Vitamin D, 3rd Edition (2-Volume Set)

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VITAMIN D THIRD EDITION

http://www.elsevierdirect.com/companion.jsp?ISBN=9780123819789

Vitamin D David Feldman, Editor-in-Chief, J. Wesley Pike and John S. Adams, Associate Editors

VITAMIN D THIRD EDITION VOLUME I Editor-in-Chief

DAVID FELDMAN Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Associate Editors

J. WESLEY PIKE Department of Biochemistry, University of Wisconsin, Madison, WI, USA

JOHN S. ADAMS UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 1997 Second edition 2005 Third edition 2011 Copyright Ó 2011, 2005, 1997 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+ 44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administrations, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-381978-9 Two Volume Set ISBN: 978-0-12-387035-3 Volume 1 ISBN: 978-0-12-387034-6 Volume 2 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by TNQ Books and Journals Printed and bound in United States of America 11 12 13 14

10 9 8 7

6 5 4 3

2 1

Contents

11. Target Genes of Vitamin D: Spatio-temporal Interaction of

Preface to the 3rd Edition ix Preface to the 2nd Edition xi Preface to the 1st Edition xiii Contributors xv Introduction xxi Abbreviations xxiii Relevant Lab Values in Adults and Children xxix

12.

13.

VOLUME I

14.

I 15.

CHEMISTRY, METABOLISM, CIRCULATION 1. Historical Overview of Vitamin D 3

Chromatin, VDR, and Response Elements 211 Carsten Carlberg Epigenetic Modifications in Vitamin D Receptor-mediated Transrepression 227 Alexander Kouzmenko, Fumiaki Ohtake, Ryoji Fujiki, Shigeaki Kato Vitamin D and Wnt/b-Catenin Signaling 235 Jose´ Manuel Gonza´lez-Sancho, Marıá Jesu´s Larriba, Alberto Munõz Vitamin D Response Element-binding Protein 251 Thomas S. Lisse, Hong Chen, Mark S. Nanes, Martin Hewison, John S. Adams Vitamin D Sterol/VDR Conformational Dynamics and Nongenomic Actions 271 Mathew T. Mizwicki, Anthony W. Norman

Hector F. Deluca

2. Photobiology of Vitamin D 13

III

Michael F. Holick

3. The Activating Enzymes of Vitamin D Metabolism

(25- and 1a-Hydroxylases) 23 Glenville Jones, David E. Prosser 4. CYP24A1: Structure, Function, and Physiological Role Rene´ St. Arnaud 5. The Vitamin D Binding Protein DBP 57 Roger Bouillon 6. Industrial Aspects of Vitamin D 73 Arnold Lippert Hirsch

MINERAL AND BONE HOMEOSTASIS 16. Genetic and Epigenetic Control of the Regulatory Machinery

43

17. 18.

II

19.

MECHANISMS OF ACTION 7. The Vitamin D Receptor: Biochemical, Molecular,

20.

Biological, and Genomic Era Investigations 97 J. Wesley Pike, Mark B. Meyer, Seong Min Lee 8. Nuclear Vitamin D Receptor: Natural Ligands, Molecular StructureeFunction, and Transcriptional Control of Vital Genes 137 Mark R. Haussler, G. Kerr Whitfield, Carol A. Haussler, Jui-Cheng Hsieh, Peter W. Jurutka 9. Structural Basis for Ligand Activity in VDR 171 Natacha Rochel, Dino Moras 10. Coregulators of VDR-mediated Gene Expression 193 Diane R. Dowd, Paul N. MacDonald

21. 22. 23.

v

for Skeletal Development and Bone Formation: Contributions of Vitamin D3 301 Jane B. Lian, Gary S. Stein, Martin Montecino, Janet L. Stein, Andre J. van Wijnen Vitamin D Regulation of Osteoblast Function 321 Renny T. Franceschi, Yan Li Osteoclasts 335 F. Patrick Ross Molecular Mechanisms for Regulation of Intestinal Calcium and Phosphate Absorption by Vitamin D 349 James C. Fleet, Ryan D. Schoch The Calbindins: Calbindin-D28K and Calbindin-D9K and the Epithelial Calcium Channels TRPV5 and TRPV6 363 Sylvia Christakos, Leila J. Mady, Puneet Dhawan Mineralization 381 Eve Donnelly, Adele L. Boskey Vitamin D Regulation of Type I Collagen Expression in Bone 403 Barbara E. Kream, Alexander C. Lichtler Target Genes: Bone Proteins 411 Gerald J. Atkins, David M. Findlay, Paul H. Anderson, Howard A. Morris

vi

CONTENTS

24. Vitamin D and the Calcium-Sensing Receptor 425 Edward M. Brown 25. Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in Osteoblast Differentiation and Morphology 457 William R. Thompson, Mary C. Farach-Carson

40. Vitamin D and the Renin-Angiotensin System 707 Yan Chun Li

41. Parathyroid Hormone, Parathyroid Hormone-Related Protein, 42. 43.

IV TARGETS 26. Vitamin D and the Kidney 471 27. 28. 29. 30. 31. 32.

33.

Peter Tebben, Rajiv Kumar Vitamin D and the Parathyroids 493 Justin Silver, Tally Naveh-Many Cartilage 507 Barbara D. Boyan, Maryam Doroudi, Zvi Schwartz Vitamin D and Oral Health 521 Ariane Berdal, Muriel Molla, Vianney Descroix The Role of Vitamin D and its Receptor in Skin and Hair Follicle Biology 533 Marie B. Demay Vitamin D and the Cardiovascular System 541 David G. Gardner, Songcang Chen, Denis J. Glenn, Wei Ni Vitamin D: A Neurosteroid Affecting Brain Development and Function; Implications for Neurological and Psychiatric Disorders 565 Darryl Eyles, Thomas Burne, John McGrath Contributions of Genetically Modified Mouse Models to Understanding the Physiology and Pathophysiology of the 25Hydroxyvitamin D-1-Alpha Hydroxylase Enzyme (1a(OH) ase) and the Vitamin D Receptor (VDR) 583 Geoffrey N. Hendy, Richard Kremer, David Goltzman

44. 45.

and Calcitonin 725 Elizabeth Holt, John J. Wysolmerski FGF23/Klotho New Regulators of Vitamin D Metabolism 747 Valentin David, L. Darryl Quarles The Role of the Vitamin D Receptor in Bile Acid Homeostasis 763 Daniel R. Schmidt, Steven A. Kliewer, David J. Mangelsdorf Vitamin D and Fat 769 Francisco J.A. de Paula, Clifford J. Rosen Extrarenal 1a-Hydroxylase 777 Martin Hewison, John S. Adams

VI DIAGNOSIS AND MANAGEMENT 46. Approach to the Patient with Metabolic Bone Disease 807 Michael P. Whyte

47. Detection of Vitamin D and Its Major Metabolites 823 Bruce W. Hollis

48. Bone Histomorphometry 845 Linda Skingle, Juliet Compston

49. Radiology of Rickets and Osteomalacia 861 Judith E. Adams

50. High-Resolution Imaging Techniques for Bone Quality Assessment 891 Andrew J. Burghardt, Roland Krug, Sharmila Majumdar 51. The Role of Vitamin D in Orthopedic Surgery 927 Aasis Unnanuntana, Brian J. Rebolledo, Joseph M. Lane

VII V HUMAN PHYSIOLOGY 34. Vitamin D: Role in the Calcium and Phosphorus 35. 36. 37. 38. 39.

Economies 607 Robert P. Heaney Fetus, Neonate and Infant 625 Christopher S. Kovacs Vitamin D Deficiency and Calcium Absorption during Childhood 647 Steven A. Abrams Adolescence and Acquisition of Peak Bone Mass 657 Connie Weaver, Richard Lewis, Emma Laing Vitamin D Metabolism in Pregnancy and Lactation 679 Natalie W. Thiex, Heidi J. Kalkwarf, Bonny L. Specker Vitamin D: Relevance in Reproductive Biology and Pathophysiological Implications in Reproductive Dysfunction 695 Lubna Pal, Hugh S. Taylor

NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY 52. Worldwide Vitamin D Status 947 Paul Lips, Natasja van Schoor

53. Sunlight, Vitamin D and Prostate Cancer Epidemiology 965 Gary G. Schwartz

54. Nutrition and Lifestyle Effects on Vitamin D Status 979 Susan J. Whiting, Mona S. Calvo

55. Bone Loss, Vitamin D and Bariatric Surgery: Nutrition and Obesity 1009 Lenore Arab, Ian Yip 56. Genetics of the Vitamin D Endocrine System 1025 Andre´ G. Uitterlinden 57. The Pharmacology of Vitamin D 1041 Reinhold Vieth 58. How to Define Optimal Vitamin D Status 1067 Roger Bouillon

Volume I Color Plate Section

vii

CONTENTS

76. Analogs of Calcitriol 1461

VOLUME II

VIII

77.

DISORDERS

78.

59. The Hypocalcemic Disorders: Differential Diagnosis and 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.

Therapeutic Use of Vitamin D 1091 Thomas O. Carpenter, Karl L. Insogna Vitamin D Deficiency and Nutritional Rickets in Children 1107 John M. Pettifor Vitamin D and Osteoporosis 1129 Peter R. Ebeling, John A. Eisman Relevance of Vitamin D Deficiency in Adult Fracture and Fall Prevention 1145 Heike Bischoff-Ferrari, Bess Dawson-Hughes Clinical Disorders of Phosphate Homeostasis 1155 Karen E. Hansen, Marc K. Drezner Pseudo-vitamin D Deficiency 1187 Francis H. Glorieux, Thomas Edouard, Rene´ St-Arnaud Hereditary 1,25-Dihydroxyvitamin-D-Resistant Rickets 1197 Peter J. Malloy, Dov Tiosano, David Feldman Glucocorticoids and Vitamin D 1233 Philip Sambrook Drug and Hormone Effects on Vitamin D Metabolism Barrie M. Weinstein, Sol Epstein Vitamin D and Organ Transplantation 1291 Emily M. Stein, Elizabeth Shane Vitamin D and Bone Mineral Metabolism in Hepatogastrointestinal Diseases 1299 Daniel D. Bikle Vitamin D and Renal Disease 1325 Adriana S. Dusso, Eduardo Slatopolsky Idiopathic Hypercalciuria and Nephrolithiasis 1359 Murray J. Favus, Fredric L. Coe Hypercalcemia Due to Vitamin D Toxicity 1381 Natalie E. Cusano, Susan Thys-Jacobs, John P. Bilezikian Vitamin D: Cardiovascular Effects and Vascular Calcification 1403 Dwight A. Towler

79.

80. 81.

Lieve Verlinden, Guy Eelen, Roger Bouillon, Maurits Vandewalle, Pierre De Clercq, Annemieke Verstuyf Analogs for the Treatment of Osteoporosis 1489 Noboru Kubodera, Fumiaki Takahashi Non-secosteroidal Ligands and Modulators 1497 Keith R. Stayrook, Matthew W. Carson, Yanfei L. Ma, Jeffrey A. Dodge The Bile Acid Derivatives Lithocholic Acid Acetate and Lithocholic Acid Propionate are Functionally Selective Vitamin D Receptor Ligands 1509 Makoto Makishima, Sachiko Yamada CYP24A1 Regulation in Health and Disease 1525 Martin Petkovich, Christian Helvig, Tina Epps Calcitriol and Analogs in the Treatment of Chronic Kidney Disease 1555 Ishir Bhan, Ravi Thadhani

X CANCER 82. The Epidemiology of Vitamin D and Cancer Risk 1569 Edward Giovannucci

83. Vitamin D: Cancer and Differentiation 1591 1245

84. 85. 86. 87. 88. 89. 90.

Johannes P.T.M. van Leeuwen, Marjolein van Driel, David Feldman, Alberto Munõz Vitamin D Effects on Differentiation and Cell Cycle 1625 George P. Studzinski, Elzbieta Gocek, Michael Danilenko Vitamin D Actions in Mammary Gland and Breast Cancer 1657 JoEllen Welsh Vitamin D and Prostate Cancer 1675 Aruna V. Krishnan, David Feldman The Vitamin D System and Colorectal Cancer Prevention 1711 Heide S. Cross Hematological Malignancy 1731 Ryoko Okamoto, H. Phillip Koeffler Vitamin D and Skin Cancer 1751 Jean Y. Tang, Ervin H. Epstein, Jr. The Anti-tumor Effects of Vitamin D in Other Cancers 1763 Donald L. Trump, Candace S. Johnson

IX ANALOGS 74. Alterations in 1,25-Dihydroxyvitamin D3

Structure that Produce Profound Changes in in Vivo Activity 1429 Hector F. DeLuca, Lori A. Plum 75. Mechanisms for the Selective Actions of Vitamin D Analogs 1437 Alex J. Brown

XI IMMUNITY, INFLAMMATION, AND DISEASE 91. Vitamin D and Innate Immunity 1777 John H. White

92. Control of Adaptive Immunity by Vitamin D Receptor Agonists 1789 Luciano Adorini

viii

CONTENTS

93. The Role of Vitamin D in Innate Immunity: Antimicrobial 94. 95. 96. 97.

Activity, Oxidative Stress and Barrier Function 1811 Philip T. Liu Vitamin D and Diabetes 1825 Conny Gysemans, Hannelie Korf, Chantal Mathieu Vitamin D and Multiple Sclerosis 1843 Colleen E. Hayes, Faye E. Nashold, Christopher G. Mayne, Justin A. Spanier, Corwin D. Nelson Vitamin D and Inflammatory Bowel Disease 1879 Danny Bruce, Margherita T. Cantorna Psoriasis and Other Skin Diseases 1891 Jo¨rg Reichrath, Michael F. Holick

99. Vitamin D Receptor Agonists in the Treatment of Benign 100. 101. 102. 103. 104.

XII THERAPEUTIC APPLICATIONS AND NEW ADVANCES 98. The Role of Vitamin D in Type 2 Diabetes and Hypertension 1907 Anastassios G. Pittas, Bess Dawson-Hughes

105.

Prostatic Hyperplasia 1931 Annamaria Morelli, Mario Maggi, Luciano Adorini Sunlight Protection by Vitamin D Compounds 1943 Rebecca S. Mason, Katie M. Dixon, Vanessa B. Sequeira, Clare Gordon-Thomson The Role of Vitamin D in Osteoarthritis and Rheumatic Disease 1955 M. Kyla Shea, Timothy E. McAlindon Vitamin D and Cardiovascular Disease 1973 Harald Sourij, Harald Dobnig Vitamin D, Childhood Wheezing, Asthma, and Chronic Obstructive Pulmonary Disease 1999 Carlos A. Camargo Jr., Adit A. Ginde, Jonathan M. Mansbach Vitamin D and Skeletal Muscle Function 2023 Lisa Ceglia, Robert U. Simpson The VITamin D and OmegA-3 TriaL (VITAL): Rationale and Design of a Large-Scale Randomized Controlled Trial 2043 Olivia I. Okereke, JoAnn E. Manson

Index 2057 Volume II Color Plate Section

Preface to the 3rd Edition The 3rd edition of Vitamin D was written at a time of great interest, exuberant hype, and even commotion in the public and lay press about vitamin D as a potential drug to treat and/or prevent multiple important and common diseases. Recent noteworthy events impacting the vitamin D field were the launching of the VITAL trial to discover whether vitamin D supplementation can reduce the risk of severe and life-threatening disease and the Institute of Medicine (IOM) report setting new dietary reference intakes (DRIs) for calcium and vitamin D. The IOM report expressed doubt on how well current data supported the beneficial actions of vitamin D on nonskeletal sites and called for more research to prove the hypothesis. This volume marshals the currently available data on basic mechanisms, normal physiology, and effects on disease and lays out for the reader up-todate and expert information on the role of vitamin D in health and many disorders. These and other current trends in vitamin D research are extensively covered in this new edition. The editors have continued our basic plan to constantly renew and remodel this book with each successive edition. To this end, we have added a new editor, Dr. John Adams, who has broad skill and knowledge in many areas of vitamin D research at both the basic science and clinical levels. John replaces Francis Glorieux who has undertaken to edit a separate book on pediatric bone disease. We thank Francis for his years of exemplary service to this book and wish him well in his new endeavors. John adds new energy and expertise to the editorial team. The 3rd edition has 105 chapters, making the book approximately the same size as the 2nd edition. However, the editors have worked very hard to revise and update this edition with new material and the presentation of fresh and different perspectives from respected authors. Some chapters covered in the 2nd edition have not been continued in this edition because relatively little new research was added in those areas. We thank the authors who are no longer contributing to this edition for their previous efforts. They may well be asked to write in the next edition as we continue our strategy of rotating authors. All chapters have been revised and updated and new references added. In our revitalization of the material in the book we

have added 32 new chapters to cover previously uncovered areas of research. In addition, we have changed the authorship of 20 additional chapters that are now written by different authors who have been charged with revising and updating previous chapters. These extensive modifications, with major updates and expansion of the content and the addition of totally new material in half of the chapters, has resulted in a substantially reorganized, modified, and modernized book compared to the 2nd edition. Finally, the expanded internet availability of the text and the figures will make access to the material easier and more flexible. Among the areas given new emphasis are nutrition, additional diseases that may be affected by vitamin D, and newly recognized biological pathways that regulate or are regulated by vitamin D. As we appreciate the full scope of vitamin D action, it has become clearer that the vitamin D endocrine system affects most if not all tissues in the body. We have tried to keep up with these advances in the state of knowledge about vitamin D by increasing our coverage of these newly recognized areas. We have enlisted the leading investigators in each area to provide truly expert opinion about each field. We would like to thank the excellent team at Elsevier/Academic Press for their outstanding support of our efforts to produce this new edition. We especially thank Mara Conner and Megan Wickline for their indispensable contributions to make this edition possible. We also want to extend our thanks and appreciation to the many authors who contributed to this volume. Without their hard work there of course would be no new edition. We therefore wish to express our gratitude for their willingness to offer their time and knowledge to make this book a success. Finally, we hope that this book will provide for our readers the authoritative information that they seek about the significance and importance of vitamin D in health and disease and serve as the means to keep their knowledge current about the continuing growth of the field of vitamin D biology. David Feldman J. Wesley Pike John S. Adams

ix

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Preface to the 2nd Edition Those interested in the vitamin D field will not be surprised that this second edition is considerably larger than the first edition. A great deal of progress has been made since the first edition was published in 1997. However, our goal in planning this updated version remains the same. We have endeavored to provide investigators, clinicians, and students with a comprehensive, definitive, and up-to-date compendium of the diverse scientific and clinical aspects of vitamin D, each area covered by experts in the field. Our hope for the second edition is that this book will continue to serve as both a resource for current researchers, as well as a guide to assist those in related disciplines to enter the realm of vitamin D research. We hope that this book will illuminate the vitamin D field and help investigators identify areas where new research is needed as well as educate them about what is currently known. We believe that the first edition succeeded in stimulating interactions between researchers and clinicians from different disciplines and that it facilitated collaborations. As we move from basic science and physiology to the use of vitamin D and its analogs as pharmacological agents to treat various diseases, the need for crosscollaborations between researchers and clinicians from different disciplines will increase. We hope that this new volume will continue to be a valuable resource that plays a role in this advancement and stimulates and facilitates these interactions. Enormous progress in the study of vitamin D has been made in the approximately eight years since the first edition was written and we hope that this book has contributed in some way to this progress. The first edition proved to be highly valuable to its readers and the chapters have been cited frequently as authoritative reviews of the field. However, it became clear to us that the time was ripe to organize a second edition. Building on the original, we hope this second edition will

incorporate all of the progress made in the field since the first edition was published so that our objective of an up-to-date compendium containing everything you wanted to know about vitamin D will continue. The second edition is essentially a new and reinvigorated book. We have changed the symbol on the cover to reflect its updated content and the field’s continued evolution into the molecular world. This new edition now includes 104 chapters. In order to cover the growth of new information on vitamin D, we have had to publish this new edition in two volumes. In addition, the book has undergone some major remodeling. There are 33 completely new chapters and 18 other chapters have had major changes in authorship and are totally rewritten. While approximately half of the chapters have some of the same authors, all have had major updates and many have new co-authors with new perspectives. We have endeavored to attract the leading investigators in each field to author the chapter covering their area. We are especially pleased with the roster of authors who have written for the second edition. They really are the leaders in their respective fields. We wish to express our thanks to Tari Paschall, Judy Meyer, and Sarah Hajduk as well as the rest of the Elsevier/Academic Press staff for their expertise and indispensable contribution to bringing this revised edition to fruition. Most of all, we thank the authors for their contributions. We hope that our readers will find this updated volume useful and informative and that it will contribute to the burgeoning growth of the vitamin D field. David Feldman J. Wesley Pike Francis H. Glorieux

xi


Preface to the 1st Edition Our reasons for deciding to publish an entire book devoted to vitamin D can be found in the rapid and extensive advances currently being made in this important field of research. Enormous progress in investigating many aspects of vitamin D, from basic science to clinical medicine, has been made in recent years. The ever-widening scope of vitamin D research has created new areas of inquiry so that even workers immersed in the field are not fully aware of the entire spectrum of current investigation. Our goal in planning this book was to bring the diverse scientific and clinical fields together in one definitive and up-to-date volume. It is our hope that this compendium on vitamin D will serve as both a resource for current researchers and a guide to stimulate and assist those in related disciplines to enter this field of research. In addition, we hope to provide clinicians and students with a comprehensive source of information for the varied and extensive material related to vitamin D. The explosion of information in the vitamin D sphere has led to new insights into many different areas, and in our treatment of each subject in this book we have tried to emphasize the recent advances as well as the established concepts. The classic view of vitamin D action, as a hormone limited to calcium metabolism and bone homeostasis, has undergone extensive revision and amplification in the past few years. We now know that the vitamin D receptor (VDR) is present in most tissues of the body and that vitamin D actions, in addition to the classic ones, include important effects on an extensive array of other target organs. To cover this large number of subjects, we have organized the book in the following manner: Section I, the enzymes involved in vitamin D metabolism and the activities of the various metabolites; Section II, the mechanism of action of vitamin D, including rapid, nongenomic actions and the role of the VDR in health and disease; Section III, the effects of vitamin D and its metabolites on the various elements that constitute bone and the expanded understanding of vitamin D actions in multiple target organs, both classic and nonclassic; Section IV, the role of vitamin D in the physiology and regulation of calcium and phosphate metabolism and the multiplicity of hormonal, environmental, and other factors influencing vitamin D metabolism and action; and Sections V and VI, the role of

vitamin D in the etiology and treatment of rickets, osteomalacia, and osteoporosis and the pathophysiological basis, diagnosis, and management of numerous clinical disorders involving vitamin D. The recent recognition of an expanded scope of vitamin D action and the new investigational approaches it has generated were part of the impetus for developing this volume on vitamin D. It has become clear that in addition to the classic vitamin D actions, a new spectrum of vitamin D activities that include important effects on cellular proliferation, differentiation, and the immune system has been identified. This new information has greatly expanded our understanding of the breadth of vitamin D action and has opened for investigation a large number of new avenues of research that are covered in Sections VII and VIII of this volume. Furthermore, these recently recognized nonclassic actions have led to a consideration of the potential application of vitamin D therapy to a range of diseases not previously envisioned. This therapeutic potential has spawned the search for vitamin D analogs that might have a more favorable therapeutic profile, one that is less active in causing hypercalcemia and hypercalciuria while more active in a desired application such as inducing antiproliferation, prodifferentiation, or immunosuppresion. Since 1a,25dihydroxyvitamin D [1,25(OH)2D] and its analogs are all presumed to act via a single VDR, a few years ago most of us probably would have thought that it was impossible to achieve a separation of these activities. Yet today, many analogs that exhibit different profiles of activity relative to 1,25(OH)2D have already been produced and extensively studied. The development of analogs with an improved therapeutic index has opened another large and complex area of vitamin D research. This work currently encompasses three domains: (1) the design and synthesis of vitamin D analogs exhibiting a separation of actions with less hypercalcemic and more antiproliferative or immunosuppressive activity, (2) the interesting biological question of the mechanism(s) by which these analogs achieve their differential activity, and (3) the investigation of the potential therapeutic applications of these analogs to treat various disease states. These new therapeutic applications, from psoriasis to cancer, from immunosuppression to neurodegenerative diseases, have drawn into the field an expanded

xiii

xiv

PREFACE TO THE 1ST EDITION

population of scientists and physicians interested in vitamin D. Our goal in editing this book was to create a comprehensive resource on vitamin D that would be of use to a mix of researchers in different disciplines. To achieve this goal, we sought authors who had contributed greatly to their respective fields of vitamin D research. The book has a large number of chapters to accommodate many contributors and provide expertise in multiple areas. Introductory chapters in each section of the book are designed to furnish an overview of that area of vitamin D research, with other chapters devoted to a narrowly focused subject. Adjacent and closely related subjects are often covered in separate chapters by other authors. While this intensive style may occasionally create some redundancy, it has the advantage of allowing the reader to view the diverse perspectives

of the different authors working in overlapping fields. In this regard, we have endeavored to provide many cross-references to guide the reader to related information in different chapters. We express our thanks to Jasna Markovac (Editor-inChief), for encouraging us to develop this book and guiding us through the process; to Tari Paschall (Acquisitions Editor) and the Academic Press staff for their diligence, expertise, and patience in helping us complete this work. Most of all, we thank the authors for their contributions that have made this book possible. David Feldman Francis H. Glorieux J. Wesley Pike

Contributors Nutrition

Mona S. Calvo US Food and Drug Administration, Laurel, MD, USA (979)

John S. Adams UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777)

Carlos A. Camargo Jr. Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (1999)

Judith E. Adams Manchester Royal Infirmary, Manchester, UK and Imaging Science and Biomedical Engineering, The University, Manchester, UK (861)

Margherita Cantorna The Pennsylvania State University, University Park, PA, USA (1879)

Steven A. Abrams USDA/ARS Children’s Research Center, Houston, TX, USA (647)

Luciano Adorini (1789, 1931)

Carsten Carlberg University of Luxembourg, Luxembourg and University of Eastern Finland, Kuopio, Finland (211)

Intercept Pharmaceuticals, Perugia, Italy

Thomas O. Carpenter Yale University School of Medicine, New Haven, CT, USA (1091)

Paul H. Anderson SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)

Matthew W. Carson Lilly Indianapolis, IN, USA (1497)

Lenore Arab David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)

Lisa Ceglia

Research

Laboratories,

Tufts University, Boston, MA, USA (2023)

Hong Chen Emory University School of Medicine, Atlanta, GA, USA (251)

Gerald J. Atkins University of Adelaide, Adelaide, South Australia, Australia (411)

Songcang Chen University of California at San Francisco, San Francisco, CA, USA (541)

Ariane Berdal Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521)

Sylvia Christakos University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)

Ishir Bhan Massachusetts General Hospital, Boston, MA, USA (1555)

Fredric L. Coe The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359)

Daniel Bikle Veterans Affairs Medical Center and University of California, San Francisco, CA, USA (1299)

Juliet Compston Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK (845)

John P. Bilezikian Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)

Heide S. Cross Retired, Medical University of Vienna, Austria (1711)

Heike Bischoff-Ferrari University of Zurich, Switzerland and University Hospital Zurich, Switzerland (1145)

