renal medulla) (118,120). Accordingly, mice that inherit the defective Gnas gene maternally express only that allele in imprinted tissues, such as the PTH-sensitive renal proximal tubule, and therefore have no functional GsOLprotein. By contrast, the 50% reduction in Gsc~ expression that occurs in nonimprinted tissues, which express both Gnas alleles, may account for more variable and moderate h o r m o n e resistance in these sites (e.g., the thyroid). Thus, variable hormonal responsiveness implies that haploinsufficiency of GsoLis tissue specific; that is, in some tissues a 50% reduction in GsOLis still sufficient to facilitate normal signal transduction. Confirmation of this proposed mechanism in patients with AHO will require demonstration that the h u m a n GsoL transcript is paternally imprinted in the renal cortex. In AHO, inherited GNAS1 gene mutations reduce expression or function of GsoL protein. By contrast, in the McCune-Albright syndrome, postzygotic somatic mutations in the GNAS1 gene enhance activity of the

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protein (121,122). These mutations lead to constitutive activation of adenylyl cyclase and result in proliferation and autonomous hyperfunction of hormonally responsive cells. The clinical significance of Gse~ activity as a determinant of h o r m o n e action is emphasized by the description by Iiri et al. (44) of two males with both precocious puberty and PHP type la. These two unrelated boys had identical GNAS1 gene mutations in exon 13 (A366S) that resulted in a temperature-sensitive form of Gse~. This GsoL is constitutively active in the cooler environment of the testis, while being rapidly degraded in other tissues at normal body temperature. Thus, different tissues in these two individuals could show h o r m o n e resistance (to PTH and TSH), h o r m o n e responsiveness (to ACTH), or h o r m o n e - i n d e p e n d e n t activation (to LH).

PSEUDOHYPOPARATHYROIDISM

TYPE lb

Subjects with PHP type 1 who lack features of AHO, manifest h o r m o n e resistance that is limited to PTH target organs (Table 1), and have normal Gse~ activity (Fig. 5) (69) are variants of PHP termed PHP type lb (123). Although patients with PHP type lb fail to show a n e p h r o g e n o u s cAMP response to PTH, they often manifest osteopenia or skeletal lesions similar to those that occur in patients with hyperparathyroidism, including osteitis fibrosa cystica (Fig. 4) (124). Cultured bone cells from one patient with PHP type l b and osteitis fibrosa cystica were shown to have normal adenylyl cyclase responsiveness to PTH in vitro (10). These observations have suggested that at least one intracellular signaling pathway coupled to the PTH receptor may be intact in patients with PHP type lb. Specific resistance of target tissues to PTH, and normal activity of Gsc~, had implicated decreased expression or function of the P T H / P T H r P receptor as the cause for h o r m o n e resistance. In addition, cultured fibroblasts from some, but not all, PHP type 1b patients were shown to accumulate reduced levels of cAMP in response to PTH (125) and to contain decreased levels of mRNA encoding the P T H / P T H r P receptor (126). Several lines of evidence suggest that the primary defect in PHP type l b is not in the gene encoding the P T H / P T H r P receptor, however. First, pretreatment of cultured fibroblasts from subjects with PHP type l b with dexamethsone was found to normalize the PTHinduced cAMP response and to increase expression of P T H / P T H r P receptor mRNA (126). Second, molecular studies have failed to disclose mutations in the coding exons (127) and p r o m o t e r regions (128) of the P T H / P T H r P receptor gene or its mRNA (129). Furthermore, linkage studies have demonstrated discordance between inheritance of PHP type lb and alleles for the P T H / P T H r P receptor, thus excluding

defects even in regions of the gene that have not yet been examined by sequence analysis (130). Third, mice ( 131 ) and humans (132) that are heterozygous for inactivation of the gene encoding the P T H / P T H r P receptor do not manifest PTH resistance or hypocalcemia. And finally, inheritance of two defective P T H / P T H r P receptor genes results in Blomstrand chondrodysplasia, a lethal genetic disorder characterized b y advanced e n d o c h o n d r a l bone maturation (132). Thus, it is likely that the molecular defect in PHP type l b resides in other genes that regulate expression or activity of the P T H / P T H r P receptor. Although most cases of PHP type l b appear to be sporadic, familial cases have been described in which transmission of the defect is most consistent with an autosomal dominant pattern (133,134). In some of these kindreds, there appears to be a pattern of inheritance consistent with imprinting as observed in PHP type la. Studies have used gene mapping to identify the location of the PHP type lb gene (135). The PHP type lb gene was mapped to a small region of chromosome 20ql 3.3 near the GNAS1 gene, thus raising the possibility that some patients with PHP type l b have inherited a defective promoter or enhancer that regulates expression of GsoL in the kidney (135). Furthermore, an imprinting defect that impairs the switching of the paternal-to-maternal imprint in the female germ line, resulting in offspring with two paternally imprinted, transcriptionally silent genes, could result in renal Gse~ deficiency in PHP type lb. Alternatively, the chromosomal region demonstrating the greatest linkage to PHP type l b is centromeric to GNAS1, thus raising the intriguing possibility that a second gene responsible for mineral homeostasis resides close to GNAS1 and may be defective in PHP type 1b (135).