Natalie E. Cusano Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)

Adele L. Boskey Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381)

Bess Dawson-Hughes USA (1145, 1907)

Roger Bouillon Laboratory of Experimental Medicine and Endocrinology, K.U. Leuven, Leuven, Belgium (57, 1067, 1461)

Tufts

University,

Boston,

MA,

Pierre De Clercq Universiteit Gent, Vakgroep Organische Chemie, Gent, Belgium (1461)

voor

Hector DeLuca University of Wisconsin-Madison, WI, USA (1429)

Barbara D. Boyan Georgia Institute of Technology, Atlanta, GA, USA (507)

Michael Danilenko Ben-Gurion University of the Negev, Beer-Sheva, Israel (1625)

Alex J. Brown Washington University School of Medicine, St. Louis, MO, USA (1437)

Valentin David University of Tennessee Health Science Center, Memphis, TN, USA (747)

Edward M. Brown Brigham and Women’s Hospital, Boston, MA, USA (425)

Hector F. Deluca University Madison, WI, USA (3)

Danny Bruce The Pennsylvania State University, University Park, PA, USA (1879)

of

Wisconsin-Madison,

San

Marie B. Demay Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (533)

Thomas Burne The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565)

Francisco J.A. de Paula Maine Medical Center Research Institute and Department of Internal Medicine, School of Medicine of Ribeiraõ Preto, USP, Ribeiraõ Preto, SP, Brazil (769)

Andrew J. Burghardt University Francisco, CA, USA (891)

of

California,

xv

xvi

CONTRIBUTORS

Vianney Descroix Universities Paris 5, Paris 6 and Paris 7, Paris, France and Pitie´-Salpeˆtrie`re Hospital, Assistance Publique-Hoˆpitaux de Paris, Paris France (521) Puneet Dhawan University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)

David G. Gardner University of California at San Francisco, San Francisco, CA, USA (541) Adit A. Ginde University of Colorado Denver School of Medicine, Aurora, CO, USA (1999)

Katie M. Dixon University of Sydney, NSW, Australia (1943)

Edward Giovannucci Harvard School of Public Health, Boston, MA, USA and Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (1569)

Harald Dobnig Medical University of Graz, Graz, Austria (1973)

Denis J. Glenn University of California at San Francisco, San Francisco, CA, USA (541)

Jeffrey A. Dodge Lilly Research Laboratories, Indianapolis, IN, USA (1497) Eve Donnelly Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381) Maryam Doroudi Georgia Institute of Technology, Atlanta, GA, USA (507) Diane R. Dowd Case Western Cleveland, OH, USA (193)

Reserve

University,

Marc K. Drezner William H. Middleton Veterans Administration Medical Center, Madison, WI, USA (1155) Adriana S. Dusso Experimental Nephrology Laboratory, Lleida, Spain (1325) Peter R. Ebeling Australia (1129)

University

of

Melbourne,

Victoria,

Thomas Edouard Shriners Hospital for Children, Montreal, Quebec, Canada (1187) Guy Eelen Katholieke Belgium (1461)

Universiteit

Leuven,

Leuven,

John A. Eisman Garvan Institute of Medical Research, Sydney, Australia (1129) Tina Epps (1525)

Cytochroma Inc., Markham, Ontario, Canada

Ervin H. Epstein Jr. Children’s Hospital Oakland Research Institute, Oakland, CA, USA (1751) Sol Epstein Mount Sinai School of Medicine, New York, NY, USA (1245) Darryl Eyles The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mary C. Farach-Carson USA (457)

Rice University, Houston, TX,

Murray J. Favus The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359) David Feldman Stanford University School of Medicine, Stanford, CA, USA (1197, 1591, 1675) David M. Findlay University of Adelaide, Adelaide, South Australia, Australia (411) James C. Fleet USA (349)

Purdue University, West Lafayette, IN,

Renny T. Franceschi MI, USA (321) Ryoji Fujiki

University of Michigan, Ann Arbor,

University of Tokyo, Tokyo, Japan

(227)

Francis H. Glorieux Shriners Hospital Montreal, Quebec, Canada (1187)

for

Children,

Elzbieta Gocek University of Wroclaw, Wroclaw, Poland (1625) David Goltzman McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Jose´ Manuel Gonza´lez-Sancho Universidad Autońoma de Madrid, Madrid, Spain (235) Clare Gordon-Thomson Australia (1943)

University

of

Sydney,

NSW,

Conny Gysemans Katholieke Universiteit Leuven, Leuven, Belgium (1825) Karen E. Hansen USA (1155)

University of Wisconsin, Madison, WI,

Carol A. Haussler University of Arizona, Phoenix, AZ, USA (137) Mark R. Haussler USA (137)

University of Arizona, Phoenix, AZ,

Colleen E. Hayes University Madison, WI, USA (1843) Robert P. Heaney (607)

of

Wisconsin-Madison,

Creighton University, Omaha, NE, USA

Christian Helvig Cytochroma Inc., Markham, Ontario, Canada (1525) Geoffrey N. Hendy McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Martin Hewison UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777) Arnold Lippert Hirsch AGD Nutrition LLC, Lewisville, Texas, USA (73) Michael F. Holick Boston Medical Center and Boston University School of Medicine, Boston, MA, USA (13, 1891) Bruce W. Hollis Medical University of South Carolina, Charleston, SC, USA (823) Elizabeth Holt Yale University School of Medicine, New Haven, CT, USA (725) Jui-Cheng Hsieh USA (137)


Karl L. Insogna Yale University School of Medicine, New Haven, CT, USA (1091)

xvii

CONTRIBUTORS

Candace Johnson Roswell Park Cancer Institute, Buffalo, NY, USA (1763)

Paul Lips VU University Medical Center, Amsterdam, The Netherlands (947)

Glenville Jones Queen’s University, Kingston, Ontario, Canada (23)

Philip T. Liu University of California at Los Angeles, CA, USA (1811)

Peter W. Jurutka Arizona State University at the West Campus, Glendale, AZ, USA (137)

Thomas S. Lisse UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251)

Heidi J. Kalkwarf Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (679)

Yanfei L. Ma Lilly Research Laboratories, Indianapolis, IN, USA (1497)

Shigeaki Kato

Paul N. MacDonald Case Western Reserve University, Cleveland, OH, USA (193)


(227)

Steven A. Kliewer University of Texas Southwestern Medical Center, Dallas, TX, USA (763) H. Phillip Koeffler Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA and National University of Singapore, Singapore (1731) Hannelie Korf Katholieke Universiteit Leuven, Leuven, Belgium (1825) Alexander Kouzmenko University of Tokyo, Tokyo, Japan and Alfaisal University, Riyadh, Kingdom of Saudi Arabia (227) Christopher S. Kovacs Memorial University of Newfoundland, Health Sciences Centre, St. John’s, Newfoundland, Canada (625) Barbara E. Kream University of Connecticut Health Center, Farmington, CT, USA (403) Richard Kremer McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Aruna V. Krishnan Stanford University School of Medicine, Stanford, CA, USA (1675) Roland Krug University of California, San Francisco, CA, USA (891) Noboru Kubodera Chugai Pharmaceutical Co., Ltd, Tokyo, Japan, present address: International Institute of Active Vitamin D Analogs (1489) Rajiv Kumar Mayo Clinic and Foundation, Rochester, MN, USA (471) Emma M. Laing University of Georgia, Athens, GA, USA (657) Joseph M. Lane USA (927)

Hospital for Special Surgery, New York, NY,

Marıá Jesu´s Larriba Cientificas (235)

Consejo Superior de Investigacio nes

Seong Min Lee University of Wisconsin-Madison, Madison, WI, USA (97) Richard D. Lewis University of Georgia, Athens, GA, USA (657) Yan Li University of Michigan, Ann Arbor, MI, USA (321) Yan Chun Li (707)

The University of Chicago, Chicago, IL, USA

Jane B. Lian University of Massachusetts Medical School, Worcester, MA, USA (301) Alexander C. Lichtler University of Connecticut Health Center, Farmington, CT, USA (403)

Leila Mady University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363) Mario Maggi University of Florence, Florence, Italy (1931) Sharmila Majumdar University of California, San Francisco, CA, USA (891) Makoto Makishima Nihon University School of Medicine, Tokyo, Japan (1509) Peter J. Malloy Stanford University School of Medicine, Stanford, CA, USA (1197) David J. Mangelsdorf University of Texas Southwestern Medical Center, Dallas, TX, USA (763) Jonathan M. Mansbach Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA (1999) JoAnn E. Manson Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043) Rebecca S. Mason University of Sydney, NSW, Australia (1943) Chantal Mathieu Katholieke Universiteit Leuven, Leuven, Belgium (1825) Christopher G. Mayne Medical College of Wisconsin, Milwaukee, WI, USA (1843) Timothy M. McAlindon Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts (1955) John McGrath The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mark B. Meyer University of Wisconsin-Madison, Madison, WI, USA (97) Mathew T. Mizwicki CA, USA (271)

University of California, Riverside,

Muriel Molla Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521) Martin Montecino Chile (301)

Universidad Andres Bello, Santiago,

Dino Moras Universite´ de Strasbourg, 67404 Illkirch, France (171) Annamaria Morelli University of Florence, Florence, Italy (1931) Howard A. Morris SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)

xviii

CONTRIBUTORS

Alberto Munõz Consejo Superior Cientificas, Madrid, Spain (1591)

Investigacio nes

Gary G. Schwartz Wake Forest University School of Medicine, Winston-Salem, NC, USA (965)

Mark S. Nanes Emory University School of Medicine, Atlanta, GA, USA (251)

Zvi Schwartz Georgia Institute of Technology, Atlanta, GA, USA (507)

Faye E. Nashold University Madison, WI, USA (1843)

Vanessa Sequeira University of Sydney, NSW, Australia (1943)

of

de

Wisconsin-Madison,

Tally Naveh-Many Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)

Elizabeth Shane Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)

Wei Ni University of California at San Francisco, San Francisco, CA, USA (541)

M. Kyla Shea Wake Forest University School of Medicine, Winston-Salem, NC, USA (1955)

Corwin D. Nelson University Madison, WI, USA (1843)

Wisconsin-Madison,

Justin Silver Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)

University of California, Riverside,

Robert U. Simpson University of Michigan Medical School, Ann Arbor, MI, USA (2203)

Anthony W. Norman CA, USA (271) Fumiaki Ohtake

of


(227)

Ryoko Okamoto Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA (1731)

Linda Skingle Cambridge University Hospitals Foundation Trust, Cambridge, UK (845)

NHS

Eduardo Slatopolsky Washington University School of Medicine, St. Louis, MO, USA (1325)

Olivia I. Okereke Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043)

Harald Sourij (1973)

Medical University of Graz, Graz, Austria

Lubna Pal Yale University School of Medicine, New Haven, CT, USA (695)

Justin A. Spanier University Madison, WI, USA (1843)

Martin Petkovich Cytochroma Inc., Markham, Ontario, Canada and Queen’s University, Kingston, Ontario, Canada (1525)

Bonny L. Specker South Dakota State University, Brookings, SD, USA (679)

of

Wisconsin-Madison,

John M. Pettifor University of the Witwatersrand and Chris Hani Baragwanath Hospital, South Africa (1107)

Rene´ St-Arnaud Shriners Hospital for Children, Montreal, Quebec, Canada and McGill University, Montreal, Quebec Canada (43, 1187)

J. Wesley Pike University of Wisconsin-Madison, Madison, WI, USA (97)

Keith R. Stayrook Indiana University School of Medicine, Indianapolis, IN, USA (1497)

Anastassios G. Pittas Tufts Medical Center, Boston, MA, USA (1907)

Emily M. Stein Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)

Lori A. Plum (1429)

Gary S. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)

University of Wisconsin-Madison, WI, USA Queen’s University, Kingston, Ontario,

Janet L. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)

L. Darryl Quarles University of Tennessee Health Science Center, Memphis, TN, USA (747)

George P. Studzinski UMD-New Jersey Medical School, Newark, NJ, USA (1625)

Brian J. Rebolledo Weill Cornell Medical College, New York, NY, USA (927)

Fumiaki Takahashi Japan (1489)

Jo¨rg Reichrath Universita¨tsklinikum Homburg, Germany (1891)

Jean Y. Tang Stanford University School of Medicine, Stanford, CA, USA (1751)

David E. Prosser Canada (23)

Natacha Rochel (171)

des

Saarlandes,

Universite´ de Strasbourg, Illkirch, France

Chugai Pharmaceutical Co., Ltd, Tokyo,

Hugh S. Taylor Yale University School of Medicine, New Haven, CT, USA (695)

Clifford J. Rosen School of Medicine of Ribeiraõ Preto, USP, Ribeiraõ Preto, SP, Brazil (769)

Peter Tebben Mayo Clinic and Foundation, Rochester, MN, USA (471)

F. Patrick Ross Washington University School of Medicine, St. Louis, MO, USA (335)

Ravi Thadhani Massachusetts General Hospital, Boston, MA, USA (1555)

Philip Sambrook University of Sydney, Sydney, NSW, Australia (1233)

Natalie W. Thiex South Dakota State University, Brookings, SD, USA (679)

Daniel R. Schmidt University of Texas Southwestern Medical Center, Dallas, TX, USA (763)

William R. Thompson USA (457)

Ryan D. Schoch USA (349)

Susan Thys-Jacobs Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)

Purdue University, West Lafayette, IN,

University of Delaware, Newark, DE,

xix

CONTRIBUTORS

Dov Tiosano Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel (1197) Dwight A. Towler Washington University in St. Louis, St. Louis, MO, USA (1403) Donald Trump Roswell Park Cancer Institute, Buffalo, NY, USA (1763) Andre´ G. Uitterlinden Erasmus Medical Center, Rotterdam, The Netherlands (1025) Aasis Unnanuntana Hospital for Special Surgery, New York, NY, USA and Siriraj Hospital, Mahidol University, Bangkok, Thailand (927) Maurits Vandewalle Universiteit Gent, Vakgroep voor Organische Chemie, Gent, Belgium (1461) Marjolein van Driel Erasmus Medical Center, Rotterdam, The Netherlands (1591) Johannes P.T.M. van Leeuwen Erasmus Medical Center, Rotterdam, The Netherlands (1591) Andre J. van Wijnen University of Massachusetts Medical School, Worcester, MA, USA (301) Natasja van Schoor VU University Medical Center, Amsterdam, The Netherlands (947) Lieve Verlinden Katholieke Universiteit Leuven, Leuven, Belgium (1461) Annemieke Verstuyf Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Leuven, Belgium (1461)

Reinhold Vieth University of Toronto, Toronto, Canada and Mount Sinai Hospital, Toronto, Canada (1041) Connie M. Weaver IN, USA (657)

Purdue University, West Lafayette,

Barrie M. Weinstein Mount Sinai School of Medicine, New York, NY, USA (1245) JoEllen Welsh University at Albany, Rensselaer, NY, USA (1657) John H. White (1777)

McGill University, Montreal, Canada

G. Kerr Whitfield USA (137)


Susan J. Whiting University of Saskatchewan, Saskatoon, Saskatchewan, Canada (979) Michael P. Whyte Shriners Hospital for Children and Washington University School of Medicine at BarnesJewish Hospital, St Louis, MI USA (807) John J. Wysolmerski Yale University School of Medicine, New Haven, CT, USA (725) Sachiko Yamada Nihon University School of Medicine, Tokyo, Japan (1509) Ian Yip David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)


Introduction

On November 30, 2010, after nearly two years of deliberation, an Institute of Medicine (IOM)-appointed committee released their findings, “2011 Report on dietary reference intakes (DRIs) for calcium and vitamin D.” Among their many recommendations the important conclusions regarding vitamin D were: (1) most of the population was not currently vitamin-D-deficient: (2) that 600 IU/day for ages 1e70 and 800 IU/day if over age 70 was adequate to protect bones; and (3) that all of the other potential benefits of vitamin D, besides bone health, did not yet have compelling evidence to support advising higher doses. They concluded that higher doses of vitamin D should not be advised on a public health basis until further research was done. It should be noted that the European counterpart to this report concluded that 800 IU was the suggested daily intake. Although the IOM report was meant to provide the populations of Canada and the United States with Recommended Dietary Allowances (RDAs) and Tolerable Upper Intake Levels (ULs) for calcium and vitamin D, the committee also identified a large number of uncertainties surrounding the DRI values that they recommended. For instance, the committee expressed a need for more research into both the skeletal and nonskeletal actions of vitamin D. Despite the thousands of publications on vitamin D, the committee was clearly disappointed by the lack of rigorous randomized trials and convincing clinically applicable knowledge on the subject of vitamin D benefits beyond the skeleton. With this in mind, how does the IOM report impact what is written by the contributors to the third edition of “Vitamin D”? It is important for the readers of this book to know that the authors of each of the 105 chapters were asked to consider revising their chapters on the basis of the IOM report. For those authors contributing chapters in the book’s Sections III (Mineral and Bone Homeostasis), V (Human Physiology), VI (Diagnosis and Management), VII (Nutrition, Sunlight, Genetics and Vitamin D Deficiency), and VIII (Disorders) this

task was of particular importance, because, as mentioned above, the IOM determined there was insufficient causeand-effect evidence to support a role for vitamin D beyond its effects on bone health. That is not to say that vitamin D does not impact other human health conditions; the IOM committee simply stated that conclusive causal evidence was lacking in these areas and existing data were insufficient to support a public health statement for nonskeletal outcomes. As the authors in Sections IV (Targets), IX (Analogs), X (Cancer), XI (Immunity, Inflammation, and Disease) and XII (Therapeutic Applications and New Advances) remind us over and over again, definitive, randomized, clinical trial data supporting a role for vitamin D in the pathophysiology and/or treatment of nonskeletal human diseases are still wanting. However, as covered in essentially every chapter in the book, data highly suggestive of benefit in a multitude of diseases are so strong that many vitamin D researchers are persuaded that vitamin D will eventually be convincingly demonstrated to be efficacious in many disease states. Furthermore, many authors express the viewpoint that avoidance of vitamin D deficiency will be shown to prevent, delay, or reduce the development of numerous diseases. What is the reason convincing clinical studies are missing from the published literature? Most of the previous NIH-sponsored trials of vitamin D have focused on bone or musculoskeletal health. Moreover, there is a lack of pharmaceutical company interest in a nonpatentable small molecule like vitamin D as a therapeutic. Pharmaceutical companies are at work developing vitamin D analogs, but most of this work has not progressed beyond preclinical studies. Hopefully, much of the lack of interest in the use of vitamin D itself as a preventive or therapeutic agent for extraskeletal chronic diseases, including cardiovascular disease, cancer, diabetes, hypertension, cognitive decline, depression, lung disorders, infections, and autoimmune diseases, will be allayed by the recently initiated, NIH-funded, randomized, placebo-controlled VITamin

xxi

xxii

INTRODUCTION

D-OmegA-3 TriaL (VITAL); VITAL is reviewed by its principal investigator, Dr. JoAnn Manson, in Chapter 105 of this text. The central aim of VITAL is to determine whether the administration of 2000 IU daily with or without 1 g of marine omega-3 fatty acids (in a 2 2 factorial design) reduces the risk of developing heart disease, stroke, or cancer in those without a prior history of these illnesses. It is of note that all study participants will be allowed to take up to 800 IU of personal vitamin D supplements (a dose, when added to dietary sources, exceeds the IOM recommended daily intake). If shown to be efficacious alone or in combination with omega-3 fatty acids in preventing the leading causes of death of American men and women, then vitamin D supplementation at a daily dose higher than the IOM guidelines will be justified. However, even if a 2000 IU dose of vitamin D3 daily reduces the risk of one or more of these nonskeletal diseases, other controversies raised by the IOM report will no doubt persist or surface. For example, the IOM report claimed that most of the US population is not vitamin-D-deficient. This obviously raises the discussion of where the cut-points for deficiency should be placed. The IOM has chosen 20 ng/ml (50 nmol/L) as the cut-off, a concentration they felt was sufficient to maintain bone health. Some would argue this is not

high enough even for bone health, let alone the other potential diseases that vitamin D may benefit. There will be much continued discussion of this report in the literature and no doubt there will be spirited debate about some of its findings. It is not our intent to carry out a pro and con discussion of the report but to emphasize several points. Importantly, public health policy must be conservative and risk averse and the IOM concluded that it should await more convincing data before recommending higher vitamin D intakes. The IOM was also concerned that, on a public health level, advising millions of people to take higher doses of vitamin D for extended periods of time could raise safety issues not observed in much smaller and shorter studies. These are real concerns. Finally, the IOM called for continued research efforts to develop compelling data to demonstrate the benefits of vitamin D claimed by many researchers. Although there is disagreement about the potential risks of not instituting vigorous vitamin D supplementation now, the editors and authors agree that more and better research would be welcome. It is our hope that the compilation of evidence about vitamin D action in normal and disease states contained in this volume will help to clarify the state of the science and be of use in elucidating the role of vitamin D in health and disease.

Abbreviations

AA AC ACE ACF ACTH ADH ADHR ADP AHO AI AIDS Aj.AR ALP ANG II ANP APC APD APL AR ARC 5-ASA ATP ATRA AUC Bmax BARE bFGF BFU BGP BLM BMC BMD BMI BMP BMU bp BPH

BSA BUA [Ca2+]i

arachadonic acid adenyl cyclase angiotensin converting enzyme activation frequency adrenocorticotropin antidiuretic hormone (vasopressin) autosomal dominant hypophosphatemic rickets adenosine diphosphate Albright’s hereditary osteodystrophy adequate intake acquired immunodeficiency syndrome adjusted apposition rate alkaline phosphatase angiotensin II atrial natriuretic peptide antigen presenting cell aminohydroxypropylidene bisphosphonate atrichia with papular lesions androgen receptor activator recruited cofactor 5-aminosalicylic acid adenosine triphosphate all-trans-retinoic acid area under the curve maximum number of binding sites bile acid response element basic fibroblast growth factor burst-forming unit bone Gla protein (osteocalcin) basal lateral membrane bone mineral content bone mineral density body mass index bone morphogenetic protein basic multicellular unit base pairs benign prostatic hyperplasia

CaBP CAD CaM cAMP CaSR or CaR CAT CBG CBP CC CD CDCA CDK or Cdk cDNA CDP Cdx-2 CFU cGMP CGRP CHF CK-II CLIA cM Cm. Ln. CNS CPBA cpm CRE CREB CRF CsA CSF CT CTR

xxiii

bovine serum albumin bone ultrasound attentuation internal calcium ion molar concentration calcium-binding protein coronary artery disease calmodulin cyclic AMP calcium-sensing receptor chloramphenicol acetyltransferase corticosteroid-binding globulin competitive protein-binding assay chief complaint Crohn’s disease chenodeoxycholic acid cyclin-dependent kinase complementary DNA collagenase-digestible protein caudal-related homeodomain protein colony-forming unit cyclic GMP calcitonin gene-related peptide congestive heart failure casein kinase-II competitive chemiluminescence immunoassay centimorgans cement line central nervous system competitive protein-binding assays counts per minute cAMP response element cAMP response element binding protein chronic renal failure cyclosporin A colony-stimulating factor calcitonin or computerized tomography calcitonin receptor

xxiv CTX CVC CYP CYP24 DAG DBD DBP DBP DC DCA DCT DEXA or DXA 7-DHC DHEA DHT DIC DMSO DR DRIP DSP DSS E1 E2 EAE EBT EBV EC EC50 or ED50 ECaC ECF EDTA EGF ELISA EMSA EP1 ER ERE ERK Et

ABBREVIATIONS

cerebrotendinous xanthomatosis calcifying vascular cells cytochrome P450 cytochrome P450, 24-hydroxylase diacylglycerol DNA-binding domain diastolic blood pressure vitamin-D-binding protein dendritic cell deoxycholic acid distal convoluted tubule dual energy X-ray absorptiometry 7-dehydrocholesterol dehydroepiandrosterone dihydrotachysterol or dihydrotestosterone disseminated intravascular coagulation dimethyl sulfoxide direct repeat vitamin D receptor interacting protein dental sialoprotein dextran sodium sulfate estrone estradiol experimental autoimmune encephalitis electron-beam computed technology Epstein-Barr virus endothelial cells effective concentration (dose) to cause 50% effect epithelium calcium channel extracellular fluid ethylenediaminetetraacetic acid epidermal growth factor enzyme-linked immunosorbent assay electrophoretic mobility shift assay PG receptor-1 estrogen receptor or endoplasmic reticulum estrogen response element extracellular signal-regulated kinase endothelin

FACS FAD FCS FDA FFA FIT FMTC FP FRAP FS FSK FXR g g G0, G1, G2 GAG GC-MS G-CSF GDNF GFP GFR GH GHRH GIO GM-CSF GnRH GR GRE GRTH GWAS HAT HDAC HEK HHRH HIV HNF HPI HPLC HPV

fluorescence-activated cell sorting or sorter flavin adenine dinucleotide fetal calf serum US Food and Drug Administration free fatty acid Fracture Intervention Trial familial medullary thyroid carcinoma formation period fluorescence recovery after photobleaching Fanconi syndrome forskolin farnesoid X receptor gram acceleration due to gravity gap phases of the cell cycle glycosaminoglycan gas chromatography-mass spectrometry granulocyte colony-stimulating factor glial-cell-derived neurotrophic factor green fluorescent protein glomerular filtration rate growth hormone growth-hormone-releasing hormone glucocorticoid-induced osteoporosis granulocyte-macrophage colonystimulating factor gonadotropin-releasing hormone glucocorticoid receptor glucocorticoid response element generalized resistance to thyroid hormone genome-wide association study histone acetyltransferase histone deacetylase human embryonic kidney hereditary hypophosphatemic rickets with hypercalciuria human immunodeficiency virus hepatocyte nuclear factor history of present illness high-performance liquid chromatography human papilloma virus

ABBREVIATIONS

h HR HRE HSA Hsp HSV HVDRR HVO IBD IBMX IC50 ICA ICMA IDBP IDDM IDM IEL IFN Ig IGFBP IGF-I, -II IGF-IR IL i.m. IMCal iNKT i.p. IP3 IRMA IU IUPAC i.v. JG JNK Kd Km kb kbp kDa KO LBD LCA LDL

hour hairless hormone response element human serum albumin heat-shock protein herpes simplex virus hereditary vitamin-D-resistant rickets hypovitaminosis D osteopathy inflammatory bowel disease isobutylmethylxanthine concentration to inhibit 50% effect intestinal calcium absorption immunochemiluminometric assay intracellular vitamin-D-binding protein insulin-dependent diabetes mellitus infants of diabetic mothers intraepithelial cells interferon immunoglobulin IGF-binding protein insulin-like growth factor type I, II IGF-I receptor interleukin (e.g., IL-1, IL-1b, etc.) intramuscular intestinal membrane calciumbinding complex invariant NKT intraperitoneal inositol trisphosphate immunoradiometric assay international units International Union of Pure and Applied Chemists intravenous juxtaglomerular c-Jun NH2-terminal kinase dissociation constant Michaelis constant kilobases kilobase pairs kilodaltons knockout ligand-binding domain lithocholic acid low-density lipoprotein

Li. Ce. LIF LNH LOD LPS LT LXR M M MAPK Mab MAR MAR MARRS MCR M-CSF MEN2 MGP MHC min MIU MLR Mlt MR MRI mRNA MS MT MTC NADH NADPH NAF NBT NcAMP NCP NFkB NGF NHANES III NHL NIDDM NIH NK cell

xxv lining cell leukemia inhibitory factor late neonatal hypocalcemia logarithm of the odds lipopolysaccharide leukotriene liver X receptor mitosis phase of cell cycle molar mitogen-activated protein kinase monoclonal antibody matrix attachment region mineral apposition rate membrane-associated rapid response steroid metabolic clearance rate macrophage colony-stimulating factor multiple endocrine neoplasia type 2 matrix Gla protein major histocompatibility complex minute million international units mixed lymphocyte reaction mineralization lag time mineralocorticoid receptor magnetic resonance imaging messenger ribonucleic acid multiple sclerosis metric ton medullary thyroid carcinoma nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nuclear accessory factor nitroblue tetrazolium nephrogenous cAMP noncollagen protein nuclear factor kappa B nerve growth factor National Health and Nutrition Examination Survey III Non-Hodgkin’s lymphoma non-insulin-dependent diabetes mellitus National Institutes of Health natural killer cell

xxvi NLS NMR NOD NPT NR Ob Oc OCIF OCT ODF 1a-(OH)D3 25(OH)D3 1,25(OH)2D3 24,25(OH)2D3 OHO Omt OPG OPN OSM OVX Pi PA2 PAD PAM PBL PBMC PBS PC PCNA PCR PCT PDDR PDGF PEIT PHEX

PG PHA PHP PIC PKA PKC

ABBREVIATIONS

nuclear localization signal nuclear magnetic resonance nod-like sodium/phosphate cotransporter nuclear receptor osteoblast osteocalcin or osteoclast osteoclastogenesis inhibitory factor (same as OPG) 22-oxacalcitriol osteoclast differentiation factor (same as RANKL) 1a-hydroxyvitamin D3 25-hydroxyvitamin D3 1a,25-dihydroxyvitamin D3 24,25-dihydroxyvitamin D3 oncogenic hypophosphatemic osteomalacia osteoid maturation time osteoprotegerin osteopontin oncostatin M ovariectomy inorganic phosphate phospholipase A2 peripheral arterial vascular disease pulmonary alveolar macrophage peripheral blood lymphocyte peripheral blood mononuclear cells phosphate-buffered saline phophatidyl choline proliferating cell nuclear antigen polymerase chain reaction proximal convoluted tubule pseudovitamin D deficiency rickets platelet-derived growth factor percutaneous ethanol injection therapy phosphate regulating gene with homologies to endopeptidases on the X chromosome prostaglandin phytohemagglutinin pseudohypoparathyroidism preinitiation complex protein kinase A protein kinase C

PKI PLA2 PLC PMA PMCA PMH p.o. poly(A) PPAR PR PRA PRL PRR PSA PSI PT PTH PTHrP PTX PUVA QCT QSAR 9-cis-RA RA RA Rag RANK RANKL RAP RAR RARE RAS RBP RCI RDA RFLP RIA RID RNase ROCs ROS RPA

protein kinase inhibitor phospholipase A2 phospholipase C phorbol 12-myristate 13-acetate plasma membrane calcium pump past medical history oral polyadenosine peroxisome proliferator-activated receptor progesterone receptor plasma renin activity prolactin pattern recognition receptors prostate-specific antigen psoriasis severity index parathyroid parathyroid hormone parathyroid hormone-related peptide parathyroidectomy psoralen-ultraviolet A quantitative computerized tomography quantitative structureeactivity relationship 9-cis-retinoic acid retinoic acid rheumatoid arthritis recombination activating gene receptor activator NF-kB receptor activator NF-kB ligand receptor-associated protein retinoic acid receptor retinoic acid response element renineangiotensin system retinol-binding protein relative competitive index recommended dietary allowance restriction fragment length polymorphism radioimmunoassay receptor interacting domain ribonuclease receptor-operated calcium channels reactive oxygen species ribonuclease protection assay

ABBREVIATIONS

RRA RT-PCR RXR RXRE SBP SD SDS SE SEM SH SHBG SLE SNP SNPs SOS Sp1 SPF SRC-1 SSCP SV40 SXA t1/2 T3 T4 TBG TBP TC TF TFIIB TG TGF TIO TK TLR TmP or TmPi TNBS TNF TPA

radioreceptor assay reverse transcriptase-polymerase chain reaction retinoid X receptor retinoid X receptor response element systolic blood pressure standard deviation sodium dodecyl sulfate standard error standard error of the mean social history sex-hormone-binding globulin systematic lupus erythematosus single nucleotide polymorphism single nucleotide polymorphisms speed of sound selective promoter factor 1 sun protection factor steroid receptor coactivator-1 single-strand conformational polymorphism simian virus 40 single energy X-ray absorptiometry half-time triiodothyronine thyroxine thyroid-binding globulin TATA-binding protein tumoral calcinosis tubular fluid general transcription factor IIB transgenic transforming growth factor tumor-induced osteomalacia thymidine kinase toll-like receptor tubular absorptive maximum for phosphorus trinitrobenzene sulfonic acid tumor necrosis factor 12-O-tetradecanoylphorbol13-acetate

TPN TPTX TR TRAP TRAP TRP TRE TRE TRH Trk TSH TSS UF US USDA UTR UV VDDR-I VDDR-II VDR VDRE VDRL VEGF VERT VICCs VSMC VSSCs WHI WRE WSTF WT XLH XRD ZEB

xxvii total parenteral nutrition thyroparathyroidectomized thyroid hormone receptor tartrate-resistant acid phosphatase thyroid hormone receptor associated proteins transient receptor potential thyroid hormone response element TPA response element thyrotropin-releasing hormone tyrosine kinase thyrotropin transcription start site ultrafiltrable fluid ultrasonography US Department of Agriculture untranslated region ultraviolet vitamin-D-dependent rickets type I (see PDDR) vitamin-D-dependent rickets type II (see HVDRR) vitamin D receptor vitamin D response element vitamin D receptor ligand vascular endothelial growth factor Vertebral Efficacy with Risedronate Therapy studies voltage-insensitive calcium channels vascular smooth muscle cell voltage-senstive calcium channels Women’s Health Initiative Wilms’ tumor gene, WT1, responsive element Williams syndrome transcription factor wild-type X-linked hypophosphatemic rickets X-ray diffraction zinc finger, E box-binding transcription factor


Relevant Lab Values in Adults and Children

CRITERIA FOR VITAMIN D DEFICIENCY: 25(OH)D SERUM LEVELS Adult IOM recommendations

Deficient Normal Excessive

Conventional units

SI units

50 ng ml

125 nmol/L

Frequently used vitamin D cut-points by many laboratories

Deficient Insufficient Sufficient

Conventional units

SI units

< 20 ng/ml 20 to 29.9 ng/ml >30 ng/ml

75 nmol/L

Pediatric (The IOM and the Pediatric Endocrine Society have agreed on these cut-points.)

Deficient Normal

Conventional units

SI units

70 y

e

400 IU (10 mg)

800 IU (20 mg)

4000 IU (100 mg)

1500e2000

10 000

14e18 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

19e30 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

31e50 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

14e18 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

19e30 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

31e50 y

e

400 IU (10 mg)

600 IU (15 mg)

4000 IU (100 mg)

1500e2000

10 000

UL (IU)

Children

Males

Females

Pregnancy

Lactation*

deficiency. Data to support or refute the vitamin De cancer link is carefully analyzed in Chapter 82. Some experts advocate the use of vitamin D supplements to reduce these risks rather than attempting to balance the amount of sunlight needed to supply adequate vitamin D. There is concern in many quarters about advocating tanning beds as a source of vitamin D

because of the risk of over-usage and over-exposure to harmful UV-A rays (see also Chapter 89). Additional opinions about the risks of vitamin D deficiency are discussed in many chapters in this volume. Discussion of the optimum target levels for serum 25(OH)D are also discussed in Chapters 57 and 58, where aggressive and conservative views are presented.

I. CHEMISTRY, METABOLISM, CIRCULATION

20

2. PHOTOBIOLOGY OF VITAMIN D

CONCLUSION This chapter presents the opinions of this author and to obtain a balanced view of the subject, the reader is advised to also read the opinions expressed by other authors in various chapters throughout this volume. Of particular relevance will be different opinions on the subjects of: risks of vitamin D deficiency, safety of sunlight, the use of UV exposure vs supplements to achieve vitamin D sufficiency, and optimum target levels of serum 25(OH)D. The skin has a huge capacity to produce vitamin D [3,36,65]. Children and adults exposed to natural or artificial ultraviolet B radiation can satisfy their vitamin D requirement [3,65] (Fig 2.9). Exposure of a healthy adult in a bathing suit to one minimal erythemal dose (a light pinkness to the skin) of sunlight or tanning bed UV radiation is equivalent to taking between 10 000 IU and 20 000 IU of vitamin D [1,3] (Fig. 2.10). Because melanin is such an effective sunscreen, African Americans require 5e10 times the exposure that a white person requires to satisfy their body’s vitamin D requirement [19]. Although aging markedly diminishes the capacity of the skin to produce vitamin D3, because its capacity is so high, elders exposed to sunlight can still raise their blood levels of 25(OH)D into a satisfactory range [70e72]. Thus, there needs to be a reexamination of the message that any exposure to sunlight requires some type of sunscreen or sun protection. This extreme position has pervaded the psyche of the population at large. It unfortunately has put many people at risk for vitamin

Comparison of serum vitamin D3 levels after a whole-body exposure (in a bathing suit; bikini for women) to 1 MED (minimal erythemal dose) of simulated sunlight compared with a single oral dose of either 10 000 or 25 000 IU of vitamin D2. Reproduced with permission from Holick [76].

FIGURE 2.10

D deficiency and many of the serious chronic diseases that have been associated with inadequate sun exposure and vitamin D deficiency. Sensible and limited exposure to sunlight, typically no more than 5e15 minutes a day on arms and legs (depending on time of day, season, skin sensitivity, latitude) between 10 a.m. and 3 p.m. during seasons when vitamin D can be produced in the skin will satisfy most people’s vitamin D requirement [3,68e75]. Always protect the face. It is the most sun-exposed area on the body and only represents 5e9% of the body surface area. Taking a multi-vitamin containing 400 IU of vitamin D will satisfy approximately 20% of an adult’s requirement. Thus, there is a need for additional supplementation and/or foods that contain vitamin D to satisfy the 1000e2000 IU and 400e1000 IU of vitamin D that adults and children respectively require to raise their blood levels of 25 (OH)D above 30 ng/ml, which is considered to be a healthy level [36] (Table 2.1).

Acknowledgment This work was supported in part by the NIH CTSI Grant # UL1RR025771 and the UV Foundation.

References

FIGURE 2.9 The serum 25-hydroxyvitamin D levels in healthy adults with skin types II, III, and IV exposed to 0.75 MEDs of simulated sunlight in a bathing suit three times a week for 12 weeks compared to healthy adults receiving a daily dose of 1000 IU of vitamin D3 daily for 12 weeks. )p < 0.01. Copyright Holick 2010; reproduced with permission.

[1] M.F. Holick, 2004 Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Robert H. Herman Memorial Award in Clinical Nutrition Lecture, Am. J. Clin. Nutr. 79 (2003) 362e371. [2] M.F. Holick, Phylogenetic and evolutionary aspects of vitamin D from phytoplankton to humans, in: P.K.T. Pang, M.P. Schreibman (Eds.), Vertebrate Endocrinology: Fundamentals and Biomedical Implications, vol. 3, Academic Press, Orlando, FL, 1989, pp. 7e43. [3] M.F. Holick, Vitamin D deficiency. New Engl, J. Med. 357 (2007) 266e281.


21

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[4] J. Sniadecki, cited by W Mozolowski, Jerdrzej Sniadecki (1768e1883) on the cure of rickets, Nature 143 (1939) 121. [5] T.A. Palm, The geographic distribution and etiology of rickets, Practitioner 45 (1980) 270e279. 421e442. [6] K. Huldschinsky, Curing rickets by artificial UV-radiation (Heilung von Rachitis durch kunstliche Hohensonne), Deut. Med. Wochenschr. 45 (1919) 712e713 (in German). [7] A.F. Hess, L.F. Unger, Cure of infantile rickets by sunlight, JAMA 77 (1921) 39. [8] J. Mayer, Armand Trousseau and the arrow of time, Nutr. Rev. 15 (1957) 321e323. [9] T. Mellanby, The part played by an accessory factor in the production of experimental rickets, J. Physiol. 52 (1918) 11e14. [10] E.F. McCollum, N. Simmonds, J.E. Becker, P.G. Shipley, Studies on experimental rickets; and experimental demonstration of the existence of a vitamin which promotes calcium deposition, J. Biol. Chem. 53 (1922) 293e312. [11] H. Steenbock, A. Black, The reduction of growth-promoting and calcifying properties in a ration by exposure to ultraviolet light, J. Biol. Chem. 61 (1924) 408e411. [12] A.F. Hess, M. Weinstock, Antirachitic properties imparted to inert fluids and green vegetables by ultraviolet irradiation, J. Biol. Chem. 62 (1924) 301e313. [13] H. Steenbock, The induction of growth-prompting and calcifying properties in a ration exposed to light, Science 60 (1924) 224e225. [14] J.A. MacLaughlin, R.R. Anderson, M.F. Holick, Spectral character of sunlight modulates photosynthesis of previtamin D3 and its photoisomers in human skin, Science 216 (1982) 1001e1003. [15] M.F. Holick, X.Q. Tian, M. Allen, Evolutionary importance for the membrane enhancement of the production of vitamin D3 in the skin of poikilothermic animals, Proc. Natl. Acad. Sci. USA 92 (1995) 3124e3126. [16] F. Loomis, Skin-pigment regulation of vitamin D biosynthesis in man, Science 157 (1967) 501e506. [17] M.F. Holick, J.A. MacLaughlin, S.H. Doppelt, Factors that influence the cutaneous photosynthesis of previtamin D3, Science 211 (1981) 590e593. [18] A.R. Webb, B.R. deCosta, M.F. Holick, Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation, J. Clin. Endocrinol. Metab. 68 (1989) 882e887. [19] T.L. Clemens, J.S. Adams, S.L. Henderson, M.F. Holick, Increased skin pigment reduces the capacity of the skin to synthesize vitamin D, Lancet 1 (1982) 74e76. [20] N.H. Bell, A. Greene, S. Epstein, M.J. Oexmann, W. Shaw, J. Shary, Evidence for alteration of the vitamin D endocrine system in Blacks, J. Pediatr. 76 (1985) 470e473. [21] M. Kassowitz, Tetany and autointoxication in infants (Tetani and autointoxication in kindersalter), Wien. Med. Presse. 97 (1987) 139 (in Dutch). [22] A.R. Webb, L. Kline, M.F. Holick, Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin, J. Clin. Endocrinol. Metab. 67 (1988) 373e378. [23] L.Y. Matsuoka, L. Ide, J. Wortsman, J.A. MacLaughlin, M.F. Holick, Sunscreens suppress cutaneous vitamin D3 synthesis, J. Clin. Endocrinol. Metab. 64 (1987) 1165e1168. [24] L.Y. Matsuoka, J. Wortsman, M.J. Dannenberg, B.W. Hollis, Z. Lu, M.F. Holick, Clothing prevents ultraviolet-B radiationdependent photosynthesis of vitamin D3, J. Clin. Endocrinol. Metab. 75 (1992) 1099e1103. [25] L.Y. Matsuoka, J. Wortsman, N. Hanifan, M.F. Holick, Chronic sunscreen use decreases circulating concentrations of

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25-hydroxyvitamin D: a preliminary study, Arch. Dermatol. 124 (1988) 1802e1804. S.H. Sedrani, K.M. Al-Arabi, A. Abanny, A. Elidrissy, Frequency of vitamin D deficiency rickets in Riyadh, in: Study of Vitamin D Status and Factors Leading to Its Deficiency in Saudi Arabia, King Saudi Univ. Press, Riyadh, 1990, pp. 281e285. F.M. Gloth, C.M. Gundberg, B.W. Hollis, H.G. Haddad, J.D. Tobin, Vitamin D deficiency in homebound elderly persons, JAMA 274 (1995) 1683e1686. A. Malabanan, I.E. Veronikis, M.F. Holick, Redefining vitamin D insufficiency, Lancet 351 (1998) 805e806. P. Lips, Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications, Endocr. Rev. 22 (2001) 477e501. M.C. Chapuy, P. Preziosi, M. Maaner, S. Arnaud, P. Galan, S. Hercberg, et al., Prevalence of vitamin D insufficiency in an adult normal population, Osteopor. Int. 7 (1997) 439e443. V. Tangpricha, E.N. Pearce, T.C. Chen, M.F. Holick, Vitamin D insufficiency among free-living healthy young adults, Am. J. Med. 112 (2002) 659e662. S.R. Kreiter, R.P. Schwartz, H.N. Kirkman, P.A. Charlton, A.S. Calikoglu, M. Davenport, Nutritional rickets in African American breast-fed infants, J. Pediatr. 137 (2000) 2e6. T.R. Welch, Vitamin D deficient rickets: the reemergence of a once-conquered disease, J. Pediatr. 137 (2000) 143e145. M.K. Thomas, D.M. Lloyd-Jones, R.I. Thadhani, A.C. Shaw, D.J. Deraska, B.T. Kitch, Hypovitaminosis D in medical inpatients, N. Engl. J. Med. 338 (1998) 777e783. S.S. Sullivan, C.J. Rosen, T.C. Chen, M.F. Holick, 2003 Seasonal changes in serum 25(OH)D in adolescent girls in Maine. J Bone Mine Res (Proceedings for Twenty-Fifth Annual Meeting of the American Society for Bone and Mineral Research), S407. M.F. Holick, Vitamin D and health: evolution, biologic functions, and recommended dietary intakes for vitamin D, Clin. Rev. Bone. Miner. Metab. 7 (1) (2009) 2e19. R.P. Heaney, K.M. Davies, T.C. Chen, M.F. Holick, M.J. BargerLux, Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol, Am. J. Clin. Nutr. 77 (2003) 204e210. M.F. Holick, R.M. Biancuzzo, T.C. Chen, E.K. Klein, A. Young, D. Bibuld, et al., Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D, J. Clin. Endo. Metab. 93 (2008) 677e681. S. Nesby-O’Dell, K.S. Scanlon, M.E. Cogswell, C. Gillespie, B.W. Hollis, A.C. Looker, Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third national health and nutrition examination survey, 1988e1994, Am. J. Clin. Nutr. 76 (2002) 187e192. A.J. Rovner, K.O. O’Brien, Hypovitaminosis D among healthy children in the United States: a review of current evidence, Arch. Pediatr. Adolesc. Med. (2008) 513e519. J. Kumar, P. Muntner, F.J. Kaskel, S.M. Hailpern, M.L. Melamed, Prevalence and associations of 25-hydroxyvitamin D deficiency in US Children: NHANES, Pediatrics (2001e2004). B.W. Hollis, C.L. Wagner, Assessment of dietary vitamin D requirements during pregnancy and lactation, Am. J. Clin. Nut. 79 (2004) 717e726. L.M. Bodnar, J.M. Catov, H.N. Simhan, M.F. Holick, R.W. Powers, J.M. Roberts, Maternal vitamin D deficiency increases the risk of preeclampsia, J. Clin. Endo. Metab. 92 (2007) 3517e3522. A. Merewood, S.D. Mehta, T.C. Chen, M.F. Holick, H. Bauchner, Association between severe vitamin D deficiency and primary caesarean section, J. Clin. Endo. Metab. 94 (3) (2009) 940e945.


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[45] M.F. Holick, Sunlight “D”ilemma: risk of skin cancer or bone disease and muscle weakness, Lancet 357 (2001) 4e6. [46] H. Glerup, K. Mikkelsen, L. Poulsen, E. Hass, S. Overbeck, H. Andersen, et al., Hypovitaminosis D myopathy without osteomalacic bone involvement, Calcif. Tissue Int. 66 (2000) 419e424. [47] A.O. Malabanan, A.K. Turner, M.F. Holick, Severe generalized bone pain and osteoporosis in a premenopausal black female: effect of vitamin D replacement, J. Clin. Densitometr. 1 (1998) 201e204. [48] C.F. Garland, F.C. Garland, E.K. Shaw, G.W. Comstock, K.J. Helsing, E.D. Gorham, Serum 25-hydroxyvitamin D and colon cancer: eight-year prospective study, Lancet 18 (1989) 1176e1178. [49] C.F. Garland, F.C. Garland, E.D. Gorham, J. Raffa, Sunlight, Vitamin D, and Mortality from Breast and Colorectal Cancer in Italy. Biologic Effects of Light, Walter de Gruyter, New York, 1992. 39e43. [50] C.L. Hanchette, G.G. Schwartz, Geographic patterns of prostate cancer mortality, Cancer 70 (1992) 2861e2869. [51] M.H. Ahonen, L. Tenkanen, L. Teppo, M. Hakama, P. Tuohimaa, Prostate cancer risk and prediagnostic serum 25-hydroxy-vitamin D levels (Finland), Cancer Causes Control 11 (2000) 847e852. [52] W.B. Grant, An ecologic study of dietary and solar ultra-violet-B links to breast carcinoma mortality rates, Am. Cancer Soc. 94 (2002) 272e281. [53] H.A. Bischoff-Ferrari, E. Giovannucci, W.C. Willett, T. Dietrich, B. Dawson-Hughes, Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes, Am. J. Clin. Nutr. 84 (2006) 18e28. [54] E. Hypponen, E. Laara, M.-R. Jarvelin, S.M. Virtanen, Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study, Lancet 358 (2001) 1500e1503. [55] S.G. Rostand, Ultraviolet light may contribute to geographic and racial blood pressure differences, Hypertension 30 (1979) 150e156. [56] Y.C. Li, J. Kong, M. Wei, Z.F. Chen, S.Q. Liu, L.P. Cao, 1,25dihydroxyvitamin D3 is a negative endocrine regulator of the renineangiotensin system, J. Clin. Invest. 110 (2002) 229e238. [57] R. Scragg, R. Jackson, I.M. Holdaway, T. Lim, R. Beaglehole, Myocardial infarction is inversely associated with plasma 25hydroxyvitamin D3 levels: a community-based study, Int. J. Epidemiol. 19 (1990) 559e563. [58] A. Zitterman, S. Schulze Schleithoff, C. Tenderich, H. Berthold, R. Koefer, P. Stehle, Low vitamin D status: a contributing factor in the pathogenesis of congestive heart failure? J. Am. Coll. Cardiol. 41 (2003) 105e112. [59] J. Moan, A.C. Porojnicu, A. Dahlback, R.B. Setlow, Addressing the health benefits and risks, involving vitamin D or skin cancer, of increased sun exposure, Proc. Natl. Acad. Sci. USA 105 (2008) 668e673. [60] T.J. Wang, M.J. Pencina, S.L. Booth, P.F. Jacques, E. Ingelsson, K. Lanier, Vitamin D deficiency and risk of cardiovascular disease, Circulation 117 (2008) 503e511. [61] A.A. Ginde, J.M. Mansbach, C.A. Camargo, Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey, Arch. Intern. Med. 169 (2009) 384e390. [62] M. Urashima, T. Segawa, M. Okazaki, M. Kurihara, Y. Wada, H. Ida, Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren, Am. J. Clin. Nutr. 91 (2010) 1255e1260. [63] T.S. Housman, S.R. Feldman, P.M. Williford, A.B. Fleischer Jr., N.D. Goldman, J.M. Acostamadiedo, Skin cancer is among the

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most costly of all cancers to treat for the Medicare population, J. Am. Acad. Dermatol. 48 (2003) 425e429. C. Kennedy, C.D. Bajdik, R. Willemze, F.R. de Gruijl, J.N. Bavinck, The influence of painful sunburns and lifetime of sun exposure on the risk of actinic keratoses, seborrheic warts, melanocytic nevi, atypical nevi and skin cancer, J. Invest. Dermatol. 120 (2003) 1087e1093. C.F. Garland, F.C. Garland, E.D. Gorham, Rising trends in melanoma. A hypothesis concerning sunscreen effectiveness, Ann. Epidemiol. 3 (1993) 103e110. M.B. Veierod, E. Weiderpass, M. Thorn, J. Hansson, E. Lund, B. Armstrong, et al., A prospective study of pigmentation, sun exposure, and risk of cutaneous malignant melanoma in women, J. Nat. Can. Inst. 95 (2003) 1530e1538. W.B. Grant, An estimate of premature cancer mortality in the U.S. due to inadequate doses of solar ultraviolet-B radiation, Cancer 70 (2002) 2861e2869. M.F. Holick, The Vitamin D Solution, Hudson Street Press, New York NY, 2010. M.F. Holick, T.C. Chen, E.R. Sauter, Vitamin D and Skin Physiology: A D-Lightful Story, J. Bone. Miner. Res. 22 (2007) S2. V28-V33. V.G.M. Chel, M.E. Ooms, C. Popp-Snijders, S. Pavel, A.A. Schothorst, C.C.E. Meulemans, et al., Ultraviolet irradiation corrects vitamin D deficiency and suppresses secondary hyperparathyroidism in the elderly, J. Bone. Miner. Res. 13 (1998) 1238e1242. A. Chuck, J. Todd, B. Diffey, Subliminal ultraviolet-B irradiation for the prevention of vitamin D deficiency in the elderly: a feasibility study, Photochem. Photoimmun. Photomed. 17 (2001) 168e171. B. Lund, O.H. Sorensen, Measurement of 25-hydroxy-vitamin D in serum and its relation to sunshine, age, and vitamin D intake in the Danish population, Scand. J. Clin. Lab. Invest. 39 (1979) 23e30. J.A. Adams, T.L. Clemens, J.A. Parrish, M.F. Holick, Vitamin-D synthesis and metabolism after ultraviolet irradiation of normal and vitamin-D-deficient subjects, N. Engl. J. Med. 306 (1982) 722e725. P. Koutkia, Z. Lu, T.C. Chen, M.F. Holick, Treatment of vitamin D deficiency due to Crohn’s disease with tanning bed ultraviolet B radiation, Gastroenterology 121 (2001) 1485e1488. I.R. Reid, D.J.A. Gallagher, J. Bosworth, Prophylaxis against vitamin D deficiency in the elderly by regular sunlight exposure, Age Ageing 15 (1985) 35e40. M.F. Holick, McCollum Award Lecture, Vitamin D e New horizons for the 21st century, Am. J. Clin. Nutr. 60 (1994) 619e630. D.M. Freedman, A.C. Looker, C. Shih-Chen, B.I. Graubard, Prospective Study of Serum Vitamin D and Cancer Mortality in the United States, J. Natl. Cancer Inst. 99 (21) (2007) 1594e1602. C.C. Abnet, Y. Chen, C. Wong-Ho, G. Yu-Tang, K.J. Helzlsouer, L.L. Marchand, et al., Circulating 25-hydroxyvitamin D and risk of esophageal and gastric cancer, Am. J. Epidemiol. 172 (1) (2010) 94e106. K. Michae¨lsson, J.A. Baron, G. Snellman, R. Gedeborg, L. Byberg, J. Sundstro¨m, et al., Plasma vitamin D and mortality in older men: a community based prospective cohort study, Am. J. Clin. Nutr. 92 (4) (2010) 841.8. L. Yin, N. Grandi, E. Raum, U. Haug, V. Arndt, H. Brenner, Meta-analysis: serum vitamin D and breast cancer risk, Eur. J. Cancer 46 (12) (2010) 2196e2205.