PSEUDOHYPOPARATHYROIDISM

TYPE lc

Resistance to multiple hormones has been described in several patients with AHO who have do not have a demonstrable defect in G s or G~ (Table 1) (69,99,136). This disorder is termed PHP type l c. The nature of the lesion in such patients is unclear, but it could be related to some other general c o m p o n e n t of the receptor-adenylyl cyclase system, such as the catalytic unit (137). Alternatively, these patients could have functional defects of G s (or Gi) that do not become apparent in the assays presently available.

PSEUDOHYPOPARATHYROIDISM

TYPE 2

PHP type 2 is the least c o m m o n form of PHE This variant of PHP is typically a sporadic disorder, although one case of familial PHP type 2 has been reported

PSEUDOHYPOPARATHYROIDISM / (138). Patients do not have features of AHO. Renal resistance to PTH in PHP type 2 patients is manifested by a reduced phosphaturic response to administration of PTH, despite a normal increase in urinary cAMP excretion (Table 1) (9). These observations suggest that the PTH receptor-adenylyl cyclase complex functions normally to increase cyclic AMP in response to PTH, and are consistent with a model in which PTH resistance arises from an inability of intracellular cAMP to initiate the chain of metabolic events that result in the ultimate expression of PTH action. Although supportive data are not yet available, a defect in cAMP-dependent protein kinase A has been proposed as the basis for this disorder (9). Alternatively, the defect in PHP type 2 may not reside in an inability to generate a physiologic response to intracellular cAMP: a defect in another PTH-sensitive signal transduction pathway may explain the lack of a phosphaturic response. One candidate is the PTH-sensitive phospholipase C pathway that leads to increased concentrations of the intracellular second messengers inositol 1,4,5-trisphosphate and diacylglycerol (139,140) and cytosolic calcium (141-144). In some patients with PHP type 2 the phosphaturic response to PTH has been restored to normal after serum levels of calcium have been normalized by treatm e n t with calcium infusion or vitamin D (145). These results point to the importance of Ca 2+ as an intracellular second messenger. Finally, a similar dissociation between the effects of PTH on generation of cAMP and tubular reabsorption of phosphate has been observed in patients with profound hypocalcemia due to vitamin D deficiency (146), suggesting that some cases of PHP type 2 may in fact represent vitamin D deficiency.

DIAGNOSIS Natural History The natural history of PHP is quite variable. Although PHP is congenital, hypocalcemia is not present from birth, and the biochemical defects arise gradually during childhood. The initial manifestations of tetany typically occur between 3 and 8 years of age, but the significance of these findings may not be appreciated and the diagnosis of hypocalcemia may be delayed for months or even years. A progressive decline in serum calcium, preceded by increasing levels of serum phosphate, PTH, and 1,25(OH)2D ~, has been documented in one child as he advanced from 3 to 31⁄2 years of age (147). In a second report serial PTH infusions were used to evaluate h o r m o n e responsiveness in an infant from an AHO family. This child was shown to have a normal cAMP response at age 3 months when serum levels of calcium, phosphorous, and PTH were normal, but was found to have an abnormal cAMP

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response when retested at age 2.6 years after he had developed tetany and was found to be hypocalcemic (25). At the time of his second PTH infusion, the child had markedly elevated serum concentrations of phosphorous and PTH and was receiving thyroxine for recently diagnosed primary hypothyroidism (25). Some affected children show few symptoms of tetany, and the diagnosis of PHP is recognized only later in life after hypocalcemia is discovered serendipitously or when features of AHO become obvious. Hypocalcemia may not always provide a clue to the clinical diagnosis of PHP, however, because some PHP patients are able to maintain a normal serum calcium level without treatment (i.e., normocalcemic PHP) (16).

General Physical, Laboratory, and Radiographic Features The diagnosis of PHP should be considered in any patient with hypocalcemia and hyperphosphatemia. A low serum calcium level may be found during an evaluation of unexplained paresthesias or seizures, or may be discovered after multichannel analysis of a blood specimen obtained as part of a routine examination. PHP should be strongly suspected if the serum concentration of PTH is elevated, although occasionally serum levels of PTH are "inappropriately" normal in subjects with PHP owing to confounding hypomagnesemia (133) or other factors (148). Hypocalcemia may be precipitated or worsened during times of "stress" on calcium homeostasis, such as during early pregnancy, lactation, or during an episode of acute pancreatitis. Although hypocalcemia is present in most patients with hypoparathyroidism b y the end of the first decade of life, this biochemical finding may go undetected for many years. Cataracts and intracranial calcification, particularly of the basal ganglion, are c o m m o n in patients with all forms of chronic hypoparathyroidism. Thus, the presence of these ectopic or metastatic calcifications does not help to discriminate a m o n g the various causes of hypocalcemia and hyperphosphatemia (149). Intracranial calcifications are readily detected when CT scanning is employed (150,151), and may occasionally be associated with symptoms such as Parkinson's disease (152). Unusual presenting manifestations of PHP include neonatal hypothyroidism (71,72), unexplained cardiac failure (153), Parkinson's disease (152) and spinal cord compression (154). A diagnosis of PHP should be suspected in any patient with hypocalcemia and elevated serum level of phosphorous and PTH, particularly when clinical features of AHO are present. Further corroboration of the diagnosis of PHP requires demonstration of normal renal function and normal serum levels of magnesium and 25-hydroxyvitamin D. The presence of AHO a n d / o r manifestations of m u l t i h o r m o n e resistance,