C H A P T E R

3 The Activating Enzymes of Vitamin D Metabolism (25- and 1a-Hydroxylases) Glenville Jones 1, 2, David E. Prosser 1 1

Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada 2 Department of Medicine, Queen’s University, Kingston, Ontario, Canada

covered in the first and second editions of this book [9,10] and in reviews of the subject [11,12]. Also, while it is not the purview of this chapter to review knowledge of the vitamin-D-inactivating enzyme, 25(OH)D3-24hydroxylase, also known as CYP24A1 (covered in Chapter 4), the similarities of its structure to the activating enzymes make it inevitable that there will be occasional references to both activating and inactivating enzymes.

INTRODUCTION The activation of vitamin D3 is accomplished by sequential steps of 25-hydroxylation to produce the main circulating form, 25-hydroxyvitamin D [25(OH) D3] followed by 1a-hydroxylation to produce the hormonal form, 1a,25-dihydroxyvitamin D3 [1,25 (OH)2D3] [1] (Fig. 3.1). The initial step of 25-hydroxylation occurs in the liver [2], while the second step occurs both in the kidney and extra-renal sites [3,4]. While work performed over the past four decades in humans and a variety of animal species has revealed that several cytochrome P450 enzymes (CYPs): CYP2R1, CYP27A1, CYP3A4, CYP2D25, and perhaps others, are capable of 25-hydroxylation of vitamin D3 or related compounds and are thus referred to as vitamin D3-25-hydroxylase, it is CYP2R1 that is emerging as the physiologically relevant enzyme [5]. (The nomenclature of all cytochromes P450, including those involved in vitamin D metabolism, is the responsibility of an internationally acknowledged group headed by D. Nelson [8]. Individual CYP names are based upon sequence similarity, function, and other considerations.) On the other hand there is no ambiguity over the second step of 1a-hydroxylation or the 25(OH) D3-1a-hydroxylase enzyme responsible, which is carried out by a single cytochrome P450 named CYP27B1 [6,7]. This chapter assembles the most currently pertinent literature on the activating enzymes of vitamin D metabolism: protein structure and enzymatic properties, crystal structures, gene organization, mutational analysis and regulation. Due to space restrictions, this overview will not cover all of the rich history which went into the early enzymology or cloning of these cytochrome-P450-containing enzymes, much of which has been extensively

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10003-4

GENERAL INFORMATION REGARDING VITAMIN D HYDROXYLASES Table 3.1 summarizes pertinent information about all of the vitamin-D-metabolizing CYPs including both the activating and inactivating enzymes. CYPs are classified into two main subtypes based upon their subcellular location: microsomal or mitochondrial; vitamin D metabolism featuring both subtypes. Both microsomal and mitochondrial CYP subtypes do not function alone but are components of electron transport chains, microsomal CYPs (e.g., CYP2R1) requiring a single generalpurpose protein NADPH-cytochrome P450 reductase (Fig. 3.2A). As with all mitochondrial CYPs, the functional enzyme activity for mitochondrial vitaminD-related CYPs (e.g., CYP27A1, CYP27B1, CYP24A1) requires the assistance of two additional electron-transporting proteins consisting of a general purpose ferredoxin reductase, a general purpose ferredoxin and a highly specific CYP (Fig. 3.2B). All of the vitamin-Drelated CYPs catalyze single or multiple hydroxylation reactions on specific carbons of the vitamin D substrate using a transient Fe-O intermediate. The exact site of hydroxylation, termed regioselectivity, can be somewhat

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Copyright Ó 2011 Elsevier Inc. All rights reserved.

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3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)

FIGURE 3.1 Pathways of vitamin D activation and inactivation. Vitamin D3 absorbed from the diet or synthesized in the skin on exposure to

UVB is stored in the liver and fat. The first step in activation, 25-hydroxylation, occurs primarily in the liver and is mediated either by a lowcapacity, high-specificity microsomal 25-hydroxylase, CYP2R1, or a high-capacity, low-affinity mitochondrial hydroxylase, CYP27A1. The 25(OH)D3 is transported from the liver to the kidney bound to the plasma vitamin-D-binding protein (DBP, also known as Gc protein) where it is internalized by megalin-dependent cubilin-mediated endocytosis in the renal proximal tubule [125]. The second step in activation occurs primarily in the proximal tubule through the action of the 1a-hydroxylase, CYP27B1, to yield the active seco-steroid hormone, 1a,25(OH)2D3. The active hormone is transported to the kidney, intestine, bone, and vitamin D target tissues where it binds to the nuclear vitamin D receptor (VDR). Ligand-bound VDR heterodimerizes with retinoid-X receptor (RXR) and modulates the expression of 200e800 genes, including up-regulation of the 24-hydroxylase, CYP24A1, which degrades 1a,25(OH)2D3. The 24-hydroxylase and the 1a-hydroxylase are reciprocally expressed to regulate 1a,25(OH)2D3 levels. Expression of 24-hydroxylase activity produces 24,25(OH)2D3 from 25(OH)D3 which is putatively involved in bone fracture healing, but CYP24A1 predominantly inactivates 1a,25(OH)2D3 by a series of sequential hydroxylation and oxidation reactions to yield either calcitroic acid, which is excreted in the bile, or the 1a,25(OH)2D3-26,23-lactone, which itself is a weak VDR antagonist.

variable with vitamin-D-related CYPs, human CYP24A1 being documented to hydroxylate at C23, C24, or C26. From alignments of the vitamin-D-related CYPs (Fig. 3.3), it is immediately apparent that all CYP proteins possess around 500 amino acids and a size of 50e55 kDa, featuring abundant highly conserved residues which suggest a common secondary structure with multiple highly conserved helices (designated AeL) connected by loops and b-sheet structures. All

CYPs possess a cysteine residue and two other residues near to the C terminus which covalently bind and align the heme group, in addition to several other domains for interaction with the electron-transferring machinery, such as ferredoxin or NADPH-cytochrome P450 reductase. The N-terminus is thought to insert into the endoplasmic reticular membrane for microsomal CYPs or the inner mitochondrial membrane for mitochondrial CYPs. The substrate-binding pocket is formed by several


TABLE 3.1 Vitamin D Metabolizing CYPs Subcellular location

Size (aa)

Human gene locus

Mouse gene locus

Activity

Human >47 species

Liver

micro.

501

11p15.2 [23]

7-7E3 [23]

25-hydroxylation of D3 25-hydroxylation of D2

CYP27A1

Human >56 species

Liver Macrophage

mito.

531

2q33-qter [30]


CYP2C11

Rat

Liver (male)

micro.

500


[135]

CYP2D25

Pig

Liver

micro.

500

25-hydroxylation of D3

[136]

CYP2J2 CYP2J3

Human Rat

Liver

micro.

502

1p31.3-p31.2 [134]


[137]

CYP3A4

Human

Liver Intestine

micro.

503

7q22.1 [138]

25-hydroxylation of D2

[138] [43,44]

CYP27B1

Human >39 species

Kidney

mito.

508

12q13.1-q13.3 [65]

1a-hydroxylation of D3 1a-hydroxylation of D2

CYP24A1

Human >51 species

Target tissue

mito.

514

20q13.2-q13.3 [140]

23- & 24-hydroxylation of 25(OH)D/1,25 (OH)2D

Species

CYP2R1*

Disease state human or (mouse XO)

Function

Ref.

VDDR-1B (unknown)

Physiological 25-hydroxylase

[5] [132] [133] [134]

CTX

Pharmacological 25-hydroxylase

[27] [128]

VDDR-1A (Rickets)

1a-hydroxylase

[65] [75] [69] [139]

24-hydroxylase

[140e142]

GENERAL INFORMATION REGARDING VITAMIN D HYDROXYLASES


Tissue location

P450

CYP2R1 maps near PTH (11p15.3-p15.1), calcitonin and calcitonin-related polypeptide (11p15.2-p15.1), and insulin and insulin-like growth factor 2 (11p15.5).

25

26


(A) Microsomal NADP

+

NADPH

NADPH P450 Reductase

_ 2e

_ 2e e.g. CYP2R1 Fe

P450 Product R-OH + H2O

Endoplasmic Reticulum NADP

+

NADPH

3+

FPred

P450-Fe

P450 Reductase

Cytochrome P450

FPox

P450-Fe

2H

+

VITAMIN D3-25-HYDROXYLASES

2+

R-H + O 2 substrate

(B) Mitochondrial Ferredoxin Reductase

NADP

+

_ 1e

_ 2e

(twice)

_ 1e

Ferredoxin

NADPH

e.g. CYP27A1 CYP27B1 CYP24A1

Fe

P450 Product R-OH + H 2O

Inner Mitochondrial Membrane NADPH

NADP

+

FPox

crystallography-derived models of CYP2R1 [18] and CYP24A1 [19]. Mutational analyses to pinpoint residues involved in contact with the main functional groups (hydroxyls) or hydrophobic cis-triene of the vitamin D substrate, as well as define those amino acid residues which are closest to the hydroxylation-sensitive 1aposition in CYP27B1 or the side-chain C-23 to 27 carbons in the side-chain hydroxylases (CYP2R1, CYP24A1, and CYP27A1), are currently in progress in various laboratories.

2+

NHI-Fe

Reductase

Ferredoxin

FPred

NHI-Fe

3+

3+

P450-Fe

Cytochrome P450

2H

+

2+

P450-Fe

R-H + O 2 substrate

Electron transport chains and protein components of the vitamin D hydroxylases. (A) In the endoplasmic reticulum, electron equivalents from nicotinamide adenine dinucleotide phosphate (NADPH) are captured by the NADPH P450 reductase (also known as P450 oxidoreductase, POR). The two electrons from NADPH are transferred sequentially to the microsomal P450 (e.g., CYP2R1). (B) In mitochondria, NADPH is oxidized by the flavoprotein, ferredoxin reductase, which transfers single electrons through a pool of ferredoxin ironesulfur proteins to the mitochondrial P450s on the inner membrane. In both systems, the P450þ electron-transfer protein interaction is not well characterized, but is presumed to occur through close proximity of the heme with the redox center. The electron equivalents are used in the two-electron reduction of molecular oxygen at the heme iron atom, consuming two protons to liberate H2O and to generate a reactive ironeoxygen intermediate capable of regio- and stereo-selectively abstracting a hydrogen atom from a substrate ligand and replacing it with a hydroxyl group or, more generally, mediating P450-specific oxidation reactions.

FIGURE 3.2

secondary structures folded around the distal face of the heme group so that the substrate can be brought to ˚ of the iron atom for hydroxylation. within 3.2A Attempts to identify the key substrate-binding residues were originally guided by homology models [13e17] based upon 10e20 available crystal structures from unrelated soluble prokaryotic CYPs. Recently, the study of the active site of vitamin-D-related CYPs has been further advanced by the emergence of X-ray

As outlined above, there has been no shortage of CYPs proposed as candidates for the title of physiologically relevant vitamin D3-25-hydroxylase. Early work suggested that there were both mitochondrial and microsomal 25-hydroxylase enzyme activities [20,21], and experiments with the perfused rat liver suggested that these might be a low-affinity, high-capacity mitochondrial enzyme and a high-affinity, low-capacity microsomal enzyme [11,22]. More than three decades later we can use several criteria to decide the answer to the question: which CYP is the physiologically important 25-hydroxylase in vivo? These criteria include: a. substrate specificity towards D3 and D2 substrates b. Km and Vmax and enzymatic properties of the expressed enzyme c. tissue and subcellular location d. occurrence of natural mutations e. disease consequence of gene deletion or mutation in human and animal models. Currently, based upon available data for these criteria, we can conclude that the answer to the above question is still not fully resolved, since there is still insufficient evidence that deletion of any single CYP results in a rickets phenotype in the mouse or vitamin D deficiency/rickets in humans. Indeed, it is possible that in vivo several CYPs could contribute to 25-hydroxylation of vitamin D and its analogs under a broad substrate concentration range. However, all available evidence suggests that CYP2R1 is probably the physiologically relevant enzyme at normal vitamin D concentrations (nM) but that it is possibly backed up by others that operate when substrate concentrations rise into the pharmacological range (high nMelow mM). Consequently, we have reviewed relevant information firstly on CYP2R1 and then the other candidate CYPs.

CYP2R1 The discovery of CYP2R1 in 2003 by a research group headed by David Russell and David Mangelsdorf [23]



27

Sequence alignments of structurally determined or predicted secondary structures for vitamin D hydroxylases. Residues conserved in both mitochondrial and microsomal P450s (shaded) are structurally or functionally important, although elements of substrate recognition, binding, and specificity are inherently less conserved. The locations of missense mutations leading to autosomal CYP27A1 deficiency (cerebrotendinous xanthomatosis, CTX) and CYP27B1 deficiency (vitamin-D-dependent rickets type IA) are indicated by the dark shading. Heme-binding residues and ERR-triad residues are also indicated.

FIGURE 3.3


28


arguably ended a three-decade-long search for the elusive physiologically relevant vitamin D3-25-hydroxylase. CYP2R1 satisfies most, if not all, of the criteria listed above to describe the location and properties of the enzyme activity first defined by Bhattacharrya and DeLuca in the early 1970s [20,21]. CYP2R1 is a liver microsomal cytochrome P450 that is 501 amino acids in size and was cloned from mouse and human and shown by real-time PCR to be primarily expressed in liver and testis [23]. The full amino acid sequence of hCYP2R1 is shown in Figure 3.3 and alignments of all known CYP2R1 isoforms (current databases hold 47 species) reveal that it is highly conserved in comparison to other CYP2 family members which are not highly conserved between species presumably because they are usually broad-specificity, xenobioticmetabolizing enzymes [24]. The initial Cheng et al. studies demonstrated that CYP2R1, unlike all other putative 25hydroxylases, would 25-hydroxylate both vitamin D2 and vitamin D3 equally well at physiologically relevant substrate concentrations [23]. Subsequent work [25] using nanomolar substrate concentrations of [3H]1a(OH)D2, a vitamin D2 analog, has reinforced the finding that transfected mouse and human CYP2R1 enzymes are able to synthesize the predominant in vivo metabolite 1,25(OH)2D2, and not 1,24(OH)2D2, the minor in vivo product of 1a(OH)D2 which is also the major in vitro product of 1a(OH)D2 incubated with CYP27A1. Recent work [18] using bacterially expressed human CYP2R1 protein in a solubilized system revealed enzyme kinetic properties consistent with both of the earlier studies. hCYP2R1 showed Km values of 4.4, 11.3, and 15.8 mM for vitamin D3, 1a-OHD2, and 1a-OH-D3 respectively, while Kcat values of 0.48, 0.45, and 1.17 mol/min/mol P450 were observed for the same three substrates. As defined in the associated LC analysis (Fig. 3.4), the regioselectivity of hCYP2R1 was clearly confined to the C-25 position with no peaks corresponding to 24- or 26-hydroxylated products, this being in sharp contrast to the findings with CYP27A1 [18, supplemental material]. Furthermore, CYP2R1 failed to metabolize 25(OH)D3, cholesterol, or 7-dehydrocholesterol thereby demonstrating a high specificity for the C-25 position on vitamin D and not other sterol substrates. Thus, the evidence suggests that CYP2R1 has the enzymatic properties needed for a vitamin D-25-hydroxylase capable of appropriately activating known vitamin D precursors in vivo. Strushkevich et al. [18] also solved the crystal structure of a functional form of CYP2R1 in complex with vitamin D3, this representing the first crystal structure of a vitamin-D-related CYP. The crystal structure generally confirmed the helical nature and binding pocket of CYP2R1 predicted from other CYPs using homology modeling [13]. Co-crystallized vitamin D3 in the CYP2R1 occupied a position with the side chain pointing

towards the heme group, but somewhat paradoxically, it was not optimally placed for hydroxylation, since the C˚ from the heme iron. It is unclear at 25 carbon was 6.5 A this point if the substrate was trapped in an ingress channel or if there is some other explanation for the data. Another piece of evidence that strengthens the case for CYP2R1 being the vitamin D3-25-hydroxylase is the finding of a human Leu99Pro mutation in a Nigerian family which results in vitamin-D-dependent rickets, type 1B [26]. This disease was postulated four decades ago [27] following the elucidation of vitamin D metabolism. Unfortunately, the genetic nature of the Leu99Pro mutation of CYP2R1 was determined by Cheng et al. [5], a decade after the initial identification of the Nigerian rachitic patient, making patient and family follow-up difficult. However, subsequent genetic analysis of exon 2 of CYP2R1 by Cheng et al. [5] in 50 Nigerian individuals revealed one heterozygote with the leu99pro mutation suggesting that there may be a founder gene effect in the Nigerian population, and where vitamin D deficiency is quite prevalent [28]. Though the Leu99 residue is not in a region of the CYP2R1 coding for substratebinding domain, it is involved in water-mediated hydrogen bonding to the Arg445 amide nitrogen located three residues from the heme coordinating Cys448, and thus a Leu99Pro mutation probably results in a misfolded protein with little or no enzyme activity. Numerous attempts to bacterially express both hCYP2R1 with a Leu99Pro mutation and wild-type hCYP2R1 simultaneously failed, leading Strushkevich et al. [18] to conclude that CYP2R1 with Leu99Pro is misfolded or shows poor protein stability. To date there is no animal model or mouse knockout with defective CYP2R1 reported in the literature to confirm that CYP2R1 is essential and thus the possibility that there is some redundancy in the liver vitamin D3-25-hydroxylase “family” of enzymes that can compensate for the deletion of CYP2R1 cannot be confirmed. Recently, a genome-wide association study of the genetic determinants of serum 25-hydroxyvitamin D concentrations [29] concluded that variants at the chromosomal locus for CYP2R1 (11p15) were the second strongest association of only four sites, DBP or Gc, CYP24A1 and 7-dehydrocholesterol reductase (DHR7) being the others. Notably, variants of the other 25-hydroxylases such as CYP27A1 were not identified to be associated with serum 25(OH) D concentrations, again arguing for the fact that they play no role in 25-hydroxylation of vitamin D at physiological substrate concentrations.

CYP27A1 This was the first cloned vitamin D-25-hydroxylase in the early 1990s again discovered by David Russell’s group. CYP27A1 is a liver mitochondrial cytochrome


29


0.05

0.05 1 (OH)D3

1 (OH)D2

0.04

Absorbance (265nm)

0.04 1 ,25(OH)2D2

0.03

0.03

0.02

0.02

0.01

0.01

0.00

0.00

0

5

Comparison of the enzymatic properties of two vitamin D-25-hydroxylases: CYP2R1 and CYP27A1. The substrates used are the prodrugs, 1a(OH)D2 and 1a(OH)D3, to gauge the hydroxylation site and efficiency of the two vitamin D 25-hydroxylases towards D2 and D3 family members. Straight-phase chromatograms of metabolites from (A) in vitro reconstituted CYP2R1 enzyme [18] and (B) transiently transfected CYP27A1 in COS-1 cells [31]. The lack of CYP27A1-mediated 25-hydroxylation towards 1a(OH)D2 is evident, although the major product, 1a,24(OH)2D2, is detectable in serum of animals given large doses of vitamin D2 [33] and is a known VDR agonist [126].

FIGURE 3.4

(A) CYP2R1

10

15

20

1 ,25(OH)2D3

0

5

10

15

20

Absorbance (265nm)

(B) CYP27A1

Time (minutes)

Time (minutes)

P450 with a homolog in >56 species that is 531 amino acids in size in the human. It was originally cloned from rabbit but also from human [30,31]. Even the earliest claims, that CYP27A1 was a vitamin D25-hydroxylase, were controversial as the purified liver enzyme seemed to be a better cholesterol-26-hydroxylase than vitamin D-25-hydroxylase. Thus, it was proposed as a bifunctional enzyme involved in both bile acid and vitamin D metabolism [32]. Work with the recombinant protein demonstrated that CYP27A1 is a low-affinity, high-capacity vitamin D3-25-hydroxylase that also 25-hydroxylates 1a-OH-D3 but seems incapable of the 25-hydroxylation of vitamin D2 or 1a-OH-D2 catalyzing 24-hydroxylation to 24-OH-D2 or 1,24S-(OH)2D2 instead (Fig. 3.4) [31,33]. Figure 3.4 shows that while CYP27A1 exhibits the ability to 24- and 26(27)-hydroxylate 1a-

OH-D3, its primary site of hydroxylation is C25; whereas with 1a-OH-D2, this switches to C24-hydroxylation with a small amount of 26(27)-hydroxylation. The inability of CYP27A1 enzymatic properties to explain the formation of 25(OH)D2 in animals in vivo became the main impetus for the search for an alternative 25-hydroxylase that culminated in CYP2R1 [23]. Parallel enzymatic work with bile acid substrates clearly showed that CYP27A1 could 25- and 27-hydroxylate the side chain of cholesterol and play a role in the trimming of C-27 sterols without the secosteroid, open B-ring nucleus [34]. The same workers performed mutagenesis studies which established the important residues involved in ferredoxin interaction. Although there is currently no crystal structure of CYP27A1, numerous models have been proposed for the enzyme


30


predicted from other CYPs using homology modeling [13,35]. Until the recent emergence of crystal structures of CYP2R1 and CYP24A1, these models offered the best structural insights into the general structure and substrate-binding pockets of vitamin-D-related CYPs. Several pieces of biological or clinical information argue against CYP27A1 being the physiologically relevant vitamin D-25-hydroxylase. Firstly, the CYP27A1null mouse phenotype does not include rickets or any other bone lesion [36]. However, this animal model is complicated by the absence of any significant bile acid defect either. Secondly, though human CYP27A1 mutations have been documented in the literature, these result in a bile-acid-related condition known as cerebrotendinous xanthomatosis (CTX) rather than rickets [37]. Affected individuals usually have normal serum 25(OH) D, though some of these individuals can exhibit low serum 25(OH)D and a type of osteoporosis [38]. Current opinion is that CTX is a defect in bile acid metabolism and that the bone disease is the result of malabsorption of dietary vitamin D caused by bile acid insufficiency rather than an inadequate 25-hydroxylase enzyme activity [39]. Thirdly, the genome-wide association study of the determinants of serum 25-hydroxyvitamin D concentrations [29] concluded that variants at the locus for CYP2R1 (11p15) but not CYP27A1 are associated with serum 25(OH)D concentrations arguing for the fact that CYP27A1 plays no role in 25-hydroxylation of vitamin D at physiological substrate concentrations. A more likely possibility for an in vivo role for CYP27A1 in vitamin D metabolism is as a pharmacologically relevant 25-hydroxylase, in the activation of the prodrugs, 1a-OH-D3 and 1a-OH-D2. The 1a-hydroxylated vitamin D analogs are popular prodrugs in the treatment of osteoporosis and metabolic bone disease or the secondary hyperparathyroidism associated with chronic kidney disease (CKD) [40]. Thus establishing the activating enzyme needed to convert them into active 25-hydroxylated products has some clinical importance. It is worth noting that in vitro CYP27A1 synthesizes 1,25-(OH)2D3 and 1,24S-(OH)2D2, metabolites from 1a-OH-D3 and 1a-OH-D2 respectively, 24-hydroxylated compounds such as 1,24S-(OH)2D2 are also observed in vivo following administration of pharmacological amounts of vitamin D2 compounds [33,41,42]. Thus, CYP27A1 may contribute to the metabolism of vitamin D compounds, including the 1a-hydroxylated compounds when they are present at high concentrations, but seems unlikely to be involved in vitamin D metabolism at physiologically relevant concentrations.

Other CYPs Over the past three decades, there have been numerous reports that in addition to CYP2R1 and

CYP27A1, a number of other specific microsomal CYPs, partially purified from tissues or cells, or studied in bacterial or mammalian expression systems can 25-hydroxylate spectrum vitamin D substrates, but only at micromolar substrate concentrations. These include: CYP2D11, CYP2D25, CYP2J2&3, and CYP3A4 (see Table 3.1). Some of these are expressed in one mammalian species (e.g., pig or rat) and have no obvious human equivalent, show gender differences not observed for human vitamin D-25-hydroxylation in vivo or fail to 25-hydroxylate vitamin D2 or 1a-OH-D2. Again, as with CYP27A1, lack of regioselectivity for the C-25 position surfaces as an important distinguishing feature compared with CYP2R1, as many other microsomal CYPs (e.g., CYP3A4) catalyze the 24hydroxylation of vitamin D2 and D3 compounds [43e45]. Based upon the emergence of the strong case for CYP2R1 being the vitamin D-25-hydroxylase, the pursuit of these other nonspecific CYPs is becoming less urgent, but at least one of these CYP3A4 deserves special mention. A multifunctional nonspecific enzyme such as CYP3A4, which is estimated to metabolize up to 50% of known drugs, would probably not attract special interest here were it not for the fact that recently it been shown to be selectively induced by 1a,25(OH)2D3 in the intestine [45e47]. CYP3A4 has been shown to 24- and 25-hydroxylate vitamin D2 substrates more efficiently than vitamin D3 substrates [43,44], and also 23R- and 24S-hydroxylates the already 25-hydroxylated 1a,25-(OH)2D3 [45]. However, CYP3A4 is known to have Km values for vitamin D compounds in the micromolar range, a property that questions its physiological but not pharmacological relevance. Recent work [48,49] has demonstrated that both human intestinal microsomes and recombinant CYP3A4 breakdown 1a,25-(OH)2D2 at a significantly faster rate than 1a,25(OH)2D3 suggesting that this nonspecific cytochrome P450 might limit vitamin D2 action preferentially in selective target cells where it is expressed (e.g., intestine), particularly in the pharmacological dose range. Such an observation may also offer an explanation for the welldocumented lower toxicity of vitamin D2 compounds as compared to vitamin D3 compounds in vivo, the vitamin D2 compounds not causing such severe hypercalcemia by virtue of reduced effects on intestinal calcium absorption. The same type of mechanism involving differential induction of nonspecific CYPs, such as CYP3A4, may also underlie the occasional reports of drugedrug interactions involving vitamin D, where co-administered drug classes, e.g. anticonvulsants [50,51], causing accelerated degradation of vitamin D2 over vitamin D3. Thus, while CYP3A4 might be occasionally considered as a vitamin D-25-hydroxylase, its main relevance to vitamin D metabolism may lie in its


25-HYDROXYVITAMIN D-1a-HYDROXYLASE

involvement in inactivation of vitamin D compounds at high concentrations.

25-HYDROXYVITAMIN D-1a-HYDROXYLASE The 25-hydroxyvitamin D-1a-hydroxylase has been investigated virtually from the day that the hormone 1a,25-(OH)2D3 was discovered [3,52]. As soon as it became apparent that the 25-hydroxyvitamin D-1ahydroxylase was a central regulatory axis of the calcium and phosphate homeostatic systems, subject to up-regulation by PTH, low Ca2þ and low PO3e 4 levels [1,53,54], the need to define its activity in biochemical terms was apparent. It was quickly recognized that serum 1a,25(OH)2D3 was predominantly made in the kidney [3,55] with a PTH-regulated form located in the proximal convoluted tubule and a calcitonin-regulated form in the proximal straight tubule [56e59]. Biochemical investigations showed that the enzyme involved was a mixed-function oxidase with a cytochrome P450 component [60], but the exact molecular description of this enzyme took another 25 years to unravel. During this time, there have been emerging reports of so-called “extra-renal” 25-hydroxyvitamin D-1a-hydroxylase activity in several sites including placenta, bone, and macrophage [61e66], which evoked the question of whether there was more than one cytochrome P450 with 25-hydroxyvitamin D-1a-hydroxylase activity. Unlike with the liver vitamin D-25-hydroxylase, this does not appear to be the case and with the cloning of CYP27B1 as a single gene, this story has become much simpler. In 1997, several groups coincidentally cloned, sequenced, and characterized CYP27B1 from rat, mouse, and human species [67e69]. Though many of these groups used kidney libraries as the source of the enzyme, interestingly other groups reported finding the same CYP27B1mRNA in keratinocyte [70] and human colonic cell HT-29 [71] libraries, suggesting that the enzyme was identical in all locations. Subsequently, it has been confirmed that the CYP27B1 protein is identical in all locations [4,71,72], whether renal or extrarenal, though the regulation in these different tissue locations almost certainly involves different hormones and effectors. hCYP27B1 is a 507-amino-acid protein with a molecular mass of ~55 kDa. Best available information suggests that the enzyme 1a-hydroxylates 25(OH)D2 and 25(OH)D3 equally efficiently to give the active metabolite of each form of the vitamin. The genetic rachitic condition termed vitamin D dependency rickets (VDDR Type I), in which the 1a-hydroxylase enzyme was absent or defective, presumably due to mutation of CYP27B1, had been recognized in the early 1970s by

31

Fraser and colleagues [27,73]. These authors showed that patients had low or absent serum 1,25-(OH)2D and they could be successfully treated using small amounts of synthetic 1,25-(OH)2D3. VDDR Type IA involves a resistant-rickets phenotype, characterized by hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and undermineralized bone. It is essentially cured by physiological (microgram) amounts of 1,25-(OH)2D3 or pharmacological (milligram) amounts of 25(OH)D3 or vitamin D, which is consistent with a block in 1a-hydroxylation activity [27]. Subsequent work mapped the CYP27B1 gene to 12q13.1eq13.3, which is the same location established for the VDDR Type I disease [67]. Human CYP27B1 mutations occur throughout the gene (Fig. 3.5) resulting in defective and misfolded proteins with little or no activity [74e78]. At least two groups have created CYP27B1-null mice [79,80], which exhibit a lack of 1a-hydroxylated metabolites in the blood and tissues, revealing that CYP27B1 is the sole source of 1,25-(OH)2D in the body. The mouse phenotype mirrors human VDDR Type IA in terms of resistant rickets. The animals also show a reduction in CD4- and CD8-positive peripheral lymphocytes and female mice are infertile [79]. Detailed bone histomorphometric analyses of the CYP27B1 and CYP27B1/ PTH double knockout mice established that 1,25(OH)2D3 deficiency resulted in epiphyseal dysgenesis and only minor changes in trabecular bone volume [81]. Bikle and colleagues showed that CYP27B1 is also required for optimal epidermal differentiation and permeability barrier homeostasis in the skin of mice [82]. Administration of a normal diet supplemented with either small amounts of 1,25-(OH)2D3 or use of a high-calcium “rescue diet” largely corrects the mineral metabolism and bone defects seen in the CYP27B1-null mouse [83e85]. Tissue-specific knockout of the mouse CYP27B1 gene in chondrocytes has been achieved and suggests that local production of 1,25-(OH)2D3 plays a role in growth plate development [87,88]. The availability of specific CYP27B1mRNA and antiCYP27B1 protein antibodies has allowed for a more rigorous exploration of the extrarenal expression of the enzyme. Diaz et al. [89] used Northern analysis and RT-PCR to examine mRNA expression in human synctiotrophoblasts and concluded that there was CYP27B1 expression in human placenta. Using similar techniques, several groups reported low but detectable expression of CYP27B1 in a variety of cultured cell lines and freshly isolated cell explants, e.g. prostate and colonic cells [90e92]. Immunohistochemistry data from analysis of animal and human tissues has revealed the presence of the CYP27B1 protein in several tissues purported to express 1ahydroxylase activity, e.g. skin, colon, macrophage, prostate, breast [4,72]. Not all studies have supported the


32


L99P

CYP2R1 -helix

ER

A’

B

A

B’

C

D

E

F

G

H

I

J

A216P

K259R

ER

B

A

B’

C

D

E

F

G

H

I

J

G102E R107H P112L G125E P143L D164N E189GL

Q65H

-strand

B

A

B’

C

D

E

F

G

H

I

J

R

K

508

L 3a 4 3b

1

5

P382S R389CGH T409I R429P V478G R453C P497R

T321R S323Y R335P L343F

ER

A’

531

L 3a 4 3b

CYP27B1 -helix

R

K

1

-strand

5

P384L R395CHQS P401R G472A N403K P441S R474QW T339M D354G R405QT R479CGS

CYP27A1 A’

501

L 3a 4 3b

R127QW R137QW G145ER

-helix

K

1

-strand

R

5

Missense mutations identified in patients with 25-hydroxylase deficiency rickets (VDDR-Type IB, CYP2R1), the cholesterol and bile acid metabolism disorder, cerebrotendinous xanthomatosis (CTX, CYP27A1), and vitamin-D-dependent rickets type I (VDDR Type IA, CYP27B1). The relative positions of the conserved a-helices and b-strands are indicated. Heme-binding residues are denoted by the open diamonds. The positions of the ERR-triad residues in the K-helix (E R R) and meander region (E R R) form the core of a structurally rigid motif which may stabilize ferredoxin binding and electron transfer to the heme iron. Secondary structures positioning determinants of substrate orientation in P450 crystal structures include the b-1, A-helix, B’-helix, B’/C loop, F/G loop, I-helix, b-3a, b-3b, and b-5 structures. Figure adapted from Malloy and Feldman [78], previously tabulated mutations [113], and other literature [76,77,127e131].

FIGURE 3.5

conclusion that CYP27B1 is expressed outside of the kidney in normal, nonpregnant animals. Using a b-galactosidase reporter system, Vanhooke et al. [86] found no evidence for expression of CYP27B1 in murine skin or primary keratinocytes, although there was expression in kidney and placenta. It is possible that the lack of detection of low-abundance extrarenal CYP27B1 transcripts is due to some inherent insensitivity of the b-galactosidase reporter system, whereas it is sufficiently sensitive to detect abundant renal CYP27B1 transcripts. Despite the fact that the existence of the extrarenal 1ahydroxylase remains tentative, there has been much speculation about the role of this enzyme in health and disease [93e95]. It is now widely believed the enzyme exists in nonrenal tissues to boost local production of cellular 1,25-(OH)2D3 in a paracrine/intracrine system. Such a role would suggest that cellular 1,25-(OH)2D3 concentrations in extrarenal CYP27B1 tissues might be higher than in the tissues of the classical endocrine system which depend upon renally synthesized, blood-derived 1,25-(OH)2D3 at a concentration around

10e10 to 10e9M (e.g., intestine, bone, parathyroid gland). In turn, the genes regulated in extrarenal tissues (e.g., macrophage, colon, prostate) might be a less-sensitive cell differentiation and antiproliferative subset, known to be regulated in cancer cell lines at 1,25-(OH)2D3 concentrations of 10e8 to 10e7 M under cell culture conditions. A role for the extrarenal CYP27B1 is also consistent with the finding that serum 25(OH)D levels are associated with various health outcomes from bone health to cardiovascular health. In particular, low serum 25(OH)D levels are associated with increased mortality for colon, breast, and prostate cancer; increased autoimmune diseases and greater susceptibility to tuberculosis; increased cardiovascular diseases and hypertension. The presence of CYP27B1 in cells of the colon, breast, prostate, monocytes/macrophages, and vasculature could explain why serum 25(OH)D levels are so critical to the normal functioning of these tissues. Chronic kidney disease (CKD) with five stages defined by decreasing glomerular filtration rate (GFR) is well established to be accompanied by a gradual fall


25-HYDROXYVITAMIN D-1a-HYDROXYLASE

in serum 1,25-(OH)2D3 (normal range ¼ 20e60 pg/mL), widely assumed to be due to a gradual decline in CYP27B1 activity [96]. Whether this is in turn due to loss of the CYP27B1 protein mass caused by renal damage is debatable. It is possible that the fall in serum 1,25-(OH)2D3 to values below 20 pg/mL by the end of CKD Stage 2 could be due in part to increased FGF23 levels, a known down-regulator of CYP27B1 expression in normal kidney cells [97]. Recent reports of marked increases in FGF23 levels in CKD Stage 5 dialysis patients with phosphate retention are consistent with FGF23 playing a major role in vitamin D dysregulation and mortality in chronic kidney disease [98]. The regulation of CYP27B1 (summarized in Fig. 3.6) has been a major focus ever since the enzyme’s discovery in the early 1970s [1]. Ca2þ and PO3 4 ions, probably through the hormones, PTH, calcitonin, and FGF23, regulate CYP27B1 expression through complex signal transduction processes [59,99,100,101], while 1,25-(OH)2D3, the end-product of the enzyme, downregulates its own synthesis at the transcriptional level by VDR-mediated action at the level of the CYP27B1 gene promoter [100,102,103]. Evidence is also accumulating that CYP27B1 expression is down-regulated through DNA methylation and up-regulated through DNA demethylation [103,104]. While it is logical to isolate CYP27B1 from the rest of the calcium/phosphate homeostatic system, in practice there is a reciprocity between CYP27B1 and CYP24A1 that suggests that the factors up-regulating one enzyme, down-regulate the other. This is evident in the isolated perfused kidney from the rat fed a low-Ca vitamin-D-deficient diet, or low-PO4 vitamin-D-deficient diet which is in the 1ahydroxylation mode, and which over a 4-hour perfusion period after being exposed to its 25(OH)D3 substrate turns off CYP27B1 expression and 1a-hydroxylation and turns on CYP24A1 and 24-hydroxylation [105]. The vitamin D metabolic system seems ideally designed to avoid synthesis of excessive amounts of the hormone IFN TLR2/4 Low

Other inflammatory Cytokines: IL-1 , IL-15, IGF-I, EGF, TGF-

Ca2+

PTH

CYP27B1

Epigenetic

1 ,25(OH)2D3

Low PO43-

Calcitonin

FGF23, leptin

NFRegulation of CYP27B1. Factors identified which regulate positively or negatively the expression of renal or extrarenal CYP27B1.

FIGURE 3.6

33

and also to degrade the hormone, or even its substrate, by superinduction of catabolic processes including CYP24A1. In the VDR-null mouse, we see a complete breakdown of this autoregulation process because CYP27B1 is not suppressed by excessive 1,25-(OH)2D3 production and CYP24A1 is not actively stimulated, both steps requiring VDR-mediated events. The regulation of the extrarenal 1a-hydroxylase has also received attention over the last couple of decades. What is clear is that the renal and extrarenal enzymes are regulated by different factors: the kidney CYP27B1 by calcium and phosphate homeostatic hormones described above; while the extrarenal enzyme is regulated by tissue-specific factors, including cytokines. Adams et al. [106] have shown that macrophages in the granulomatous condition, sarcoidosis, are driven by proinflammatory cytokines, such as g-interferon, which also stimulate extrarenal CYP27B1 activity, that can cause excessive serum 1,25-(OH)2D3, which left unchecked results in hypercalciuria and hypercalcemia. The mechanism of g-interferon-mediated up-regulation of CYP27B1 appears to involve the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta [107,108]. Also, the usual CYP24A1 counter-regulatory mechanism seems to have been replaced in the monocyte/macrophage system by an inactive splice-variant of CYP24A1 allowing the CYP27B1 activity to go largely unchecked and cellular 1,25-(OH)2D3 to rise to super-normal levels [109]. The nature of the down-regulator(s) of the extrarenal CYP27B1 in these and other cells of the immune system remains largely unknown. Recently, the normal regulation of the monocyte/ macrophage CYP27B1 system was elucidated [95,110,111]. Toll-like receptors (TLRs) on the cell surface respond to the presence of bacteria (e.g., M. tuberculosis) with a signal transduction process which results in upregulation of VDR and CYP27B1. Uptake of 25(OH)D bound to its blood carrier DBP, allows the cells to then manufacture 1,25-(OH)2D3, which in turn stimulates VDR-mediated gene transcription of cathelicidin. Cathelicidin is an antimicrobial peptide, which specifically kills M. tuberculosis. Stubbs et al. [112] have demonstrated the existence of a high-VDR, CYP27B1expressing subpopulation of immune cells making cathelicidin that can be selected by cell-sorting techniques in CKD Stage 5 dialysis patients treated with high doses of cholecalciferol (40 000 IU twice per week). Since these patients are virtually devoid of circulating 1,25-(OH)2D3 because of their low renal CYP27B1 activity, the data suggest that the monocyte/macrophage extrarenal CYP27B1 survives during renal failure and is responsible for cathelicidin production.


34


ADDITIONAL TOPICS Crystal Structures and Homology Models of Vitamin D-related CYPs Dating back to their first description as enzyme activities in the 1970s, biochemists have wondered about the structures of the family of vitamin-D-related hydroxylases. The cloning and characterization of these CYPs during the 1990e2003 period opened the door to defining the structures of these proteins, particularly the nature of their substrate-binding domains. Though the elucidation of these structures has been a significant challenge it has been aided by the fact that the vitaminD-related cytochrome P450s are part of a large group of highly conserved proteins across all phyla that metabolize both endogenous and exogenous small-molecule substrates. Consequently, structural information gleaned from the study of all cytochrome P450s has established that though the amino-acid sequences show wide variability, the secondary and tertiary structures have been highly conserved across phyla. Over the past 20 years, genome sequencing projects have yielded dozens of species orthologs for each of the various cytochrome P450s and the number of available crystal structures has increased impressively, starting with soluble bacterial CYPs but reaching over the past few years to membrane-bound mammalian CYP structures including some of the 59 human CYPs. These crystal structures together with the parallel advancement in protein homology modeling led to the emergence of a number of useful homology models for vitamin-D-related CYPs, first for CYP27A1 then for CYP24A1 [13e17,113]. In the last 2 years, the first crystal structures of vitamin-D-related CYPs in the form of the human microsomal CYP2R1 and the rat mitochondrial CYP24A1 have been released [18,19] and these have validated the homology modeling approach very well. In addition, two bacterial vitamin D hydroxylases capable of sequentially hydroxylating vitamin D3 to 1a,25 (OH)2D3 at production levels, CYP105A1 from Streptomyces griseolus (2zbz.pdb) [114] and P450 Vdh from Pseudonocardia autotrophica (3a4g.pdb) [115], have been determined. In general, the structures of all cytochrome P450s are similar and comprise a series of highly conserved helices (designated AeL) connected by loops and b-sheet structures (see Fig. 3.7, top). Though sequence homology is poorly conserved in the cytochrome P450 superfamily, the presence of various highly conserved heme-binding and structurally or functionally important amino acids and crystallographically determined secondary structure make it relatively easy to prepare useful sequence alignments for secondary structure prediction, model building, and site-directed mutagenesis studies. All

CYPs contain a cysteine residue near to the C-terminus (see Fig. 3.3) to which the heme group attaches. Heme binding is stabilized by an additional 4e5 amino acid sidechains which hydrogen bond with the two heme propionic acid groups. The heme-binding domain is further stabilized by a number of structurally implicit water molecules and protein backbone interactions which extend a large measure of structural stability into the surrounding secondary structures. Other structural motifs such as b-sheets, hydrophobic clusters, the ERR-triad, the ferredoxin, or NADPH-P450 reductase binding site, and remarkably well-conserved sidechain hydrogen bonds between secondary structures further maintain the folded protein and the characteristic structure of a cytochrome P450 [13]. The N-terminus of the mitochondrial CYPs contains an ~30-amino-acid targeting sequence which is cleaved during mitochondrial import. The N-terminus of the mature mitochondrial and the microsomal CYPs is thought to be membraneassociating. The substrate-binding pocket is formed by several secondary structures folded around the distal face of the heme group that precisely position the ˚ of the heme iron atom. An analsubstrate within ~3.2 A ysis of the heme-ligand geometry of 49 substrate-bound crystal structures revealed the hydroxylation target carbons actually adopt a spatially conserved orientation to the heme iron and this can be triangulated (as shown in Fig. 3.7, middle) for use in docking studies [16]. Somewhat paradoxically, none of the published crystal structures of the vitamin D hydroxylases have been very useful in understanding how a vitamin D substrate leaves the hydrophobic membrane, gains entry to the active site through a substrate access channel, and is precisely positioned in the active site. Given that the cytochrome P450 is associated with the membrane, the prevailing paradigm is that the lipid bilayer of the membrane flows into the substrate access channel and the substrate floats or diffuses in. Unfortunately, it has been difficult to ascertain exactly where the substrate access channel is located because it is usually closed in CYP crystal structures (and therefore not apparent) or it is situated in different positions in different CYP subfamilies [116]. In some cases, such as CYP3A4 and CYP24A1, the active site is so widely open that one could wonder how the substrate could spend any time in one place. Of the existing vitamin D hydroxylases, only two CYPs have been co-crystallized with vitamin D. In the bacterial CYP105A1 [114] the protein was cocrystallized with its product 1a,25(OH)2D3 in a transient, nonproductive orientation too distant from the heme iron. The vitamin D 3b-hydroxyl was hydrogen bonded to Ser236 (I-helix) and Arg193 (G-helix) which were bridged by a water molecule to Glu232 (I-helix). It was also bound to a water molecule networked to a B0 /C loop backbone carbonyl and a second water molecule


35

ADDITIONAL TOPICS

Modeling, docking, and crystal structure studies of the vitamin D hydroxylases. The top panel depicts a stereographic view of a model of CYP27A1 with 1a(OH)D3 in the heme distal cavity active site. An analysis of hemeligand geometry in cytochrome P450s revealed the existence of a preferred binding orientation for the hydroxylation target carbon (49 crystal structures) and the position of azole nitrogen interacting with the heme iron (18 crystal structures). The atoms of interest were triangulated by calculating the distances to the heme methyl carbons CMA, CMB, and CMD and the averages used to dock 1a (OH)D3 in CYP27A1. A similar process was used to dock 1a,25(OH)2D3 [16] and ketoconazole in CYP24A1. The middle panel shows the triangulated distances to the average substrate target carbon and inhibitor azole nitrogen above the heme. The lower panel depicts 1a(OH)D2 binding in CYP2R1 crystal structure (3dl9.pdb) [18]. Several key hydrogen bonds in the substrate access channel of CYP2R1 are directed towards the A-ring hydroxyls to position the substrate in a hydrophobic active site. These hydrogen bonds are directed towards the backbone carbonyls of Ala250 (G-helix) and Ile301 (I-helix) and to a water molecule (marked with an *) coordinated by Asn217 (F-helix). The substrate access channel opening between the B’-, G-, and I-helices is extensively stabilized by a network of water molecules centered in the B’/C loop and stabilized by Glu306 (I-helix) and Asn126 (B’/C loop).

FIGURE 3.7


36


hydrogen bonded to the 1a-OH. This is the only example, outside of the VDR, where a vitamin D Aring hydroxyl was hydrogen bonded to an amino acid side-chain. In CYP2R1 [18], the A-ring hydroxyls of bound vitamin D structures were hydrogen bonded to the backbone carbonyls at Ala250 (G-helix) and Ile301 (I-helix) (see Fig. 3.8, bottom). As shown in Figure 3.7, bottom and Figure 3.8, bottom, the 3b-OH was also hydrogen-bonded to a water molecule stabilized by Asn217 (F-helix). This emphasizes that the acidebase chemistry of the vitamin D hydroxyls appears to be directed towards networked water molecules in the active site and backbone amide bond carbonyls and nitrogen atoms. If this is true, it will be difficult to predict how vitamin D substrates are positioned in CYP active sites without high-resolution crystal structures showing the positions of structurally implicit water molecules. Even with such data, the CYP2R1 and CYP105A1 structures would not reveal mechanistically how the hydrophobic parts of vitamin D substrates interact with the predominantly hydrophobic residues lining the active site to position the hydroxylation target carbon so precisely for reaction. Hopefully, a clearer picture of substrate binding will be forthcoming in substrate-bound crystal structures of the mammalian vitamin D hydroxylases or the pending release of P450 Vdh mutants obtained by directed evolution co-crystallized with vitamin D3 (3a50.pdb) and 25(OH)D3 (3a51.pdb) [115]. In the meantime, continuing studies of vitamin D docking in CYP27A1, CYP27B1, and CYP24A1 will focus on naturally occurring autosomal mutations [e.g., 117] (Fig. 3.5) and on site-directed mutagenesis studies of hydrophobic contact residues in the active site. The most successful applications of the latter approach were the identification of an Ala-Gly polymorphism at Ala326 in CYP24A1 which affected C24 and C23 pathway catabolism of 1a,25(OH)2D3 [16], a Thr416Met mutation in ratCYP24A1 which had a similar (smaller) effect [14], CYP24A1 mutations at Ile131, Trp134, Leu148, Met246, and Gly499 [15], Phe249 mutations in rat CYP24A1 [118], and Phe248, Ile514, Val515, Leu516 mutations in CYP27A1 [13] among others. However, even with these data sets, a deterministic explanation of substrate binding will be difficult. What can be said for certain is that the hydrophobic core and active site residues are extensively conserved across 40e50 species orthologs in the mammalian CYPs (see Fig. 3.8) and that current research will identify hydrophobic substrate contact residues but will have difficulty in explaining how they work.

Inhibitors of Vitamin-D-related CYP Enzymes The structural analysis of the vitamin-D-related CYPs has been performed in parallel to a search for inhibitors

of these enzymes. Because of the timing of the cloning work and the perception of the relative importance of these vitamin-D-related CYPs most of this work has been directed towards CYP24A1 and to a lesser extent CYP27B1. CYP24A1 inhibitors offer the promise that they would raise intracellular 1,25-(OH)2D3 levels and might have utility as treatments for psoriasis, in cancer therapy, and in the treatment of secondary hyperparathyroidism in CKD. CYP27B1 inhibitors offered the promise that they would lower serum 1,25-(OH)2D3 levels and might have utility as agents to treat forms of vitamin-D-related hypercalcemia: possibly nephrolithiasis, idiopathic hypercalcemia. Sandoz/Novartis initiated a program to synthesize a family of CYP24A1/ CYP27B1 inhibitors based upon chemical modification of the common azole template used successfully for other CYP family members (e.g., general P450 inhibitor, ketoconazole or aromatase inhibitor, letrazole). Schuster et al. succeeded in developing both CYP27B1- and CYP24A1selective inhibitors [119e121] though these were never developed clinically. These inhibitors, most notably VID-400, have been used with some degree of success in animal research settings [122] to try to establish the relative importance of CYP27B1 and CYP24A1 in a target cell context. Another commercial company, Cytochroma Inc., has also developed inhibitors to vitamin-D-related CYPs based upon a vitamin D template and tuning out the VDR-binding properties [123]. Several of these molecules, including CTA-018, are under development as CYP24A1 inhibitors for potential clinical use in CKD, psoriasis, and cancer [124]. All of these vitamin-D-related CYP inhibitors are usually specific enough for use as single-purpose drugs and rarely inhibit other unrelated CYPs and occasionally trigger VDR-mediated gene transcription. Accordingly, their properties indicate that the vitamin-D-related CYPs must have a binding pocket that retains some specificity for its vitamin D substrate but also shares a degree of similarity to the ligandbinding pocket of the VDR and the substrate-binding pockets of nonspecific CYPs.

Future Directions In addition to the predictable improvement in the resolution of the structures of vitamin-D-related CYPs over the coming years, several other future developments can be speculated upon. Firstly, there will be a continued search for natural mutations of any of the known CYPs. Loss of function mutations have manifested themselves as VDDR Type 1A and 1B, but other variants can be predicted as the techniques for exploring the human genome become more powerful and more widely used. It is possible that some of these variants are associated with previously unconnected disease states. Secondly, one can predict that the concept of


37

ADDITIONAL TOPICS

Crystal-structure-based analysis of the conservation of amino acid sequences in mitochondrial vitamin D hydroxylases and substrate binding in the crystal structure of CYP2R1 [18]. The top panel depicts the frequency of conserved residues in CYP27A1 (41 species, excluding nine fish and frog) mapped from sequence alignments onto the crystal structure of CYP24A1. The extensively open active site seen in the crystal structure of CYP24A1 is very likely conserved in CYP27A1 and CYP27B1 and provides a clear view through to the heme. The positions of two mutations causing autosomal CYP27A1 deficiency (cerebrotendinous xanthomatosis, CTX) are indicated. The first residue, Thr339, is highly conserved (with only minor exceptions) in the I-helix of cytochrome P450s and is thought to structurally distort a helical turn necessary for molecular oxygen staging during the redox cycle. The second residue, Arg405, forms a hydrogen bond with the heme A-ring propionate to structurally stabilize the active site. Many microsomal P450s, including CYP2R1, use histidine for the same purpose. In the middle panel, mutation of corresponding residues causes CYP27B1 deficiency (vitamin-D-dependent rickets type I, VDDR-I). The frequency of conserved residues in CYP27B1 (39 species including five fish and frog) appears to be marginally better than CYP27A1. Approximately 40% of the residues are >95% conserved (green) across species and these are mostly in the core of the enzyme and on the proximal heme ferredoxin-binding surface. The less conserved yellow (80e95%), orange (60e80%), and blue ( G, identified by resequencing of the gene in Caucasian samples, showed a statistically significant protective association with risk of colon cancer overall, and particularly for proximal colon cancer [97]. Three other CYP24A1 polymorphisms showed statistically significant association with risk of distal colon cancer: IVS4 þ 1653C > T (lower risk); IVS9 þ 198T > C (increased risk); and þ4125bp 30 of STPC > G (higher risk, in whites only) [97]. Of these, IVS9 þ 198T > C could be involved in the regulation of splicing of an alternate exon [97], a mechanism involved with different patterns of constitutive and inducible CYP24A1 activity [100e102]. The þ4125bp 30 of STPC > G variant, located in the 30 -untranslated region, could modulate mRNA stability [103]. Future studies will be required to confirm the risk associations and to determine the functional significance of the sequence variations.