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such as hypothyroidism or hypogonadism, favors a diagnosis of PHP type la (69). When most or all of these features are present, more sophisticated tests may not be necessary to confirm the clinical diagnosis. Serum calcium levels can fluctuate in patients with PHP, and may spontaneously change from low to normal and vice versa, thus contributing to the confusion regarding the distinction between PHP and pseudoPHP (16,155). However, the abnormal cAMP response to administered PTH (see below) does not become normal in PHP patients who become normocalcemic with or without treatment. Thus, the PTH infusion remains the most reliable test to distinguish between these two variants (Fig. 2).

Specialized Tests The biochemical hallmark of PHP is the failure of the target organs--bone, and kidney--to respond normally to PTH. Additional tests have been developed to identify subjects with PHP type la; these research tests, which are based on analysis of G~ot protein or the GNAS1 gene, are only rarely indicated u n d e r typical clinical circumstances. The classic tests of Ellsworth and Howard, and of Chase, Melson, and Aurbach, involved the intravenous infusion of 200-300 USP units of bovine parathyroid extract (parathyroid injection, USP; Lilly) and subsequent measurement of urinary excretion of n e p h r o g e n o u s (or total) cAMP (Fig. 2) and phosphate. This relatively crude PTH preparation is no longer available, and has been replaced by synthetic peptides corresponding to the amino-terminal region of h u m a n PTH (e.g., hPTH(1-34) (156-158). A standard protocol involves the infusion of synthetic hPTH(1-34) peptide, 200 units in an adult and 3 u n i t s / k g body weight (200 units maximum) in children over the age of 3 years, intravenously over 10 minutes (156,159). Test subjects should be in a fasting state and active urine output should be initiated and maintained by the ingestion of 200 ml of water per hour, 2 hours prior to the infusion of PTH and continuing through the study. A base line urine collection should be made in a 60-minute period preceding the PTH infusion. Starting at time 0, urine should be collected in separate collections at three time periods: 0-30, 30-60, and 60-120 minutes. Blood samples should be obtained at time 0 and at 2 hours after the start of PTH infusion for m e a s u r e m e n t of serum creatinine and phosphorus concentrations. Urine samples should be analyzed for cAMP, phosphorus, and creatinine concentrations. The preferred unit for expression of urinary cAMP is n m o l / 1 0 0 ml (or per liter) of glomerular filtrate ( n m o l / d l GF). The cAMP response during the first 30 minutes after the start of PTH infusion best differentiates patients with PHP type 1 from those with

hypoparathyroidism and from normal subjects, compared to other parameters of cAMP metabolism (159). Normal subjects and patients with h o r m o n o p e n i c hypoparathyroidism usually display a 10- to 20-fold increase in urinary cAMP excretion, whereas patients with PHP type 1 (types la and lb) show a markedly blunted response regardless of their serum calcium concentration. The urinary cAMP response to infusion of synthetic h P T H fragments in patients with PHP type 1 is unrelated to serum calcium levels, but may be related to endogenous serum PTH levels. The maximal urinary cAMP response to PTH increases after suppression of endogenous PTH in patients with PHP type 1, but nevertheless does not reach that of the normal range (8). Thus, this test can distinguish patients with so-called "normocalcemic" PHP (i.e., patients with PTH resistance who are able to maintain normal serum calcium levels without treatment) from subjects with pseudoPHP (who will have a normal urinary cAMP response to PTH (4,23) (Fig. 2). Subjects with PHP type 2 typically manifest a normal urinary cAMP response to infused PTH but fail to demonstrate an appropriate phosphaturic response. Several metabolic abnormalities, such as hypo- and hypermagnesemia and metabolic acidosis, may interfere with the renal generation and excretion of cAMP in response to PTH (160-163). These abnormalities should be corrected if possible, but probably do not interfere with the interpretation of the test. Calculation of the phosphaturic response to PTH as the percent decrease in tubular maximum for phosphate reabsorption (percent decrease in TmP/GFR) during the first hour after PTH infusion yields the best separation between normal subjects and patients with PHP or hypoparathyroidism (159). However, distinction between groups is also possible when the results are expressed as the decrease in percent tubular reabsorption of phosphorus (decrease in % TRP). A nomogram has been developed that facilitates calculation of T m P / G F R (164). T m P / G F R is elevated in patients with PHP and hypoparathyroidism. Patients with hormonedeficient hypoparathyroidism have a steep decrease in T m P / G F R during the first h o u r after beginning the infusion of PTH. This decrease does not occur in patients with PHP [for further details see references by Mallette et al. (156,159)]. Although a normal phosphate response may occur in PHP type 1 patients with serum calcium or PTH levels in the normal range (8), in patients with PHP type 2 the phosphaturic response to PTH is not changed despite at least a 10-fold increase in cAMP excretion. Unfortunately, interpretation of the phosphaturic response to PTH is often complicated by r a n d o m variations in phosphate clearance, and it is sometimes not possible to classify a phosphaturic response as normal or subnormal regardless of the