CYP24A1 in Chronic Kidney Disease Chronic kidney disease (CKD) shows steadily increasing worldwide incidence due to an aging population and to augmenting obesity with its associated complications of hypertension and adult-onset diabetes [104]. Stages 3 and 4 CKD (moderate) are characterized by progressively decreasing kidney function as assessed by glomerular filtration rate. Severe CKD (stage 5) is associated with minimal or altogether absent kidney function and patients require regular dialysis or kidney transplant for survival [105]. With declining renal function, kidney failure patients experience declining 1,25 (OH)2D levels, which causes decreased systemic calcium levels and leads to the development of secondary hyperparathyroidism (SHPT), a disorder characterized by elevated serum levels of parathyroid hormone (PTH) [106e108]. Patients with SHPT of renal failure also develop disorders of bone termed uremic osteodystrophy, characterized by disorganized bone


50

4. CYP24A1: STRUCTURE, FUNCTION, AND PHYSIOLOGICAL ROLE

remodelling and accompanied by loss of bone strength and integrity, with associated morbidity [109e111]. Untreated, SHPT of renal failure has also been associated with increased mortality through increased cardiovascular calcification and associated ischemic events [112e114]. Treatment with vitamin D analogs is advocated for the clinical care of renal patients [115,116]. These compounds act to lower PTH levels close to the normal range and help to maintain normocalcemia, bone health, and cardiovascular integrity [117e120]. Recent results suggest that in CKD, CYP24A1 expression can be aberrantly elevated [121,122]. This may be due in part to processes related to kidney damage, as well as to hormonal replacement therapeutic regimen with current vitamin D analogs. As CYP24A1 inactivates pro-hormonal, hormonal, and analog forms of vitamin D, the aberrant elevation in CYP24A1 expression may contribute to vitamin D insufficiency and exacerbate SHPT in CKD patients. These novel findings suggest that treatment of CKD patients might be another clinical management situation that could benefit from the availability of specific inhibitors of CYP24A1 [123].

24,25(OH)2D and Fracture Repair It has been proposed that 24,25(OH)2D, the enzymatic product of the CYP24A1 activity on the 25(OH)D substrate, might also play a role in fracture repair, but there is limited information available on this putative function of the metabolite. The healing of fractures is a unique postnatal biological repair process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Fracture repair involves a complex multistep process that involves response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation, and bone remodeling. Several studies have described a complex pattern of gene expression that occurs during the course of these events [124e127]. Taken together, results from gene expression monitoring during bone repair suggest that the molecular regulation of fracture healing is complex but recapitulates some aspects of embryonic skeletal formation [128,129]. A role for 24,25(OH)2D in fracture repair is supported by the observation that the circulating levels of 24,25 (OH)2D increase during fracture repair in chickens due to an increase in CYP24A1 activity [130] (Fig. 4.3, top panel). When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25(OH)2D alone resulted in poor healing [131]. However, the strength of healed bones in animals fed 24,25(OH)2D in combination with 1,25 (OH)2D was equivalent to that measured in a control population fed 25(OH)D [131]. These results support

Cyp24a1 expression during fracture healing in chicks and mice. Top panel: the changes in CYP24A1 activity and circulating 24,25(OH)2D concentrations are listed besides the temporal sequence of fracture healing. [, increase; N.D., not determined. The putative expression of a receptor/binding protein for 24,25(OH)2D at day 10 post-fracture is expressed by a question mark. Based on the data described in references 130e133. Bottom panel: quantitative reversetranscription PCR (RT-qPCR) on mRNA extracted from the callus of the fractured right tibia (Fracture) and a diaphysial section of the left nonfractured tibia (Contralateral) of wild-type mice at 14 days postfracture. The expression of Cyp24a1 was significantly increased in the fractured bone, confirming the observations previously obtained in chicks. *, pq13.3, Cytogenet. Cell Genet. 62 (1993) 192e193. [73] D.G. Albertson, B. Ylstra, R. Segraves, C. Collins, S.H. Dairkee, D. Kowbel, et al., Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene, Nat. Genet. 25 (2000) 144e146. [74] S. Fukushige, F.M. Waldman, M. Kimura, T. Abe, T. Furukawa, M. Sunamura, et al., Frequent gain of copy number on the long

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arm of chromosome 20 in human pancreatic adenocarcinoma, Genes Chromosomes Cancer 19 (1997) 161e169. W.M. Korn, T. Yasutake, W.L. Kuo, R.S. Warren, C. Collins, M. Tomita, et al., Chromosome arm 20q gains and other genomic alterations in colorectal cancer metastatic to liver, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization, Genes Chromosomes Cancer 25 (1999) 82e90. G.A. Meijer, M.A. Hermsen, J.P. Baak, P.J. van Diest, S.G. Meuwissen, J.A. Belien, et al., Progression from colorectal adenoma to carcinoma is associated with non-random chromosomal gains as detected by comparative genomic hybridisation, J. Clin. Pathol. 51 (1998) 901e909. G. Sonoda, J. Palazzo, S. du Manoir, A.K. Godwin, M. Feder, M. Yakushiji, et al., Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas, Genes Chromosomes Cancer 20 (1997) 320e328. M.M. Weiss, A.M. Snijders, E.J. Kuipers, B. Ylstra, D. Pinkel, S.G. Meuwissen, et al., Determination of amplicon boundaries at 20q13.2 in tissue samples of human gastric adenocarcinomas by high-resolution microarray comparative genomic hybridization, J. Pathol. 200 (2003) 320e326. M. Werner, A. Mattis, M. Aubele, M. Cummings, H. Zitzelsberger, P. Hutzler, et al., 20q13.2 amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast, Virchows. Arch. 435 (1999) 469e472. M.M. Tanner, S. Grenman, A. Koul, O. Johannsson, P. Meltzer, T. Pejovic, et al., Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer, Clin. Cancer Res. 6 (2000) 1833e1839. H. Wolter, H.W. Gottfried, T. Mattfeldt, Genetic changes in stage pT2N0 prostate cancer studied by comparative genomic hybridization, BJU. Int. 89 (2002) 310e316. M.D. Hogarty, G.M. Brodeur, Gene amplification in human cancers: biological and clinical significance, in: B. Vogelstein, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, second ed., McGraw-Hill, New York, 2002, pp. 115e128. K. Mimori, Y. Tanaka, K. Yoshinaga, T. Masuda, K. Yamashita, M. Okamoto, et al., Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer, Ann. Oncol. 15 (2004) 236e241. J. Reichrath, L. Rafi, M. Rech, T. Mitschele, V. Meineke, B.C. Gartner, et al., Analysis of the vitamin D system in cutaneous squamous cell carcinomas, J. Cutan. Pathol. 31 (2004) 224e231. T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R.M. Maas, B.C. Gartner, et al., Analysis of the vitamin D system in basal cell carcinomas (BCCs), Lab. Invest. 84 (2004) 693e702. M.G. Anderson, M. Nakane, X. Ruan, P.E. Kroeger, J.R. WuWong, Expression of VDR and CYP24A1 mRNA in human tumors, Cancer Chemother. Pharmacol. 57 (2006) 234e240. H.S. Cross, G. Bises, D. Lechner, T. Manhardt, E. Kallay, The Vitamin D endocrine system of the gut e its possible role in colorectal cancer prevention, J. Steroid Biochem. Mol. Biol. 97 (2005) 121e128. R.A. Parise, M.J. Egorin, B. Kanterewicz, M. Taimi, M. Petkovich, A.M. Lew, et al., CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer, Int. J. Cancer 119 (2006) 1819e1828. G. Jones, H. Ramshaw, A. Zhang, R. Cook, V. Byford, J. White, et al., Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1alpha and CYP24) in human nonsmall cell lung carcinomas, Endocrinology 140 (1999) 3303e3310.


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C H A P T E R

5 The Vitamin D Binding Protein DBP Roger Bouillon Laboratory of Experimental Medicine and Endocrinology (Legendo), K.U.Leuven, Herestraat 49 ON1 bus 902, 3000 Leuven, Belgium

the fast-growing side of actin monomers is blocked completely through a perfect structural fit with DBP, demonstrating how DBP effectively interferes with actin-filament formation and this could have major implications for maintaining the microcirculation. Comparisons of the DBP structures with the structure of serum albumin, another family member, reveal a similar topology but also significant differences in local folding. These structural differences explain the unique vitamin-D3-binding and actin-binding properties of DBP. DBP can also bind to fatty acid at a binding site different from the cleft for vitamin D metabolites. Finally DBP can be enzymatically transformed in DBPmacrophage activating factor (DPB-MAF) and thereby influence the function of macrophages and osteoclasts. The different functions of DBP have been reviewed extensively in the previous edition of Vitamin D [10e12] and in several reviews dating back several years [13]. Therefore, this chapter will only summarize its main functions and specifically deal with recent advances over the last 5 years.

INTRODUCTION Group-specific component of serum (Gc globulin) was originally identified in 1959 by serum electrophoresis as a polymorphic protein [1]. At that time its function was not known, although it became useful in population genetics [2] and forensic medicine [3]. Its function as binding protein for all vitamin D metabolites in serum was first discovered by Daiger while looking for polymorphic proteins [4] and independently confirmed by several groups that isolated this binding protein [5,6]. The human serum vitamin-D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to possible roles in inflammation and in the immune system. The functional implications of the polymorphic nature of DBP are still largely unknown. DBP is structurally related to albumin and a-fetoprotein, and the DBP gene is a member of the albumin and a-fetoprotein gene family. Despite large-scale human studies no cases of complete absence of DBP have yet been identified whereas this has been well documented for albumin. While this suggests that DBP may be essential for normal development and survival, DBP-null mice are viable and do not display an obvious phenotype when fed a normal diet [7]. The crystal structure of DBP in complex with the vitamin D3 metabolite 25hydroxyvitamin D3 (25(OH)D3) was solved, as well as the structure of DBP in complex with a vitamin D3 analog. Both structures reveal the vitamin-D-binding site in the N-terminal part of domain I [8]. DBP is also capable of binding monomeric actin and thereby facilitates the depolymerization of actin filaments [9]. The crystal structure of the DBPeactin complex was deter˚ resolution. This structure reveals that mined at 2.4 A


VITAMIN D BINDING PROTEIN: PROTEIN AND GENE STRUCTURE Gene The human DBP gene is located on chromosome 4q11-q13 as revealed by in situ hybridization to metaphase chromosome spreads [14]. This sublocalization overlaps with the known positions of albumin and a-fetoprotein [15]. These three genes have been assigned to chromosome 13 in the rat [16] and to chromosome 5 in the mouse [17]. The rodent chromosomes encoding DBP are syntenic with human chromosome 4, thus

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demonstrating conservation of the DBPea-fetoproteinealbumin linkage in three species. A linkage between DBP and albumin has also been noted in horses and in chickens [18,19]. A fourth gene in the DBP/ albumin/a-fetoprotein family has been described by two groups and named afamin by one group [20] and a-albumin by the other [21]. a-Albumin is selectively expressed in the liver during the late stages of development, suggesting that it may be a phylogenetic intermediate between a-fetoprotein and albumin and may share some of the functions of these proteins [21,22]. Gene analysis indicates that DBP is the oldest member of the family whereas albumin and a-fetoprotein are more recent members. Indeed in zebrafish the only member of this family, based on genome analysis, is DBP and thus preceedes albumin and a-fetoprotein in vertebrate evolution [23]. Physical and meiotic mapping has determined that the organization of the gene family in humans is centromereeDBPealbuminea-fetoproteine a-albuminetelomere. Human DBP mRNA contains 1690 nucleotides and encodes a 458-amino-acid secreted protein. Rat and human DBP genes span 35 and 42 kb, respectively, and both contain 13 exons. The signal sequence of the protein is encoded on the first exon, whereas the last exon contains the entire noncoding 30 -untranslated region. Moreover, DBP cDNAs from the mouse [17], rabbit [24], chicken [25], a turtle, Trachemys scripta [26], and zebrafish [23] have been cloned and sequenced. There is a 16-amino-acid signal peptide, based upon alignment of the sequenced amino terminus of the mature protein [27,28] with the cDNA-predicted sequence of the primary translation product. A number of models have been proposed to account for the evolution of the DBP multigene family and for the shared, triplicated internal domain structure. In one model, the ancestral internal domain is encoded by four exons that subsequently triplicated, creating the present gene encoding a three-domain protein. This domain triplication likely predated vertebrate evolution [29,30]. The separation of a unique DBP gene from this precursor is estimated to have occurred 560e600 million years ago [31,32]. It has been postulated that albumin duplicated from the ancestral gene and began to diverge about 280 million years ago, just after the time of the amphibian/reptile divergence about 350 million years ago. The absence of a larval a-fetoprotein gene in amphibians and the existence of a-fetoprotein in chickens is consistent with this conclusion [31].

Protein Structure The predicted mature DBP proteins in rat, mouse, and rabbit are each 460 amino acids long, two amino acids longer than the human sequence, whereas that of T.

scripta is 466 amino acids in length. There is an N-linked glycosylation consensus sequence in human, rat, and mouse but not rabbit or T. scripta DBP. Based on the presence of sequence homology and nearly identical disulfide bridge pattern in DBP, human serum albumin (HSA), a-fetoprotein, and afamin, the overall folds of these proteins are believed to be homologous [13]. Serum DBP is a polymorphic, monomeric, serum aglobulin of approximately 58 kDa, its size being dependent upon its glycosylation state [6]. Periodic positioning of 28 cysteine residues predicts a characteristic secondary structure demarcated by internal disulfide bonds and defines the presence of three internally homologous domains [27]. The crystal structure of DBP has now been described by several groups [8,33e36]. The three domains of DBP consist entirely of a-helices, ten in domain 1, nine in domain 2, and four in domain 3. The tertiary structure of DBP can be based ˚ on the crystal structure of human DBP, solved to 2.3 A resolution [8]. This crystal consisted of two DBP molecules present in the asymmetric unit, one in complex with 25(OH)D3, and one in apoprotein form. As in human serum albumin (HSA) [37,38], DBP has an all a-helical structure and contains three structurally similar domains. DBP and the other members of its protein family (human serum albumin, afamin, and afetoprotein) are postulated to have evolved from a progenitor that arose from the triple repeat of a 192amino-acid sequence [39]. This three-domain structure has been preserved in DBP; however, the third repeat is largely truncated at the C terminus. The human albumin structure [37,38] indicates that each domain consists of ten a-helices. In the DBP structure, the first domain (residues 1e191) has this a-helical arrangement. However, the second domain (residues 192e378) has a similar topology but helix 7 is replaced by a coil folding, and the third domain (residues 379e458) contains only helices 1e4. The three domains of DBP do not pack in a spherical manner but adopt a rather peculiar shape with two large grooves (Fig. 5.1). Despite the sequence similarity, the only function DBP shares with the other family members is its fatty-acid-binding ability [40,41]. Superposition of the respective domains of the DBP and HSA structures shows similar topologies. Although the folding within each corresponding domain shows some parallels, the global orientation of the three domains in both molecules is strikingly different, resulting in two totally different structures. Residual electron density that can accommodate 25(OH)D3 is observed in DBP holoprotein, close to the biochemically identified vitamin-D-binding residues [10,11]. Residues belonging to helices 1e6 of domain I form the complete vitamin-Dbinding site (Fig. 5.1B). This binding site designation was further confirmed by the elucidation of the structure


VITAMIN D BINDING PROTEIN: PROTEIN AND GENE STRUCTURE

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FIGURE 5.1 DBP-25(OH)D in three dimensions. (A) The DBP three-dimensional structure with its three structural domains. The numbering of the helices is the same as in human serum albumin. (B) The conformation of 25(OH)D in DBP. Helices 1e6 of the apoprotein (light blue) and the holoprotein (DBP-25(OH)D) are superimposed. 25(OH)D is shown in ball-and-stick representation. Two water molecules present in the binding site are indicated as red balls. (Reproduced from figures 2a and 4a of the manuscript of Verboven et al. [8].) Please see color plate section.

of DBP in complex with a vitamin D analog, 22-(mhydroxyphenyl)-23,24,25,26,27-pentanor vitamin D3 with known high affinity for DBP. The vitaminD-binding site is lined predominantly by hydrophobic residues, allowing favorable interactions with the hydrophobic vitamin D3 ligand. Binding of 25(OH)D3 is further improved by hydrogen bond formation of the 25-hydroxyl with Tyr 32 (OeH.O distance ˚ ), and the 3-hydroxyl with Ser 76 (O.HeO 2.85 A ˚ ) and Met 107 (OeH.S distance distance 2.81 A ˚ 3.01 A). Despite the low detail in the observed electron density, the proposed orientation of the molecule in the binding site is consistent with extensive biochemical and modeling data and, therefore, is assumed to be correct. Modeling of 1,25(OH)2D3 binding to DBP illustrates that this molecule can make the same hydrogen bonds with DBP as 25(OH)D3 but also shows that the axial 1-hydroxyl causes steric hindrance with Met 107. This may explain the lower DBP binding affinity observed for 1,25(OH)2D3. The vitamin-D-binding site is a cleft; consequently, certain parts of the vitamin D3 molecule have no interaction with DBP e for example, the b-side of the C- and D-rings of the vitamin D3 molecule. Analogs substituted in these parts of the molecule display affinities similar to the affinity between 1,25 (OH)2D3 and DBP. Furthermore, modeling of the analogs with large substituents on the C ring (11bphenyl-1a,25-dihydroxyvitamin D3, 11a-phenyl-1a,25dihydroxyvitamin D3) illustrates that large substituents, such as a vinyl group or a phenyl ring, can easily fit in the binding site. These observations confirm the proposed orientation of 25(OH)D3 in the vitamin-Dbinding site (Fig. 5.1). The binding site of DBP for vitamin D metabolites is quite different from that of the vitamin D receptor VDR. In VDR, the vitamin-D-binding site is a closed pocket formed in the inner structure of the receptor, whereas in DBP it is a cleft located at the surface of the molecule and partly in contact with the surrounding solvent. Moreover, no parallel for both of these

physiologically important binding sites is found in ligand conformation, hydrogen bonding, or hydrophobic interactions. In the VDR pocket, the A-ring of the vitamin D3 ligand has a B-chair conformation (3hydroxyl axial), whereas in the DBP cleft the A-ring has an A-chair conformation (3-hydroxyl equatorial). In both binding sites, the C5eC6eC7eC8 torsion angle of the ligand is non-planar. The angle is 149 in DBP and e149 in VDR. The torsion angles responsible for the side-chain orientation of the vitamin D3 molecule are different as well (C13eC17eC20eC22 ¼ e77 in DBP and 89 in VDR; C17eC20eC22eC23 ¼ e70 in DBP and e156 in VDR). DBP (or group-specific component, Gc) was originally characterized in humans by serum electrophoresis as the product of two autosomal, codominant alleles (Gc1 and Gc2). Isoelectric focusing allowed the further characterization of slow (Gc1S) and fast (Gc1F) subtypes of Gc1, resulting in six common phenotypes. The protein isoforms produced by the Gc1F and Gc1S alleles represent a mixed population containing or lacking a single N-acetylneuraminic acid on a threonine residue at position 420. In contrast, the Gc2 allele produces a single protein that contains a nonglycosylated lysine at position 420. The DBP gene contains an Alu middle repetitive element located at the end of DBP intron 8 resulting in four gene polymorphisms created by differences in the size of the Alu poly(A) tract. Each of these polymorphisms is equally distributed among the three common alleles. Although the major alleles can be distinguished at the DNA level by restriction digestions or PCR-single-strand conformation polymorphism analysis, protein electrophoretic techniques remain the most usual initial approach to assigning protein isoform phenotypes. Although commonly used for forensic purposes in the past [3], DBP phenotyping is now more frequently used to map population dynamics, whereas its link with disease susceptibility is still unclear. In addition to the three common alleles, there are also more than 124 rare variant alleles described worldwide.


60

5. THE VITAMIN D BINDING PROTEIN DBP

The geographic occurrences of these variants often correspond to patterns of human population migrations and thus are of anthropological interest [2,10]. The molecular bases for some of these rare variants have been determined by sequencing of exons amplified by the polymerase chain reaction. A number of genetic studies have suggested the presence of a DBP-null allele [42,43], but these have not been clearly distinguished from low-expressing “pseudo-silent” alleles [44]. The lack of a naturally occurring DBP-null homozygote in the human population suggested that such a genotype would be embryonically lethal. A homozygous, DBPdeficient mouse line has, however, been generated that is viable and fertile [7]. DBP polymorphisms have also been identified in non-human primates, rodents, a variety of ungulates, domestic cats, marsupials, and birds [10]. The high degree of polymorphic variation in the human population and its widespread occurrence among vertebrates make the DBP locus among the most polymorphic known.

Synthesis and Turnover of DBP Like albumin and other serum proteins, DBP is produced primarily in the liver with virtually no production elswhere. The half-life of DBP is in the order of a few days and therefore its daily production is high, in the order of 10 mg/kg/day of DBP in humans [45]. Clearance of DBP from the plasma in the rabbit, like that of albumin, is described by a multiexponential curve with an estimated t1/2 of 1.7 days. This is less than half that of albumin with a t1/2 of 5 days, but over five times faster than that of its major ligand, 25 (OH)D, suggesting that ligand recycling is likely [46]. DBP is primarily cleared by the kidneys as a result of its uptake by megalin in the proximal tubule epithelium [45,47e49]. The relatively low concentration of hepatic DBP mRNA suggests that the DBP transcript, like that of albumin, may be very stable and exhibit a long cytosolic half-life [50]. The circulating DBP concentration in adult mammals and birds is in the micromolar range [10]. DBP concentration increases in the postnatal period and is fairly stable thereafter. Most studies have failed to link vitamin D status or disturbances of mineral homeostasis with alterations to DBP concentration [51e54]. DBP concentration increases during pregnancy [55e57], and under the influence of female sex hormones in humans and birds [58e60]. In rodents, DBP concentration increases in response to testosterone and is higher in males than in females [61]. Malnutrition [62], liver failure [55,63], and pronounced proteinuria results in a decrease in circulating DBP concentration [64,65]. Marked tissue necrosis or damage releases intracellular actin, increases circulating actineDBP complexing and decreases DBP concentrations. DBPeactin

complexes are cleared up to three times more rapidly than unliganded DBP, primarily by hepatic filtration [66,67].

FUNCTIONS OF DBP Vitamin D Transport Lipophilic steroid hormones are largely bound to plasma proteins for their extracellular transport. For cortisol this is cortisol-binding globulin or CBG, and for sex steroids sex steroid binding b-globulin or SBBG. Also thyroid hormones use specific transport proteins predominantly thyroxine-binding globulin or TBG, whereas vitamin A or retinol mainly uses retinolbinding globulin for its transport. It is therefore no surprise that all vitamin D metabolites use a similar system of plasma transport. The major transport protein is DBP transporting more than 95% and probably even 99% of 25(OH)D, whereas albumin and lipoproteins have minor transport functions as they transport a small fraction of all vitamin D metabolites despite their much higher serum concentration in comparison with DBP. Based on competition experiments and on the crystal structure of the holoprotein (DBP plus 25(OH)D or analogs) it is clear that there is only a single binding site for all the vitamin D metabolites. The affinity of DBP for these metabolites is however quite different with the highest affinity for 25(OH)D lactones, followed by 25(OH)D and its catabolic metabolites such as 24,25and 25,26-dihydroxyvitamin D, whereas 1,25(OH)2D has about a 10- to 100-fold lower affinity for DBP than 25(OH)D. These differences in affinity can be reasonably explained by the structure of the cleft-like binding site on DBP. Vitamin D itself has still a much lower affinity. Vitamin D2 metabolites bind slightly less well to human DBP than vitamin D3 metabolites, whereas chick DBP has a much lower affinity for vitamin D2 metabolites for otherwise unexplained structural reasons. It is also well known that vitamin D2 has poor antirachitic properties in birds and it is attractive to link that to differences in DBP affinities, although direct proof is missing. The vitamin D receptor VDR has a much higher (about 100-fold) affinity for 1,25(OH)2D than for 25 (OH)D with little difference between D2 and D3 metabolites whether in humans, rodents, or birds. The binding proteins for hormones in general do not all belong to the same gene family as do the CYP P450 enzymes responsible for the metabolism of vitamin D, whereas also the receptors of these hormones all belong to the class of nuclear transcription factors. However all these binding proteins have the same consequences: due to the universal law of mass action and due to the relative high affinity of these binding proteins for their


61

FUNCTIONS OF DBP

circulating ligands, most of the ligand is bound to its binding protein and only a small fraction is unbound or “free.” This resulted in the "free hormone" hypothesis [68]. Indeed, as the small lipophilic free ligands are able to cross the cell membrane and thereby gain access to the cytoplasmic or nuclear-binding proteins, whereas the protein-bound ligand cannot freely cross the cell membrane, the biologically active fraction is the free ligand. The protein-bound ligands or hormones are thereby largely excluded from direct cellular entry and thus play essentially a role as circulatory reservoir for local delivery of the free ligands. A slightly more subtle modification of this hypothesis also takes into account the total time needed for blood to cross a particular tissue so that part of the bound hormone can be released during tissue perfusion to replace the free hormone take up by cells during this tissue perfusion. This implies that the real available hormone is higher than the static free hormone concentration due to dissociation of free hormone during tissue perfusion. A large number of biochemical, cellular, and physiologic data strongly support this free hormone hypothesis. The serum transport of 25(OH)D and 1,25(OH)2D also likely behaves as the other steroid or thyroid hormones. Indeed the cellular entry of 25(OH)D into most tissues decreases in the presence of serum protein or DBP confirming the sequestering characteristics of DBP (Table 5.1). This has been clearly demonstrated in keratinocytes and monocytes. Indeed the activity of 1,25(OH)2D or 25 (OH)D in human monocytes can be evaluated by measuring the expression of cathelicidin. The biological activity was much higher when such monocytes were cultured in the presence of serum from DBP-null mice than in the presence of normal serum or DBP-enriched medium [69]. No megalin-mediated uptake of DBP could be detected in monocytes. The inhibition of the biological activity was two- to three-fold higher in the presence of Gc1F-1F compared to other DBP polymorphisms indicating that the biological activity of 1,25 (OH)2D or 25(OH)D may be different according to DBP polymorphism. This inhibitory effect has also been shown for cultured kidney cells as the presence of DBP decreases the production of 1,25(OH)2D. This phenomenon can be easily explained by the high affinity of 25(OH)D for extracellular DBP and a much lower affinity for the intracellular VDR, with the overall effect of slow entry of 25(OH)D into cells. This is confirmed by in vivo studies. Indeed, the distribution volume of 25 (OH)D as evaluated by radiotracer studies is very similar to the distribution volume of DBP and thus corresponds to the extracellular fluid compartment. This would be in line with the slow dissociation of 25 (OH)D bound to DBP and slow entry of 25(OH)D inside the intracellular compartment. The situation of 1,25 (OH)2D is more complex. Addition of serum proteins

TABLE 5.1

Characteristics of Human DBP

Features Isoelectric point

4.5e4.8

Electrophoretic migration

a-globulin

Size

58 kDa, single-chain glycoprotein

Plasma concentration

4e8 mM (232e464 mg/liter)

Plasma half-life

2.5e3.0 days

Daily production rate

~10 mg/kg

Altered plasma levels Increased

Estrogen, pregnancy

Decreased

Nephrotic syndrome, liver disease, malnutrition, acute critical illness, extensive tissue damage

Vitamin D sterol binding Plasma capacity

mol/mol (2.4 mg D sterol/liter)

Normal sterol occupancy

100 C leads to 9,10-syn isomers by disrotatory bond formation mechanism [57]

Photopyrocalciferol2 [41411-05-6]

Photopyrocalciferol3 [85320-70-3]

Ultraviolet over-irradiation

Photoisopyrocalciferol2 [26241-65-6] Photoisopyrocalciferol3 [85354-28-5] Ultraviolet over-irradiation 5,6-trans-vitamin D2 [14449-19-5]

5,6-trans-vitamin D3 [22350-41-0]

irradiation of calciferol in the presence of iodine [58,59]

Isocalciferol2 [469-05-6]

Isocalciferol3 [42607-12-5]

treatment of trans D with mineral or Lewis acids; also forms from trans D with heat

Isotachysterol2 [469-06-7]

Isotachysterol3 [22350-43-2]

from isocalciferol or vitamin D (via trans) upon treatment with acid


83

METABOLITE MANUFACTURE

METABOLITE MANUFACTURE 1a-Hydroxyvitamin D3

FIGURE 6.8 [41].