PSEUDOHYPOPARATHYROIDISM / criteria employed. More perplexing yet is the observation that biochemical findings that resemble PHP type 2 have been found in patients with various forms of vitamin D deficiency (146). In these patients, marked hypocalcemia is accompanied by hyperphosphatemia due presumably to an acquired dissociation between the a m o u n t of cAMP generated in the renal tubule and its effect on phosphate clearance. The plasma cAMP response to PTH can also be used to differentiate patients with PHP type 1 from normal subjects and from patients with hypoparathyroidism (158,165,166). Patients with PHP type 2 can be expected to have normal responsiveness, however. This test offers few advantages over protocols that assess the urinary excretion of cAMP, because changes in plasma cAMP in normal subjects and patients with hypoparathyroidism are much less dramatic than changes in urinary cAMP, and urine must still be collected if one wishes to assess the phosphaturic response to PTH. One reasonable indication for measuring the plasma cAMP response to PTH is the evaluation of patients in whom proper collection of urine is not possible, such as young children (166). The plasma 1,25(OH)zD ~ response to PTH has been used to differentiate between hormone-deficient and hormone-resistant hypoparathyroidism (157,167). In contrast to normal subjects and patients with hypoparathyroidism, patients with PHP had no significant increase in circulating levels of 1,25(OH)zD 3. This proposed test readily demonstrates the difference in the pathophysiology between hypoparathyroidism and PHE Its clinical relevance is probably limited to distinguishing type 1 from type 2 PHP, whereby the expected increase in the latter form of PHP might be a more reliable parameter than the phosphaturic response to PTH.

TREATMENT The treatment of hypocalcemia and hyperphosphatemia in patients with PHP is based on the guidelines r e c o m m e n d e d for treatment of patients with other forms of hypoparathyroidism (see Chapter 52). Urgent treatment of acute or severe symptomatic hypocalcemia in patients with PHP is best accomplished by the intravenous infusion of calcium. The goal is alleviation of symptoms and prevention of laryngeal spasm and seizures. The long-term treatment of hypocalcemia in patients with PHP requires administration of oral calcium and vitamin D or analogs. The goals of therapy are to maintain serum ionized calcium levels in the normal range, to avoid hypercalciuria, and to reduce elevated levels of circulating PTH. Patients with hypoparathyroidism have increased urinary calcium

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excretion in relation to serum calcium and are therefore prone to hypercalciuria (168). By contrast, patients with PHP have significantly lower urinary calcium in relation to serum calcium (168,169) and can tolerate serum calcium levels that are within the normal range without developing hypercalciuria (6). Once normocalcemia has been attained attention should be directed toward suppression of PTH levels to normal. This is important because elevated PTH levels in patients with PHP are frequently associated with increased bone remodeling. Hyperparathyroid bone disease, including osteitis fibrosa cystica (97,124,170) and cortical osteopenia (Fig. 4) (14), can occur in patients with PHP type lb. These subjects may have elevated serum levels of alkaline phosphatase (170) and urine hydroxyproline (14,171). In this regard calcitriol has a theoretical advantage over other vitamin D preparations (see below) because it may inhibit PTH release directly (172). Oral calcium is usually administered in amounts of 1-3 g of elemental calcium per day in divided doses. To assure optimal absorption and to maximize binding of dietary phosphorous, oral calcium supplements should be taken with water or other fluids, and with meals (173). Many considerations are involved in the selection of a calcium supplement, and none are unique to the treatment of PHP (see Chapter 52). Calcium carbonate is an inexpensive form of calcium that is very convenient owing to its high content of elemental calcium (40%). W h e n taken with food, absorption of calcium from calcium carbonate is adequate even in patients who are achlorhydric. All patients with PHP who are hypocalcemic will require vitamin D or analogs in addition to calcium. Calcitriol, the active form of vitamin D, is the most physiologic treatment choice. Patients with PHP require about 75% as m u c h calcitriol to maintain normocalcemia as do patients with hypoparathyroidism (174). Almost all patients with hypoparathyroidism or PHP can be effectively treated with calcitriol in the a m o u n t of 0.25 txg twice a day to 0.5 txg four times a day. Because of the expense of calcitriol and the need to administer the drug several times per day, other vitamin D preparations may be preferred. Patients with all forms of hypoparathyroidism and PHP will respond to pharmacologic doses of ergocalciferol and calcifidiol. Ergocalciferol is the least expensive choice for vitamin D therapy, and provides a long duration of action (with corresponding prolonged potential toxicity). Patients with PHP require lower doses of vitamin D than do patients with hypoparathyroidism (174), an observation that reflects the response of bone and renal distal tubular cells to endogenous PTH (175). Treatment with calcium and vitamin D usually decreases the elevated serum phosphate to a high normal level because of a

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favorable balance between increased urinary phosphate excretion and decreased intestinal phosphate absorption. In general, phosphate-binding gels such as alum i n u m hydroxide are not necessary. Estrogen therapy and pregnancy have particularly interesting effects on calcium homeostasis in patients with PHR Estrogen therapy can reduce serum calcium concentrations in women with PHP type 1 (176) as well as women with hypoparathyroidism (177). In contrast, at the time of the menses, when estrogen levels are low, some well-treated hypoparathyroid women may develop symptomatic hypocalcemia with the cause remaining unknown (178). The same p h e n o m e n o n occurs occasionally in women with PHR Hypocalcemic symptoms are relieved in 30-60 minutes by ingestion of 200-400 mg of elemental calcium. Paradoxically, during the high-estrogen state of pregnancy, the patients of Zerwekh and Breslau remained normocalcemic without therapeutic amounts of calcium and vitamin D (179). During pregnancy, serum 1,25(OH)2D3 concentrations increased two- to threefold but the PTH levels were nearly half of what was present before pregnancy. After delivery, serum calcium and 1,25(OH)2D 3 levels decreased and PTH rose (176). Because placental synthesis of 1,25-dihydroxyvitamin D is not compromised in patients with PHP (179), the placenta may have contributed to the maintenance of normocalcemia during pregnancy in these patients. In contrast, patients with hypoparathyroidism may require treatment with larger amounts of vitamin D and calcium in the latter half of pregnancy (180). Patients with AHO may require specific treatment for unusual problems related to their developmental and skeletal abnormalities. Patients with PHP type la should be treated for their associated hypogonadism and hypothyroidism. Ectopic ossification occurs in about 30% of patients with AHO (92), but rarely causes a problem. At times, large extraskeletal osteomas may occur that will require surgical removal to relieve pressure symptoms (98).

CONCLUSION The evolving characterization of the signal transduction pathways that regulate PTH secretion and action has led to a new understanding of the molecular basis of some forms of hypoparathyroidism and PHE H a n d in h a n d with this experimental approach have come unexpected insights gained through careful study of patients with disorders of mineral metabolism. Patients with defects in the GNAS1 gene provide us with the unanticipated opportunity to explore genomic imprinting as a genetic mechanism, and to test this model for regulating parental contributions to the developing fetus as an explanation for the functional differences

between subjects with PHP type la and pseudoPHP. Continuing work to define the molecular genetic defect in PHP type l b will no doubt provide equally exciting and novel insights that further illuminate PTH receptor signaling pathways in classic target tissues such as bone and kidney.

ACKNOWLEDGMENTS This work has been supported in part by grants from the National Institutes of Health (DK-34281 and DK56178, and GCRC M01-RR00052).

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125. Silve C, Suarez F, el Hessni A, Loiseau A, Graulet AM, Gueris J. The resistance to parathyroid hormone of fibroblasts from some patients with type Ib pseudohypoparathyroidism is reversible with dexamethasone. J Clin Endocrinol Metab 1990;71:631-638. 126. Suarez E Lebrun JJ, Lecossier D, Escoubet B, Coureau C, Silve C. Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 1995;80:965-970. 127. Schipani E, Weinstein LS, Bergwitz C, Iida-Klein A, Kong XF, Stuhrmann M, Kruse K, Whyte ME Murray T, Schmidtke J, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80:1611-1621. 128. BettounJD, Minagawa M, Kwan MY, Lee HS, Yasuda T, Hendy GN, Goltzman D, White JH. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type lb. J Clin Endocrinol Metab 1997;82:1031-1040. 129. Fukumoto S, Suzawa M, Takeuchi Y, Kodama Y, Nakayama K, Ogata E, Matsumoto T, Fujita T. Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1996;81: 2554-2558. 130. Jan de Beur SM, Ding CL, LaBuda MC, Usdin TB, Levine MA. Pseudohypoparathyroidism lb: Exclusion of parathyroid hormone and its receptors as candidate disease genes. J Clin Endocrinol Metab 2000;85:2239-2246. 131. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LK, Ho C, Mulligan RC, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663-666. 132. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, Le Merrer M, Silve C. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 133. Allen DB, Friedman AL, Greer FR, Chesney RW. Hypomagnesemia masking the appearance of elevated parathyroid hormone concentrations in familial pseudohypoparathyroidism. Am J Med Genet 1988;31:153-158. 134. Winter JSD. Hughes IA. Familial pseudohypoparathyroidism without somatic anomalies. Can Med AssocJ 1980;123:26-31. 135. Juppner H, Schipani E, Bastepe M, Cole DE, Lawson ML, Mannstadt M, Hendy GN, Plotkin H, Koshiyama H, Koh T, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci USA 1998;95: 11798-11803. 136. Farfel Z, Brothers VM, Brickman AS, Conte F, Neer R, Bourne HR. Pseudohypoparathyroidism: inheritance of deficient receptor-cyclase coupling activity. Proc Natl Acad Sci USA 1981;78: 3098-3102. 137. Barrett D, Breslau NA, Wax MB, Molinoff PB, Downs RW, Jr. New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase. AmJPhysiol 1989;257:E277-E283. 138. Van Dop C. Pseudohypoparathyroidism: Clinical and molecular aspects. Semin Nephro11989;9:168-178. 139. Civitelli R, Reid IR, Westbrook S, Avioli LV, Hruska KA. PTH elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line. A m J Physiol 1988;255:E660-E667. 140. Dunlay R. Hruska K. PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physio11990;258:F223-F231.