Approximate course of irradiation of provitamin D

Vitamin D can be crystallized from a mixture of hydrocarbon solvent and aliphatic nitrile, e.g., benzene and acetonitrile, or from methyl formate to give the USP product [63,64]. Chemical complexation as well as column chromatography is also used for purification of the resin to obtain crystalline vitamin D for food and pharmaceutical usage. Vitamin D products are formulated in a variety of matrices to protect the vitamin from exposure to air, heat, light, and minerals which cause it to degrade. These formulations also allow for the dilution of the high-potency pure product into its final dosage form with adequate distribution so as to assure uniform dosage of the food, feed, or pharmaceutical preparation. Vitamin D2 is made from ergosterol using the same type of technology. The same isomer distribution occurs and the irradiation must be carried out with similar care as described above for vitamin D3.

CH3

1a-Hydroxyvitamin D3 is becoming an important supplement in poultry diets. It has the ability to reduce tibial dyscondroplasia and is additive with phytase in promoting phosphorus utilization [65,66]. The product was originally prepared by Barton in 1973 and many preparations have been reported in the literature since. The one used most frequently is a modification of the Barton method [67,68]. The commercial process [69] starts by treating vitamin D3 with SO2 to produce two cyclic adducts. The 3-OH group is protected with a silicon protecting group. The SO2 is removed with the formation of a derivative of a single isomer (5,6transvitamin D3) followed by allylic oxidation to introduce the 1a-hydroxy function. After de-protection and crystallization, the 1a-hydroxytrans vitamin D3 is photochemically isomerized to 1a-hydroxyvitamin D3. The 1a-hydroxyvitamin D3 in addition to its many applications for pharmaceutical uses by several companies is formulated in a starch matrix for use in animal feed products at a concentration of 0.04%. The major use of the product is in animal feeds and volume currently approximates 100 kg of crystalline product per year which will produce 20 000 000 metric tons of feed (5 mg of 1a-hydroxyvitamin D3/kg of finished feed). The fact that the process starts with crystalline D3 causes the product to be substantially more expensive than the vitamin D itself. The benefits of using the product to replace vitamin D must overcome this cost differential. It is, therefore, used as a supplement to vitamin D3 to achieve its additive benefits.

25-Hydroxyvitamin D3 25-Hydroxyvitamin D3 is also of commercial use, primarily in animal nutrition but also pharmaceutically for osteoporosis and other bone disease treatments. Its

CH3

CH3

CH3

CH3

O

HO (17) Suprasterol II

OH (18) Toxisterol-E

(19) Toxisterol E1

FIGURE 6.9 Unusual by-products of heat and prolonged irradiation of vitamin D3.


84

6. INDUSTRIAL ASPECTS OF VITAMIN D

UV Absorbance Maxima of 7-Dehydrocholesterol, Pre- and Cis-Vitamin D [61]

TABLE 6.3

Absorbance 7-Dehydrocholesterol Pre vitamin D cis-Vitamin D @ (nm)

(E 1% 1 cm)

260

270

265

484

273

282

282

293

293

170

use in animal feeds is claimed to promote bone development, weight gain, and feed efficiency. It should be noted that when administered to an animal, vitamin D is hydroxylated in the liver soon after absorption to 25hydroxyvitamin D which then circulates in the blood. Feeding the metabolite bypasses this initial biological process. Therefore, one must evaluate the toxicity, cost, and benefits of this type of administration. The 1a-hydroxyvitamin D is not a naturally occurring material but upon absorption into the body undergoes 25-hydroxylation in a manner similar to (and at an equivalent rate to) the vitamin D. This process by-passes the kidney 1a-hydroxylation of the 25-hydroxyl metabolite, making the 1a,25-dihydroxyvitamin D3 hormone available at an accelerated rate. 25-Hydroxyvitamin D is made commercially primarily by a process which involves the fermentation

FIGURE 6.10

of a double mutant yeast to form 5,7,24-cholestatrienol [70e72]. The 25-hydroxyvitamin D3 is used at 62.5 mg/kg finished feed and current usage is estimated to be approximately 15 000 000 metric tons of feed per year. Thus, approximately 937.5 kg of 25-hydroxyvitamin D3 are used. The active hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 or calcitriol, can be made from the 25-hydroxyvitamin D [73]. 1,25-Dihydroxyvitamin D3 and its derivatives are used primarily in pharmaceutical preparations and are made by a variety of processes.

ANALYTICAL Vitamin D The early history of vitamin D led to the use of biological testing to determine the effectiveness of vitamin D products to reduce the effects of rickets. This ultimately led to standardized rat and chick tests in which laboratory animals are fed special diets devoid of vitamin D to produce rachitic conditions in the animal. The test animals are then dosed with the test substance and the bone growth at the proximal end of the tibia or distil end of the ulna are compared after staining with silver nitrate. Rats can be used to test vitamin D2 or D3, but the results can lead to false positives for use of the product in poultry, since chickens do not respond to

Vitamin D3 manufacturing flow diagram [61, p. 239].


85

ANALYTICAL

FIGURE 6.11 system [62].

vitamin D2. Therefore, chickens must be used to evaluate product that is to be used in poultry. This method is still approved by the AOAC (see AOAC 45.3.03 Chick Bioassay for Poultry Feed Supplement.932.16 [74], while material used for all other animals can use the AOAC Rat Bioassay 45.3.02 Rat Bioassay 936.14 [75]). These methods are slow, expensive, and lead to variable results. The elucidation of the chemical nature of the vitamins led to the use of chemical methods of analysis which were primarily dependent upon colorimetric procedures. These were approved by the major official governing organizations and used for many years as the official methods of analysis. The AOAC chemical method (Colorimetric method 975.42 45.1.17) [76] involves “saponification” of the sample (dry concentrate, premix, powder, capsule, tablet, or aqueous suspension) to release the vitamin from its matrix, with aqueous alcoholic KOH. The vitamin is then extracted using an appropriate solvent and the solvent containing the vitamin D is removed. Vitamin D is separated from extraneous ingredients by a chromatographic separation and the potency of the vitamin D is determined by a colorimetric determination with antimony trichloride in comparison with a solution of USP cholecalciferol reference standard. The procedure includes a step to treat unsaponifiable material

Commercial photochemical UV lamp

with maleic anhydride to remove any trans-isomer which may be present and lead to a falsely high result. The antimony trichloride colorimetric assay is performed on the trans-isomer-free material. This procedure cannot be used to distinguish isotachysterol and, if present, also gives rise to a falsely high result. A test must therefore be performed to check for the presence of isotachysterol. The USP XXXII [77] and AOAC 2010 (HPLC 979.24 [78]) both now recognize high-pressure liquid chromatography (HPLC) as the preferred method of analysis. HPLC allows the separation of the active pre- and cisisomers of vitamin D3 from other isomers and provides a means to analyze the active content by comparison with the chromatograph of a sample of pure reference cis-vitamin D3. Equilibration of a solution of the standard to a mixture of pre- and cis-isomers [12,13] is included in the procedure in order to evaluate the total active isomer content of the sample. The sensitivity of this method provides information on isomer distribution and allows for the accurate evaluation of the active pre- and cis-isomer content of a vitamin D sample. It is applicable to most forms of vitamin D, including the more dilute formulations, i.e., oils containing 100 000 IU cholecalciferol/g; resins 20 000 000 IU cholecalciferol/g; and powders and aqueous dispersions at


86


25 000 IU cholecalciferol/g (AOAC Methods 979.24; 980.26; 981.17; 982.29; 985.27) [79e81]. The limiting factor in the assay of low-level formulations is the isolation of the vitamin material from interfering and extraneous components which may obscure the vitamin D HPLC peak. Vitamin D products formulated in a variety of matrices are then usually mixed with carriers to form premixes and final dosage compositions. The assay of the vitamin requires the matrix to be broken in such a way as to assure complete availability of the vitamin. Additionally, the ability to separate it from the formulated mixture must be efficient. The above methods all address this issue. A particularly useful extraction procedure involves the use of dimethyl sulfoxide [82]. The usual HPLC procedures utilize UV detection for quantification of the elution peaks. Recently HPLC has been coupled with mass spectrographic detectors to enable the assay of vitamin D to much more significant detection limits [83,84]. A number of methods have been developed for the paper, thin-layer, and column chromatographic separation of vitamin D and related substances but these are more tedious and difficult to perform on low-level samples. Gas chromatography requires derivatization and has been applied to metabolite analysis as well as assay of multivitamin tablets and vitamin D2 in milk and other formulations [85e90]. The USP [91] requires the following tests for the pure crystalline vitamin D: Identity by: (a) IR; A typical infrared spectrum of cholecalciferol will have the following parameters; the IR spectrum of the sample should be identical to the IR spectrum of a USP reference standard of cholecalciferol. Wavelength

Peak Ht.

Characteristic

3305 2934 1642 1458, 1438, 1375 1053

Strong Strong Weak Medium Strong

-OH stretch -CH stretch & bend -C¼C- stretch -CH stretch & bend -C-OH stretch

(b) UV; A typical UV curve for a 10 mg/ml solution of cholecalciferol has a molar extinction coefficient of 18 692 at lmax 264.8 (E1%max ¼ 484). (c) Chemical color (with acetic acid and sulfuric acid turns bright red changing to violet and then blue green).

Thin Layer Chromatography: USP [77] (developed with SbCl3 in Acetyl Chloride which gives a yellow/orange color) with retention time compared to a USP reference standard. Specific Rotation; USP [92] aD ¼ between þ105 and þ112 . Assay: by High Pressure Liquid Chromatography (HPLC) USP [77] against a USP reference standard. The international standard for vitamin D is an oil solution of activated 7-dehydrocholesterol. The International Unit (IU) is the biological activity of 0.025 mg of pure cholecalciferol. One gram of vitamin D3 is equivalent to 40 106 IU or USP units. Samples of reference standard may be purchased from US Pharmacopeial Convention [93]. Reference standards are also available from the World Health Organization (WHO) as well as the European (EP) and British Pharmacopeia (BP). USP also issues vitamin D3 capsules for AOAC determination in rats and an oil solution for the vitamin D3 AOAC determination in chicks. The various isomers of vitamin D exhibit characteristically different UV absorption curves. Cis vitamins D2 and D3 exhibit UV absorption maximum at 265 nm with an Emax (absorbance) of 450e490 at 1% concentration (Table 6.3). Mixtures of the isomers are difficult to distinguish but the pure substances and their concentrates can be assayed using their UV absorption. When chromatographically separated by HPLC, the vitamin D peaks can be identified by stop-flow techniques based on UV absorption scanning or by photodiode-array spectroscopy as well as mass spectroscopy. The combination of elution time and characteristic UV absorption curves can be used to identify the isomers present in a sample of vitamin D. Infrared and NMR spectroscopy have been used to help distinguish between vitamins D2 and D3 [94e96].

Provitamin Assay The molecular extinction coefficient of 7-dehydrocholesterol at 282 nm is 11 300 and is used as a measure of 7dehydro isomer content of the provitamin [97,98]. High pressure liquid chromatography can also be used to analyze the provitamins. There are a variety of chemicals that show characteristic colors when reacted with the provitamins. Some of these are listed below. • The Salkowski reaction (revised) treats the provitamin with CHCl3 and H2SO4 (conc.) to give a deep red color in CHCl3 layer and green fluorescence in the acid layer which differentiates from sterols lacking a conjugated diene.


87

DIETARY REQUIREMENTS

• The Lieberman-Burchard reaction is run in CHCl3 with acetic acideH2SO4 added dropwise. A red color develops and changes to blue-violet to green. The test can be quantitative and acts similarly to the Salkowski reaction, but the red color lasts longer. • The Tortelli-Jaffe reaction is run in acetic acid with 2 wt% Br2 in CHCl3 which turns green with sterols having ditertiary double bonds including vitamin D and compounds that give similar bonds upon isomerization or reaction. • The Rosenheim reaction is run in CHCl3 with trichloroacetic acid in H2O. A red color develops and changes to light blue. When run with CHCl3 and lead tetraacetate in CH3COOH followed by the addition of trichloroacetic acid, the reaction gives a green fluorescence which is not given by esters of provitamin D and can be used to distinguish between provitamin and provitamin ester. The test is quantitative to 0.1 mg. • A mixture of crystalline provitamins and chloral hydrate heated slowly melts at 50 C and color develops and changes from red to green to deep blue while other sterols, e.g., cholesterol, do not react to give color. • The antimony trichloride reaction with CHCl3 and SbCl3 gives a red color. • The Chugaev reaction adds glacial acetic acid plus acetyl chloride and zinc chloride to the provitamin which is heated to boiling. An eosin-red greenish yellow fluorescence develops with a sensitivity of 1:80 000 [99].

Assay of 25(OH)D The extremely low levels of vitamin D and its metabolites in biological systems make it very difficult to assay the vitamin D products in these environs by traditional methods. The ability to assay these materials was initially developed by Haddad [100] with the use of a competitive protein-binding assay (CPBA) for 25-hydroxyvitamin D (25(OH)D). 25(OH)D is especially vulnerable to matrix effects in any protein-binding assay because of its lipophilic properties. These were overcome by the utilization of chromatographic sample purification prior to the complex formation with the calcium. A nonchromatographic radio immunoassay for circulating 25(OH)D was developed by Napoli and Hollis using an antigen that would generate an antibody that was cospecific for 25(OH)D2 and 25(OH)D3 [101]. The study of vitamin D and its metabolites and their effects in clinical disease over the past 30 years was made possible by the ability to assay these materials. The need to assay large numbers of samples to evaluate the

vitamin D blood levels of large populations requires an ability to perform these assays with a rapid, accurate, and valid method. In 2001, Nichols Diagnostics introduced the fully automated chemiluminescence CPBA ADVANTAGE 25(OH)D assay system [102] in which nonextracted serum or plasma is introduced directly into a mixture containing human D-binding protein (DBP), acridinium-ester-labeled anti-DBP, and 25(OH)D3-coated magnetic particles. Another chemiluminescence assay was developed in 2004 by the DiaSorin Corporation [103]. The assay, LIAISON 25(OH)D, is very similar to the ADVANTAGE assay but uses an antibody as a primary binding agent as opposed to the human DBP and is a radio immune assay (RIA) method. See Hollis [104] for a review of these methods and their application to the important assay of blood levels of 25(OH)D.

DIETARY REQUIREMENTS Humans Dietary Reference Intakes of vitamin D2 and vitamin D3 (DRIs) developed by the Food and Nutrition Board (FNB) at the Institute of Medicine of the National Academies (formerly National Academy of Sciences) were established in 1997 [105]. They recommended 200 IU/day from 1 month to 50 years of age and 400 IU/day from 51 years to 70 years and 600 IU/day after reaching the age of 71. In 2008, the American Academy of Pediatrics (AAP) issued recommendations for intakes for vitamin D that exceed those of FNB to 400 IU/day and the Surgeon General of the United States indicated that all citizens should make sure they took a minimum of 400 IU/g day to ensure good health. Many studies over the past 15 years have led to the suggestion that 1000e2000 units and as high as 5000 IU/day of vitamin D3 per day are necessary to provide enough vitamin D to maintain 25hydroxyvitamin D blood levels (37.5e50 nmole/L; 15e20 ng/ml) sufficiently high to provide all of the functions the vitamin and its metabolites serve to influence. The Food Nutrition Board established an expert committee in 2008 to review the DRIs for vitamin D (and calcium). The FNB issued its report, updating as appropriate the DRIs for vitamin D and calcium, in November 2010. The new recommendations are 600 IU/day from age 1 year to 70 years and 800 IU/day from age 70 and older. Upper level intake limits were set at 4000 IU/day for 9 year olds and older. As noted above, there is much evidence which suggests higher levels than this are needed and this problem will be one of ongoing concern [105a].


88 TABLE 6.4


Practical Feeding Levels of Vitamin D, 106 IU/ton [111]

Animal

Amount Animal

Amount

Poultry*

Dairy cattle

Chickens

Calf starter

1e2

Broilers

2e4

Calf milk replacer

2e4

Replacement birds

1e3

Replacement heifers

1e2

Layers

1e3

Dry cows

1e2

Breeding hens

2e4

Lactating cows

2e4

Bulls

2e4

Turkeys Starting

3e5

Growing

2e4

Beef cattle

Breeding

3e5

Calf starter

1e2

Replacement heifers

1e2

Ducks Market

1e3

Feedlots

2e3

Breeding

2e4

Dry pregnant cows

1e2

Lactating cows

1e2

Bulls

1e2

Swine Prestart (to 10 kg)

2e3

Starter (10e35 kg)

1e2

Sheep

Growing-finishing (35 kg to market)

1e2

Fattening lambs

2e3

Gestation

1e2

Breeding

1e2

Lactation

2e3

Boars

2e3

Other

ECONOMIC ASPECTS

Fish

Dogs

5e1

Cats

1e2

Horses

1e2

for various species: starting and growing chicks, 200 IU; laying and breeding hens, 500 IU; turkeys, 1100 IU; ducks, 200 IU; quail, 480e900 IU; geese, 200 IU; and swine 125e220 IU. Calves require 600 IU per 100 kg of body weight [107e109]. Higher levels are usually used in common practice in order to make sure the animals receive adequate dosage of the vitamin. Most species can safely tolerate four to ten times the NRC requirements during long-term feeding and short-term ( duodenum) is different from the location of improved calcium absorption that occurs following vitamin D repletion (duodenum >> jejunum). However, similar to what others have shown for calcium absorption, the beneficial effect of vitamin D3 repletion on intestinal phosphate absorption can be achieved with 1,25(OH)2D treatment. When vitaminD-deficient rats are given large doses of 1,25(OH)2D (325e650 pmoles), serum phosphate levels and intestinal phosphate absorption are significantly increased [117,118]. Similarly, in vitamin-D-replete rats, the reduction in phosphate absorption caused by thyroidparathyroidectomy is restored within 6 h after a 0.8 pmol/g body weight dose of 1,25(OH)2D [106]. Similar to the findings from rats, Davis et al. [103] showed that 1,25(OH)2D increased jejunal phosphate absorption in subjects with chronic renal failure (and presumably low serum 1,25(OH)2D). In this study 1,25 (OH)2D treatment increased the Vmax of the saturable component of phosphate transport approximately

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twofold without having a significant impact on the rate of nonsaturable phosphate transport. Similar findings of a 1,25(OH)2D-induced increase in the Vmax for phosphate transport across brush border membrane vesicles prepared from the jejunum of vitamin-D-deficient rats were reported by others [108]. The effect was not seen until 12 h after 1,25(OH)2D treatment suggesting that the effect of 1,25(OH)2D was due to an increased production of phosphate transporters. Marks et al. [104] confirmed this by demonstrating that treating vitaminD-deficient mice with 1,25(OH)2D increased the amount of NaPi IIb protein in brush border membrane vesicles. Although these data suggest that 1,25(OH)2D is a direct regulator of NaPi IIb gene expression, and by extension, phosphate absorption, other data do not support such a straightforward interpretation. For example, although 1,25(OH)2D increased NaPi IIb mRNA level in cultured rat intestinal epithelial (RIE) cells by an actinomycinD-sensitive mechanism, investigators were not able to find a classical VDRE in the NaPi IIb promoter [110]. In vivo, the ability of 1,25(OH)2D to increase NaPi IIb mRNA is present in 2-week-old mice with immature intestines [110], but it is lost in 12-week-old mice [110]. However, the effect of 1,25(OH)2D on phosphate transport into brush border membrane vesicles is still seen in vitamin-D-replete adults suggesting that the effect of the hormone is mediated through either new protein production from existing message or through the redistribution of existing protein to the apical membrane. Finally, both Davis et al. [103] in humans and Lee et al. [119] in mice found that the benefit of 1,25(OH)2D treatment on phosphate absorption is modest in vitaminD-replete subjects. This suggests that other factors may limit the effects of 1,25(OH)2D when phosphate status is already adequate. The Role of VDR and Phosphatonins in Phosphate Absorption There are other studies that further demonstrate the complexity of the role vitamin D signaling plays in the control of phosphate absorption. For example, while intestinal phosphate transport in brush border membrane vesicles is reduced by 30e70% in VDR knockout mice, it is accompanied by a reduction in NaPi IIb protein but not mRNA [120,121]. This is consistent with a model of 1,25(OH)2D-mediated regulation that is dependent on the presence of the VDR but not new gene transcription mediated through the VDR. It is not known how signaling through the VDR regulates NaPi IIb translation or trafficking. Another interesting observation is that dietary phosphate restriction can increase intestinal phosphate absorption in VDR- and CYP27B1-null mice so that the level of absorption is equivalent to that seen in phosphate-restricted wildtype mice. The ability of VDR and CYP27B1 knockout

III. MINERAL AND BONE HOMEOSTASIS

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19. MOLECULAR MECHANISMS FOR REGULATION OF INTESTINAL CALCIUM AND PHOSPHATE ABSORPTION BY VITAMIN D

mice to up-regulate phosphate absorption after dietary phosphate restriction is similar to what Lee et al. [119] previously observed in vitamin-D-deficient rats (40% increase in vitamin-D-deficient rats vs 90% increase in vitamin-D-replete rats). Collectively, these data demonstrate that vitamin D signaling through the VDR is important for normal levels of phosphate absorption but that dietary phosphate restriction regulates phosphate absorption through a vitamin-D-independent mechanism. Another mechanism mediating the effects of dietary phosphate on phosphate absorption is through phosphatonins like matrix extracellular phosphoglycoprotein (MEPE), secreted frizzled-related protein 4 (sFRP-4), dentin matrix protein 1 (DMP1), fibroblast growth factor 7 (FGF7), and the most studied phosphatonin, fibroblast growth factor 23 (FGF23) [122]. FGF23 and other phosphatonins inhibit renal phosphate reabsorption and their serum levels are directly related to dietary phosphate intake and serum phosphate levels [123,124]. In vivo injections of phosphatonins FGF23 and MEPE can inhibit intestinal phosphate absorption in mice [125,126]. FGF23 binds to the FGF receptors 1c, 3c, and 4 and requires the presence of the coreceptor, klotho, to signal through FGF receptors [127,128]. However, since the messages of the FGF receptors for FGF23 (R1c, R3c, R4) and the klotho coreceptor are expressed at very low levels in the intestine (determined from BioGPS (http://biogps.gnf.org/?referer¼symatlas #goto¼welcome)), this suggests that FGF23 works indirectly upon the intestine to alter phosphate absorption. This is consistent with the observation that FGF23 suppression of jejunal phosphate absorption is lost in VDR-null mice [129]. Renal CYP27B1 activity and serum 1,25(OH)2D levels are inversely associated with serum FGF23 levels in normal healthy men and in mice [123,124]. Implantation of cells producing FGF23 into nude mice reduces the renal expression of CYP27B1 mRNA [130] while in cultured mouse renal proximal tubule cells, FGF23 (R176Q) treatment suppresses CYP27B1 mRNA levels by a MAPK-dependent mechanism [131]. Thus, low dietary phosphate may improve intestinal phosphate transport in part through increased serum 1,25(OH)2D resulting from releasing the FGF23mediated inhibition of CYP27B1 expression.

Conclusion regarding the role of vitamin D on phosphate absorption It is clear that under normal conditions, the saturable portion of intestinal phosphate absorption is dependent upon adequate vitamin D status. However, the mechanism for that regulation is not clear. The central player in phosphate absorption is the apical membrane sodiumephosphate cotransporter, NaPi IIb. However,

while vitamin D changes the level of the NaPi IIb protein at the membrane, it does not strongly regulate the NaPi IIb gene and if it does so at all, it appears to be indirectly through an as yet unidentified factor. More importantly, even though both serum 1,25(OH)2D and intestinal phosphate absorption increase during dietary phosphate restriction, the body still has the ability to adapt to low-phosphate diets even in the absence of VDR or CYP27B1. This suggests that a major regulator of phosphate absorption is vitamin-D-independent and that there are critical regulators of intestinal phosphate absorption that have yet to be discovered. Finally, vitamin D may have a minor role in phosphate absorption in normal healthy men. This is because the saturable component of intestinal phosphate absorption that is regulated by vitamin D status may not be critical for human health under the high dietary phosphate loads of the typical Western diet.

Acknowledgments This work was supported by NIH award DK054111 to JCF.

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20 The Calbindins: Calbindin-D28K and Calbindin-D9K and the Epithelial Calcium Channels TRPV5 and TRPV6 Sylvia Christakos, Leila J. Mady, Puneet Dhawan Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA

INTRODUCTION AND GENERAL CONSIDERATIONS, THE CALBINDINS In the two major target tissues of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) action, intestine and kidney, one of the most pronounced effects of 1,25(OH)2D3 known is the induction of the calcium-binding protein, calbindin, the first identified target of 1,25(OH)2D3 action. There are two major subclasses of calbindin: a protein of approximately 28 000 molecular weight (calbindin-D28K) and a protein of approximately 9000 molecular weight (calbindin-D9K). Calbindin-D28K is present in highest concentration in avian intestine and in avian and mammalian kidney, brain, and pancreas. Calbindin-D28K has four functional high-affinity calcium-binding sites and is highly conserved in evolution. Calbindin-D9K has two calcium-binding domains, is present in highest concentration in mammalian intestine, and, unlike calbindin-D28K, is not evolutionarily conserved and has been observed only in mammals. There is no amino acid sequence similarity between calbindin-D9K and calbindin-D28K. The discussion that follows reviews the chemistry, localization, proposed functional significance, and regulation of these calcium-binding proteins. In addition, this chapter provides insight into the information obtained by studying these proteins concerning the multiple actions of the vitamin D endocrine system and the basic molecular mechanism of 1,25(OH)2D3 action. Findings indicating that calbindins can be regulated by a number of different hormones and factors are also reviewed. The study of the molecular


interactions of several members of the steroid hormonee retinoic acid family as well as the role of signal transduction pathways in the regulation of calbindin-D may be applicable to the regulation of other targets of 1,25 (OH)2D3 action. Elucidation of multiple factors and interactions regulating 1,25(OH)2D3 target genes should result in novel insights related to tissue-specific molecular mechanisms involved in calcium homeostasis. One of the most important findings in the vitamin D field has been the discovery by Wasserman and Taylor in 1966 of a 28 000 Mr vitamin-D-dependent calciumbinding protein in avian intestine [1]. Although previously known as the vitamin-D-dependent calcium-binding protein (CaBP), in 1985 it became officially known as calbindin-D28K and calbindin-D9K for the 28 000 Mr and the 9000 Mr proteins, respectively [2]. Initially identified in avian intestine [1], calbindinD28K has since been reported in many other tissues including kidney and bone, and in tissues that are not primary regulators of serum calcium such as pancreas, testes, and brain and in a variety of species [3e9] (see Christakos et al. [9] for review). The importance of the discovery of calbindin-D28K is that key advances in our understanding of the diversity of the vitamin D endocrine system have been made through the study of its tissue distribution and its colocalization with the vitamin D receptor (VDR). In addition, the biosynthesis of calbindin has provided a model for studies that have resulted in an important basic understanding of the molecular mechanism of action of 1,25(OH)2D3 in major target tissues such as intestine and kidney.