141. Gupta A, Martin KJ, Miyauchi A, Hruska KA. Regulation of cytosolic calcium by parathyroid hormone and oscillations of cytosolic calcium in fibroblasts from normal and pseudohypoparathyroid patients. Endocrinology 1991 ;128:2825-2836. 142. Civitelli R, Martin TJ, Fausto A, Gunsten SL, Hruska KA, Avioli LV. Parathyroid hormone-related peptide transiently increases cytosolic calcium in osteoblast-like cells: Comparison with parathyroid hormone. Endocrinology 1989; 125:1204-1210. 143. Reid IR, Civitelli R, Halstead LR, Avioli LV, Hruska KA. Parathyroid hormone acutely elevates intracellular calcium in osteoblastlike cells. Am J Physiol 1987;253:E45-E51. 144. Yamaguchi DT, Hahn TJ, Iida-Klein A, Kleeman CR, Muallem S. Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. J Biol Chem 1987;262:7711-7718. 145. Kruse K, Kracht U, Wohlfart K, Kruse U. Biochemical markers of bone turnover, intact serum parathyroid horn and renal calcium excretion in patients with pseudohypoparathyroidism and hypoparathyroidism before and during vitamin D treatment. EurJ Pediatr 1989;148:535-539. 146. Rao DS, Parfitt AM, Kleerekoper M, Pumo BS, Frame B. Dissociation between the effects of endogenous parathyroid hormone on adenosine 3',5'-monophosphate generation and phosphate reabsorption in hypocalcemia due to vitamin D depletion: An acquired disorder resembling pseudohypoparathyroidism type II. J Clin Endocrinol Metab 1985;61:285-290. 147. Tsang RC, Venkataraman P, Ho M, Steichen JJ, WhitsettJ, Greer E The development of pseudohypoparathyroidism. Am J Dis Child 1984;138:654-658. 148. Attanasio R, Curcio T, Giusti M, Monachesi M, Nalin R, Giordano G. Pseudohypoparathyroidism. A case report with low immunoreactive parathyroid hormone and multiple endocrine dysfunctions. Minerva Endocrinol 1986;11:267-273. 149. Litvin Y, Rosler A, Bloom RA. Extensive cerebral calcification in hypoparathyroidism. Neuroradiology 1981 ;21:271-271. 150. Sachs C, Sjoberg HE, Ericson K. Basal ganglia calcifications on CT: Relation to hypoparathyroidism. Neurology 1982;32: 779-782. 151. Korn-Lubetzki I, Rubinger D, Siew E Visualization of basal ganglion calcification by cranial computed tomography in a patient with pseudohypoparathyroidism. Isr J Med Sci 1980;16: 40-41. 152. Pearson DWM, Durward WF, Fogelman I, Boyle IT, Beastall G. Pseudohypoparathyroidism presenting as severe Parkinsonism. Postgrad MedJ 1981;57:445-447. 153. Miano A, Casadel G, Biasini G. Cardiac failure in pseudohypoparathyroidism. Helv Paediatr Acta 1981;36:191-192. 154. Cavallo A, Meyer III WJ, Bodensteiner JB, Chesson AL. Spinal cord compression: An unusual manifestation of pseudohypoparathyroidism. A m J Dis Child 1980;134:706-707. 155. Breslau NA, Notman D, CanterburyJM, Moses AM. Studies on the attainment of normocalcemia in patients with pseudohypoparathyroidism. Am J Med 1980;68:856-860. 156. Mallette LE. Synthetic human parathyroid hormone 1-34 fragment for diagnostic testing. Ann Intern Med 1988;109:800-804. 157. McElduff A, Lissner D, Wilkinson M, Cornish C, Posen S. A 6hour human parathyroid hormone (1-34) infusion protocol: Studies in normal and hypoparathyroid subjects. CalcifTissue Int 1987;41:267-273. 158. Furlong TJ, Seshadri MS, Wilkinson MR, Cornish CJ, Luttrell B, Posen S. Clinical experiences with human parathyroid hormone 1-34. Aust N ZJMed 1986;16:794-798. 159. Mallette LE, Kirkland JL, Gagel RF, Law WM, Jr, Heath III H. Synthetic human parathyroid hormone-(1-34) for the study of pseudohypoparathyroidism. J Clin Endocrinol Metab 1988;67: 964-972.

PSEUDOHYPOPARATHYROIDISM 160. Rude RK, Oldham SB, Singer FR. Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. Clin Endocrino11976;5:209-224. 161. Slatopolsky E, Mercado A, Morrison A, Yates J, Klahr S. Inhibitory effects of hypomagnesemia on the renal action of parathyroid hormone. J Clin Invest 1976;58:1273-1279. 162. Beck N. Davis BB. Impaired renal response to parathyroid hormone in potassium depletion. AmJPhysio11975;228:179-183. 163. Beck N, Kim HE Kim KS. Effect of metabolic acidosis on renal action of parathyroid hormone. AmJPhysiol 1975;228:1483-1488. 164. Walton RJ, Bijvoet OLM. Nomogram for derivation of renal threshold phosphate concentration. Lancet 1975;309:310. 165. Sohn HE, Furukawa Y, Yumita S, Miura R, Unakami H, Yoshinaga K. Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3',5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in Ellsworth-Howard test. EndocrinolJpn 1984;31:33-40. 166. Stirling HE DarlingJA, Barr DG. Plasma cyclic AMP response to intravenous parathyroid hormone in pseudohypoparathyroidism. Acta Paediatr Scand 1991;80:333-338. 167. Miura R, Yumita S, Yoshinaga K, Furukawa Y. Response of plasma 1,25-dihydroxyvitamin D in the human PTH(1-34) infusion test: An improved index for the diagnosis of idiopathic hypoparathyroidism and pseudohypoparathyroidism. Calcif Tissue Int 1990;46:309-313. 168. Litvak J, Moldawer ME Forbes AP, Henneman PH. Hypocalcemic hypercalciuria during vitamin D and dihydrotachysterol therapy of hypoparathyroidism. J Clin Endocrinol Metab 1958;18:246-252. 169. Yamamoto M, Takuwa Y, Masuko S, Ogata E. Effects of endogenous and exogenous parathyroid hormone on tubular reabsorption of calcium in pseudohypoparathyroidism. J Clin Endocrinol Metab 1988;66:618-625. 170. Kolb FO, Steinbach HL. Pseudohypoparathyroidism with secondary hyperparathyroidism and osteitis fibrosa. J Clin Endocrinol Metab 1962;22:59-64. 171. Tollin SR, Perlmutter S, AloiaJE Serial changes in bone mineral density and bone turnover after correction of secondary hyperparathyroidism in a patient with pseudohypoparathyroidism type Ib. JBone Miner Res 2000;15:1412-1416. 172. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by

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intravenous administration of 1,25-dihydroxycholecalciferol in uremia patients. J Clin Invest 1984;74:2136-2143. 173. Shangraw RF. Factors to consider in the selection of a calcium supplement. Public Health Rep Supp11989;104:46-50. 174. Okano K, Furukawa Y, Morii H, Fujita T. Comparative efficacy of various vitamin D metabolites in the treatment of various types of hypoparathyroidism. J Clin Endocrinol Metab 1982;55:238-243. 175. Breslau NA. Pseudohypoparathyroidism: Current concepts. Am J Med Sci 1989;298:130-140. 176. Breslau NA, Zerwekh JE. Relationship of estrogen and pregnancy to calcium homeostasis in pseudohypoparathyroidism. J Clin Endocrinol Metab 1986;62:45-51. 177. Verbeelen D, Fuss M. Hypercalcemia induced by estrogen withdrawal in vitamin D-treated hypoparathyroidism. Br Med J 1979;1:522-523. 178. Mallette LE. Case report: Hypoparathyroidism with mensesassociated hypocalcemia. Am J Med Sci 1992;304:32-37. 179. Zerwekh JE. Breslau NA. Human placental production of lalpha,25-dihydroxyvitamin D3: Biochemical characterization and production in normal subjects and patients with pseudohypoparathyroidism. J Clin Endocrinol Metab 1986;62:192-196. 180. Caplan RH, Beguin EA. Hypercalcemia in a calcitriol-treated hypoparathyroid woman during lactation. Obstet Gynecol 1990; 76: 485-489. 181. Jan de Beur SM, Deng Z, Ding C, Levine MA. Amplification of the GC-rich exon 1 of GNAS1 and identification of three novel nonsense mutations in Albright's hereditary osteodystrophy. Endocr Soc 1998 (abstract). 182. Jan de Beur SM, Ding C, Levine MA. GNAS1 gene mutations. Unpublished work, 2000. 183. Takeda K, Yokoyama M, Hashimoto K, Hiromatsu Y, Yamanaka H, Shimizu T, Sasaki M. Mutations in exon 7 of the GTP-binding protein Gs alpha were not a common cause of pseudohypoparathyroidism with Albright's hereditary osteodystrophy in Japanese. EndocrJ 1997;44:621-625. 184. Warner DR, Gejman PV, Collins RM, Weinstein LS. A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism. Mol Endocrinol 1997;11:1718-1727. 185. Wu W-I, Schwindinger WF, Aparicio LF, Levine MA. Selective resistance to parathyroid hormone caused by a novel uncoupling mutation in the carboxyl terminus of CotS: A cause of pseudohypoparathyroidism type lb. J Biol Chem 2001 ;276:165-171.

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CI-IAeTV52 Treatment of Hypoparathyroidism

MARC K. DREZNER University of Wisconsin-Madison, Madison, Wisconsin 53792

INTRODUCTION

can demonstrate increased intracranial pressure, frequently in association with papilledema, subcapsular cataracts, and calcification of the basal ganglia (6). Cardiovascular manifestations of a decreased serum calcium level include arrhythmias, bradycardia, hypotension, and impaired cardiac contractility (7). The hypotension is typically unresponsive to the use of fluids and pressors until calcium is administered. Similarly, impaired cardiac contractility responds poorly to inotropic agents unless the hypocalcemia is first corrected. Serum calcium concentrations in affected individuals vary from a very low value, 5.5 mg/dl, to low-normal levels, depending on the degree of PTH deficiency. Serum phosphorus concentrations usually are increased to values as high as 7.5 mg/dl.

The serum calcium concentration is normally mainmined within a very narrow range by an exquisitely sensitive homeostatic system (1). In the absence of the calcium-maintaining effects of parathyroid hormone, the hypoparathyroid disorders cause hypocalcemia in the subset of patients with this biochemical abnormality (2). The clinical manifestations in affected patients at the time of initial presentation often suggest the presence of these diseases, confirmation of which is usually easily achieved by appropriate laboratory tests. For example, a history of recent neck surgery may suggest injury to the parathyroid glands and surgical hypoparathyroidism, and the coexistence of ancillary endocrine disorders may indicate an autoimmune process that accounts for the hypoparathyroidism (3,4). The hypocalcemia secondary to these parathyroid disorders varies in its clinical presentation from an asymptomatic biochemical abnormality to a severe life-threatening condition. Patients with hypoparathyroidism generally experience an insidious onset of symptoms, with a slow increase in episodes related to the neuromuscular and neurologic systems. Common complaints include paresthesias (particularly in the oral area), muscle spasm, carpopedal spasm, facial grimacing, and, in extreme cases, laryngeal spasm and convulsion. In association, irritability, depression, impaired memory, and psychosis may be seen (5). Often, the symptoms are precipitated or intensified by physical or emotional stress-induced hyperventilation and consequent alkalosis. With long-standing hypocalcemia, patients The Parathyroids, Second Edition

MANAGEMENT Successful treatment of the hypoparathyroid disorders should lead to an increase in the extracellular fluid ionized calcium concentration sufficiently to abolish hypocalcemic symptoms and to prevent the long-term complications of hypocalcemia. Therapy is primarily dictated by the presence of acute severe symptoms of hypocalcemia, which demands use of intravenous calcium salts to restore normocalcemia, or the existence of relatively asymptomatic hypocalcemia, which requires only titration of orally administered vitamin D (or its analogs) a n d / o r calcium preparations in sufficient amounts to gradually achieve and maintain normocalcemia without undue complications. 827

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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Emergency Treatment The signs and symptoms of acute hypocalcemia primarily result from enhanced neuromuscular irritability (8). Sensations of numbness and tingling involving the fingertips, toes, and circumoral region are early symptoms. Subsequently, muscle cramps most commonly involve the lower back, legs, and feet. With severe hypocalcemia, the muscle cramps may progress to spontaneous carpopedal spasm (tetany). The carpal spasm often progresses to the classic "main d'accoucheur" posture. The hyperventilation commonly associated with tetany induces hypocapnea and increased epinephrine secretion, which worsen the muscle spasm. If the muscle spasm progresses, laryngospasm a n d / o r bronchospasm can follow. Seizures may also occur. Prolongation of the QT interval on the electrocardiogram, and rarely congestive heart failure, may also occur secondary to the acute hypocalcemia Hypokalemia, hypermagnesemia, metabolic acidosis, uremia, or hypophosphatemia may mask the tetany. Precipitation of acute hypocalcemia usually manifests in untreated patients with idiopathic or surgical hypoparathyroidism secondary to an inciting event, such as corticosteriod-induced malabsorption of calcium, diuretic-induced hypercalciuria, estrogenmediated inhibition of bone resorption, pregnancy, menstruation, infection, or withdrawal of thyroid medication. Alternatively, at presentation patients with neonatal hypocalcemia (early or late) secondary to hypoparathyroidism, those with surgical hypoparathyroidism and the hungry bone syndrome, and those with profound and progressive idiopathic hypoparathyroidism may spontaneously develop acute hypocalcemia. Although the presence of symptoms primarily reflects the magnitude of the hypocalcemia, a rapid decrease of the serum calcium concentration a n d / o r the concomitant presence of alkalosis, which enhances the binding of ionized calcium to albumin, may also precipitate severe signs and symptoms.

Therapy Any patient with hypocalcemic tetany or a relatively asymptomatic patient with serum calcium of less than 7.5 m g / d l must be treated aggressively with intravenous calcium administration. Such therapy may be necessary to treat neonatal hypocalcemia due to hypoparathyroidism in premature infants and in term infants (9) or to manage severe hypocalcemia in adults with hypoparathyroidism (10).

Newborns Serum calcium concentrations of 5-6 m g / d l in premature infants and less than 7.5 m g / d l in full-term

infants with neonatal hypocalcemia and hypoparathyroidism demand emergency therapy. Treatment at higher levels of the serum calcium concentration is often necessary if specific signs of hypocalcemia are present. Affected patients with severe hypocalcemia often present with convulsions, heightening the need for acute management. Though neonatal hypocalcemia is due to a variety of disorders, including perinatal asphyxia, preeclampsia, maternal diabetes, and maternal hyperparathyroidism, inherited forms of hypoparathyroidism (e.g., DiGeorge syndrome, familial hypoparathyroidism) often present as either "early" (