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20. THE CALBINDINS: CALBINDIN-D28K AND CALBINDIN-D9K AND THE EPITHELIAL CALCIUM CHANNELS TRPV5 AND TRPV6

Chicken and mammalian calbindin-D28K proteins contain 261 amino acid residues, have a molecular weight of approximately 28 000 (30 000 based on amino acid sequence and 28 000 based on migration on sodium dodecyl sulfateepolyacrylamide gels), and are blocked at the amino terminus [9e12]. The mammalian calbindin-D28K sequences are 98% similar to one another and 79% similar to chicken calbindin-D28K [9,11,12]. Calbindin-D28K is highly conserved in evolution, suggesting an important, fundamental role for calbindin-D28K in mediating intracellular calcium-dependent processes [9]. Unlike calbindin-D28K, calbindin-D9K is observed only in mammals. It has no amino acid sequence homology to calbindn-D28K. It is most abundant in mammalian duodenum, placenta, and uterus [13e16]. It is also present in mammalian yolk sac, lung, bone, and mouse kidney [14,17e23]. Human calbindin-D9K has 79 amino acid residues and a calculated molecular weight of 9015 [24,25]. It is 89% similar to the bovine and porcine sequences and 78% and 77% similar to rat and mouse calbindin-D9K, respectively [24,25]. Calbindin-D28K and calbindin-D9K belong to a family of high-affinity calcium (Ca2þ)-binding proteins (Kd ¼ 10e8e10e6 M) that contains more than 200 members and is characterized by the EF-hand structural motif [26] (Fig. 20.1). The EF-hand domain is an octahedral structure consisting of two alpha helices separated by a 12-amino-acid loop that contains side chain oxygens necessary for orienting the divalent calcium cation [26]. Calbindin-D28K contains six EF hands (Fig. 20.2); however, only four of these actively bind Ca2þ [27,28].

FIGURE 20.1 EF-hand structural motif (helixeloopehelix). The helices are represented by the extended forefinger and thumb. The clenched middle finger represents the loop that contains the oxygen ligands of the calcium ion. The EF hand is a recurring motif in calbindin and other calcium binding proteins. Reprinted with permission from Stryer L 1995 Biochemistry. Freeman, San Francisco, p. 1064.

Calbindin-D9K contains two calcium-binding sites [29]. Other calcium-binding proteins belonging to this family include calmodulin, parvalbumin, troponin C, calretinin, calcineurin, calpain, Spec I, myosin light chains, S100, and recoverin [26,30]. Although the structure of calbindin-D28K has yet to be elucidated by X-ray crystallography, circular dichroism experiments have shown that calbindin-D28K contains approximately 30% a helix, 20.6% b sheet, and 51% random coil [31]. The threedimensional structure of calbindin-D9K has been elucidated [32]. Calbindin-D9K has been shown to undergo limited conformational change in the presence or absence of calcium [33]. Both calbindins are heat-stable protein and acidic, having a pI value of approximately 5 [34,35]. The calbindins bind other cations in addition to calcium with reduced affinity: Ca2þ > Cd2þ > Sr2þ > Mn2þ > Zn2þ > Ba2þ > Co2þ > Mg2þ [36].

LOCALIZATION AND PROPOSED FUNCTIONAL SIGNIFICANCE OF THE CALBINDINS Intestine One of the most pronounced effects of 1,25(OH)2D3 is increased synthesis of intestinal calbindin. CalbindinD9K in mammalian intestine and calbindin-D28K in avian intestine have been localized primarily in the cytoplasm of absorptive cells [37], which supports the proposed role of calbindin in intestinal calcium absorption [38e40]. Early studies in chicks established a strong correlation between the level of calbindin and an increase in intestinal Ca2þ transport [41e43]. In the intestine, 1,25(OH)2D3 effects the transfer of Ca2þ across the luminal brush-border membrane, the transfer of calcium through the cell interior, and active calcium extrusion from the basolateral membrane. Vitamin-D-inducible apical calcium channels have been identified in intestine and kidney, suggesting, for the first time, a mechanism of calcium entry [44e46]. It is thought that calbindin acts to facilitate the diffusion of calcium through the cell interior toward the basolateral membrane [40,42]. Supporting this hypothesis are findings observed in vitamin D receptor knockout mice. In these mice, the major defect that results in rickets is in intestinal calcium absorption [47e49]. The defect in intestinal calcium absorption is accompanied by a 50% reduction in intestinal calbindin-D9K mRNA [50]. The 1,25(OH)2D3 regulation of intestinal calbindin-D9K is also evident in 25-hydroxyvitamin-D3 1a-hydroxylase knockout mice. In these mice, characterized by hypocalcemia, hyperparathyroidism, and skeletal abnormalities characteristic of rickets, intestinal calbindin-D9K mRNA is absent [51].


LOCALIZATION AND PROPOSED FUNCTIONAL SIGNIFICANCE OF THE CALBINDINS

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FIGURE 20.2 Position of intervening sequences within the structure of chicken calbindin-D28K. Locations of introns are indicated by circled numbers. Numbers above amino acids indicate codon positions. Invariant Glu/Leu and Gly amino acids are indicated by black circles. Calciumbinding domains are separated from the a-helix region by vertical lines. Reprinted with permission from Minghetti et al. [122].

Recent studies, however, using calbindin-D9k knockout (KO) mice have challenged the traditional model of vitamin-D-mediated transcellular calcium absorption in intestine. Calbindin-D9k KO mice show no difference in phenotype from wild-type mice and are able to maintain normal serum calcium levels regardless of age or sex [52]. In response to 1,25

(OH)2D3 treatment, calbindin-D9k KO mice are fully able to absorb calcium from the intestine, illustrating that calbindin-D9k is not required for 1,25(OH)2D3induced active intestinal calcium transport [53]. Furthermore, active intestinal calcium transport is similarly induced in both calbindin-D9k KO mice and wild-type mice in response to a low-calcium diet or 1,25(OH)2D3


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treatment [54]. These findings suggest that calbindinD9k may be compensated for by another factor or that calbindin-D9k may have another role in intestine, for example as a modulator of the activity of the vitamin-Dinducible epithelial calcium channel in the intestine and/or as a cytosolic buffer to prevent toxic levels of calcium from accumulating in the intestinal cell during vitamin-D-mediated translocation of calcium [40].

Kidney Immunocytochemical studies have reported the exclusive localization of calbindin-D28K in the distal nephron (distal convoluted tubule and connecting tubule) in a variety of species including mammals, chickens, and reptiles [3,9,55e57]. Renal calbindinD28K is localized in the cytosol and the nucleus and is not associated with membranes or filamentous elements. Both calbindin-D28K and calbindin-D9K are localized in mouse distal nephron and perinatal rat distal nephron [23]. Autoradiographic data indicated that the VDR is also predominantly localized in the distal nephron and both calbindins have been reported to be induced by 1,25(OH)2D3 in the kidney [58]. Although micropuncture data [59] as well as studies using a mouse distal convoluted tubule cell line [60] have indicated that vitamin D metabolites can enhance calcium transport in the distal nephron, little information is available concerning the exact role of vitamin-D-inducible renal calbindins in this process. Transcellular calcium transport in the distal convoluted tubule, similar to transcellular intestinal calcium absorption, involves calcium entry through the apical plasma membrane, diffusion of calcium across the cell, and active extrusion of calcium across the basolateral membrane mediated by a calcium-dependent ATPase [61]. The epithelial Ca2þ channel transient receptor potential vanilloid-subtype 5 (TRPV5) is coexpressed with calbindin-D28K and coregulated by 1,25(OH)2D3 and dietary Ca2þin the distal nephron and acts as a gatekeeper of apical Ca2þ entry during active reabsorption [62e64]. Calbindin-D28K directly interacts with TRPV5 at the apical membrane under conditions of low intracellular Ca2þ and has been shown to regulate the activity of the epithelial Ca2þ channel [65]. These findings suggest an additional role for renal calbindin-D28k as a modulator of calcium influx. It has also been suggested that calbindin-D28K may act to ferry calcium across the cell, as in the intestine, to buffer calcium, resulting in protection against calcium-mediated cell death [61,66]. Calbindin-D9K has been reported to have a different cellular action, i.e. to bind calcium and to stimulate ATP-dependent extrusion of calcium at the basolateral membrane [67]. The different functions of renal calbindin-D28K and

calbindin-D9K suggest different mechanisms that may be involved in the enhancement by 1,25(OH)2D3 of calcium transport in the distal nephron. Studies using immunosuppressant drugs (CsA and FK-506) that result in nephrotoxicity have noted decreases in calbindin-D28K in the rat coincident with increases in urinary calcium and intratubular calcification [68,69], providing additional evidence for a role of calbindin-D28K in distal tubular calcium reabsorption. In addition, calbindin-D28K KO mice fed a high-calcium diet were found to have significantly increased urinary calcium/creatinine ratio compared to wild-type controls [61,70]. The regulation of renal and intestinal calbindinD9K was found to be similar in wild-type and knockout mice, indicating that changes in calbindin-D9K were not compensating for the lack of calbindin-D28K and further suggesting different roles for these two vitamin-Ddependent calcium-binding proteins [61]. Serum calcium was not different in the wild-type and calbindin-D28K KO mice, suggesting compensatory changes in bone or in intestinal calcium absorption [61,70]. However, it should be noted that mechanisms within the kidney, independent of calbindin-D28K, are also associated with hypercalciuria [71]. To further elucidate the functional significance of mammalian calbindin-D28k, VDR/calbindin-D28K double KO mice were generated [72]. Mice deficient only in VDR display secondary hyperparathyroidism, rickets and osteomalacia, and a reduction of renal calbindin-D9K to 10% of typical levels (thus in these mice calbindin-D9k would not compensate for the loss of calbindin-D28k). VDR/calbindin-D28K double KO mice have increased urinary calcium excretion, more severe secondary hyperparathyroidism, lower bone mineral density, a more distorted growth plate and more osteoid formation in the trabecular region compared to VDR KO mice [72]. Although a high-calcium/lactose diet restored normal levels of serum ionized calcium in both VDR KO and double KO mice, the skeletal abnormalities were not completely corrected in the double KO mice [72]. These findings indicate the importance of renal calbindin-D28K in maintaining calcium homeostasis.

Bone Calbindin-D28K and calbindin-D9K are both present in chondrocytes of growth plate cartilage in rats and calbindin-D28K is present in the growth plate cartilage of chicks [22,73,74]. Although it is not clear whether calbindin is vitamin-D-dependent in chondrocytes, 1,25 (OH)2D3 receptors have been reported in developing chick bone, specifically in dividing chondrocytes [75]. It has been suggested that calbindin may be involved in the movement of calcium in the process of calcification in the chondrocyte [73]. Calbindin-D9K and



calbindin-D28K have also been localized to osteoblasts and ameloblasts of rodent teeth, and it has been reported that calbindin-D9K and calbindin-D28K mRNAs are induced by 1,25(OH)2D3 in these cells [20,21]. It has also been suggested that elevated expression of calbindin may phenotypically characterize cells that are involved in calcium handling during mineralization [20]. In addition to an association with mineralization, more recent evidence has indicated that calbindin-D28K is able to protect against apoptosis of bone cells. Calbindin-D28K was found to protect osteoblastic cells against tumor necrosis factor (TNF)-induced apoptosis (Fig. 20.3) as well as to prevent glucocorticoid-induced apoptosis of osteoblastic and osteocytic cells [76,77]. The protection against both TNF- and glucocorticoidinduced cell death was found to be at least partially due to the ability of calbindin-D28K to inhibit endogenous caspase-3, a key mediator of apoptosis in response to multiple signals. Calbindin-D28K was found to inhibit caspase-3 but was not cleaved by the caspase. In addition, the inhibition of caspase-3 by calbindin-D28K was reported to be independent of its calcium-binding ability. As the only other known natural nononcogenic

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inhibitor of capsase-3 besides the inhibitor of apoptotic proteins (IAPs), calbindin-D28K may be an important target in the prevention of cellular degeneration in bone cells.

Pancreas The pancreas was the first nonclassic target tissue in which receptors for 1,25(OH)2D3 were identified [78]. Although 1,25(OH)2D3 has been reported to play a role in insulin secretion, the exact mechanisms remain unclear [79e80]. An early indication that the pancreas may be a target for 1,25(OH)2D3 was the immunocytochemical study of Morrissey et al. [3], which localized calbindin-D28K to the islet. In the chick, calbindin-D28K is detected exclusively in insulin-producing b cells [81] and is responsive to vitamin D [82]. In the rat, however, calbindin-D28K has been reported to be localized in a as well as b cells of the pancreas [83]. Because autoradiographic data have indicated that 1,25(OH)2D3 receptors are localized only in rat b cells [84], and because insulin but not glucagon secretion is affected in vitamin-Ddeficient animals [79], studies in the rat suggest that

FIGURE 20.3 Overexpression of calbindin-D28K suppresses nuclear fragmentation of osteoblastic cells induced by TNFa. Cells were

transfected with the expression vector pREP4 alone (empty vector) or containing the cDNA for calbindin-D28K (calbindin-D28K) together with an expression vector containing the coding sequence of green fluorescent protein with a nuclear localization sequence. Forty-eight hours after transfection, cells were exposed to 1 nM TNFa for 16 h. Cells were fixed, mounted, and examined with a Zeiss confocal laser scanning microscope. Note the presence of apoptotic nuclei in the TNFa-treated vector-transfected cells but not in the calbindin-transfected cells similarly treated. Please see color plate section.


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b-cell calbindin-D28K may be regulated by 1,25(OH)2D3 while non-b-cell calbindin may be independent of vitamin D. Calbindin-D28K has also been identified in human pancreatic islet cells [85]. Studies using pancreatic beta cell lines as well as calbindin-D28K knockout mice have suggested that calbindin-D28K, by regulating intracellular calcium, modulates depolarization-stimulated insulin release [86]. In rat pancreatic beta cells, the alpha1 subunit of the L-type channel can interact with calbindin-D28K and in the presence of calbindinD28K, L-type channels show enhanced sensitivity to Ca2þ-dependent inactivation [87]. In addition to modulating insulin release, more recent studies have indicated that calbindin-D28K, by buffering calcium, can protect against destruction of beta cells by cytokines by preventing calcium-mediated mitochondrial damage and the resultant generation of free radicals [88]. These findings have important therapeutic implications for type 1 diabetes and the prevention of autoimmune destruction of pancreatic b-cells. Together, these findings indicate the involvement of pancreatic calbindin-D28K in intracellular Ca2þ homeostasis and modulation of Ca2þ influx.

Reproductive Tissues Calbindin-D28K has been reported in both chick and rat testes [5,6]. In chick and rat, immunocytochemical studies have revealed that calbindin-D28K is present in spermatogonia and spermatocytes of the seminiferous tubules and some interstitial Leydig cells [5,6]. It has been reported that vitamin-D-deficient chicks have significantly (threefold) lower testicular calbindin levels than vitamin-D-replete chicks [89]. As calbindin-D28K as well as VDR (which is also present in seminiferous tubules) have been shown to correlate with testicular maturation [6,90], the involvement of calbindin-D28K and vitamin D in spermatogenesis and steroidogenesis has been suggested [89]. Calbindin-D9K is present in the placenta and yolk sac of rats and mice [14,17]. Calbindin-D9K is also present in rat endometrium and myometrium [16]. In pregnant rats calbindin-D9K is also expressed in the uterine epithelium [16]. In placenta and yolk sac calbindin-D9K increases at the end of gestation, when there is increased calcium need of the fetus, suggesting a role for calbindin-D9K in the transport of calcium to the fetus [14,17]. Calbindin-D9k KO mice reproduce similar to wild-type mice and calbindin-D9k KO pups are indistinguishable from wild-type pups [52], suggesting compensation for the absence of calbindin-D9k in placenta by another factor. Unlike calbindin-D9K, calbindin-D28K is not present in rat reproductive tissue. However, calbindinD28K has been localized in the tubular gland cells of the shell gland in the chick [91], which are involved in calcium secretion during egg-shell formation.

Calbindin-D28K is also found in the reproductive tissues of female mice (endometrium and glandular epithelium of mouse uterus, mouse oviduct epithelium, and in primary follicles of mouse ovary) [92]. 1,25(OH)2D3 has no effect on calbindin in these tissues. However, calbindin-D9K and calbindin-D28K in rat and chick uterus, respectively, are under the positive control of estradiol [16,93]. In the mouse, calbindin-D28K gene expression is down-regulated in the uterus but not in the ovaries and oviduct, suggesting tissue- and species-specific regulation of calbindin-D28K by estradiol [92]. It has been suggested that transcellular calcium transport in epithelial cells of the uterus and oviduct is facilitated by calbindin [92]. The presence of calbindin in the myometrium suggests the involvement of calbindin in the modulation of intracellular calcium that may alter the frequency and strength of uterine contractions.

Nervous Tissue Calbindin-D28K is widely distributed throughout the brain of mammals, avians, reptiles, amphibians, fish, and mollusks [9]. It is present in most neuronal cell groups and fiber tracts and is localized in neuronal elements and some ependymal cells [8,94,95]. In brain, calbindin-D28K is not vitamin-D-dependent [9]. Neurons containing calbindin-D28K are found in the cerebral cortex in layers 2e4, primarily in pyramidal neurons as well as in hypothalamus, amygdala, thalamus, hippocampus, and cerebellum [8,94,95]. In the hippocampus, both basket cells and pyramidal neurons in CA1 stain positively for calbindin, as do granule cells and fibers in the dentate gyrus [8,94,95]. Purkinje cells of the cerebellum stain most intensely for calbindin-D28K [8,94,95]. It is of interest that the phenotype of the calbindin-D28K knockout mouse is impaired motor coordination [96]. It has been suggested that this phenotype may be the result of abnormal cerebellar activity due to the alteration of synaptically evoked calcium transients in the Purkinje cells in the absence of calbindin [96]. Calbindin-D28K is also present in the suprachiasmatic nucleus (SCN) which is a fundamental regulator of circadian function and entrainment in mammals [97]. During development, calbindin-D28K is markedly expressed in the mouse SCN and is coexpressed in adult melanopsin producing retinal ganglion cells [98]. Significant abnormalities are observed in circadian locomotor rhythmicity and entrainment to light/dark cycles in calbindin-D28K knockout mice, suggesting a role for calbindin-D28K in circadian organization and light transduction [98,99]. In addition, specific neuronal sensory cells have been shown to contain calbindin-D28K [100]. These cell populations include cochlear and vestibular hair cells in the inner ear [100], avian basilar papilla [101], cone but not



rod photoreceptor cells of the retina [102,103], and conelike, modified photoreceptor cells (pinealocytes) of pineal transducers [104]. The presence of calbindin in specific cells of the sensory pathway suggests the possible involvement of calbindin in mechanisms of signal transduction. In the nervous system it has been suggested that neuronal calbindin, by buffering calcium, can regulate intracellular calcium responses to physiological stimuli and can protect neurons against calcium-mediated neurotoxicity [105]. It has been demonstrated that introduction of exogenous calbindin into sensory neurons can modulate calcium signaling by decreasing the rate of rise of intracellular calcium and by changing the kinetics of decay of the calcium signal [106]. Using adenovirus as an expression vector, overexpression of calbindin-D28K in hippocampal neurons was reported to suppress post-tetanic potentiation, possibly by restricting and destabilizing the evoked calcium signal [107]. Calbindin-D28K was also reported to play a role in the control of hypothalamic neuroendocrine neuronal firing patterns [108]. The whole-cell patch-clamp method was used to introduce calbindin into rat supraoptic neurons. Calbindin-D28K suppressed Ca2þ-dependent depolarization afterpotentials and converted phasic into continuous firing [108]. As different firing patterns promote the release of different hypothalamic hormones, it was suggested that calbindin-D28K, by regulating firing patterns, may be involved in the control of hormone secretion from hypothalamic neuroendocrine neurons. These studies [106e108] indicated, directly by introduction of exogenous calbindin-D28K via the patch clamp method or by transfection and overexpression, that calbindin is an important and effective regulator of calcium-dependent aspects of neuronal function. Correlative evidence between decreases in neuronal calbindin-D28K and neurodegeneration in studies of ischemic injury [109], seizure activity [110], and chronic neurodegeneration (Alzheimer, Huntington, and Parkinson diseases) [111e113] have been reported. It has been suggested that decreased calbindin levels may lead to a loss of calcium buffering or intracellular calcium homeostasis, which leads to cytotoxic events associated with neuronal damage and cell death. Direct evidence of a protective role of neuronal calbindin-D28K against a variety of insults including exposure to hypoglycemia and IgG from amyotrophic lateral sclerosis patients has been shown in primary cultures of neuronal cells or in neuronal cell lines in which the calbindin-D28K gene has been transfected [114,115]. Expression of calbindin-D28K in neural cells was also found to suppress the proapoptotic actions of mutant presenilin 1 (PS-1), which is causally linked to about 50% of the cases of early-onset familial Alzheimer’s disease [116]. Mutant

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PS-1 has been reported to sensitize cells to apoptosis induced by amyloid b peptide (Ab), the major component of plaques in Alzheimer disease. Ab has been reported to damage neurons by a mechanism involving oxidative stress and disruption of calcium homeostasis. Calbindin-D28K protected against the proapoptotic action of mutant PS-1 by attenuating the increase in intracellular calcium and preventing the impairment of mitochondrial function [116]. In rats, pretreatment with a calbindin-D28K fusion protein (protein transduction domain (PTD)-calbindin-D28K) has been shown to attenuate neuronal insults induced by ischemia and reperfusion [117]. Protein transduction domains are small peptides that serve to deliver larger molecules into a variety of cells, including those of the bloodebrain barrier, without employing traditional endocytic mechanisms [118]. PTD-fused proteins have been shown to retain their biologic activity [119] and thus represent a potentially novel mode of neurobiological therapy. Since calbindin-D28K can prevent neuronal damage in neuropathies, including ischemic injury, these findings have important therapeutic implications.

Calbindin-D28K and Apoptosis Calcium is thought to play an important regulatory function in apoptosis, but the precise mechanism(s) by which calcium promotes cell death is unknown. The first study suggesting that calbindin-D28K plays a protective role in the process of apoptosis used subtraction analysis between the cDNA libraries of two human prostate cell lines [120]. One of the cell lines was androgen independent and the other one androgen dependent, and the results revealed that a hybrid calbindin-D28K gene was specifically expressed in the hormone-independent cell line. Apoptosis has been observed in prostate cells on androgen depletion. Androgen deprivation of prostate cells triggers an influx of calcium ions into the cells, leading to an increase in intracellular calcium. It was suggested that calbindin-D28K might buffer intracellular calcium and contribute to protection against apoptosis and thus androgen independence in the prostatic cell line. It has been reported that stable transfection and overexpression of calbindin-D28K in lymphocytes protect against apoptosis induced by calcium ionophore, cAMP, and glucocorticoid [121]. A similar protective role for calbindin-D28K has been observed in apoptosissusceptible cells in the central nervous system [114e116] as well as in human embryonic kidney cells (HEK 293), osteoblasts, and pancreatic b cells [66,76,77,88]. These findings indicate that calbindinD28K has a major role in different cell types in protecting against apoptotic cell death. A further understanding of the mechanisms involved will have important


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therapeutic implications for the prevention of a number of diseases including osteoporosis and diabetes.

REGULATION OF CALBINDIN GENE EXPRESSION Calbindin-D28K Genomic Organization of the Calbindin-D28K Gene The genomic organization of the chicken calbindinD28K gene has been elucidated [122,123] and a partial structure for the human gene was also reported [12,124]. The human gene (symbol: CALB1) is located on chromosome 8 [124,125] and is believed to consist of 11 exons, analogous to that demonstrated for the avian gene. Moreover, the total size of the gene is reported to be 18.5 kb in chicken and 24 kb in human. The protein coding region of the mouse gene shares 77% sequence similarity with the chicken gene [126]. However, no obvious sequence similarity exists between the mammalian and avian promoters except in the region of the TATA box [12,127]. Regulation by 1,25(OH)2D3 It is well known that calbindin-D28K in the avian intestine [1,128] and kidney [128] and in the mammalian kidney [129,130] is induced by 1,25(OH)2D3. In chicken, a putative calbindin-D28K vitamin D response element (VDRE) was suggested after computer analysis of the promoter sequence [131], but only a twofold response to 1,25(OH)2D3 was detected in primary kidney cells after transfection with a 2.1-kb segment of the 50 flanking region of the promoter [132]. A relatively inactive putative VDRE was also reported in the chicken calbindinD28K promoter by others [133,134] and the response of the mouse calbindin-D28K promoter to 1,25(OH)2D3 is modest (fivefold maximal induction in chloramphenicol acetyltransferase (CAT) activity) [127]. The modest response reflects previous in vivo findings that indicated a small transcriptional response to 1,25(OH)2D3 [125,130]. Similar findings were reported for the in vivo induction of the chick intestinal calbindin-D28K gene by 1,25(OH)2D3 [135]. In addition, in VDR knockout mice only a small reduction in basal levels of renal calbindin-D28K is observed. However, the response of renal calbindin-D28K in the VDR knockout mouse to 1,25(OH)2D3 is compromised [50]. Also in 25-hydroxyvitamin D3 1a-hydroxylase knockout mice the expression of renal calbindin-D28K is reduced [51]. There is now increasing evidence that the large induction of calbindin-D28K mRNA long after 1,25(OH)2D3 treatment may be due primarily to post-transcriptional mechanisms [125,130,135,136]. Exactly how this action is exerted is not known, but one report suggests that 1,25

(OH)2D3 may regulate the expression of an intermediate protein that may be involved in calbindin-D28K mRNA accumulation [136]. These studies suggest that the mechanism of action of 1,25(OH)2D3 on calbindin-D28K regulation is more complicated than the conventional hormone receptoretranscriptional activation model, and that this regulation may involve other factors and is mostly post-transcriptional. Regulation by other Steroids and Factors Further studies in intestine and kidney have provided evidence that the calbindin-D28K gene is not exclusively regulated by 1,25(OH)2D3 and that other factors can modulate gene expression. It has been reported that glucocorticoids can inhibit the levels of calbindin-D28K mRNA and protein in intestine of vitamin-D-treated chicks, resulting in a comparable decrease in intestinal calcium absorption [137,138]. These findings suggest the involvement of the inhibition of intestinal calbindin in the clinically important hypocalcemic action of glucocorticoids. Alterations in dietary Ca2þ and phosphorus have also been shown to modulate avian intestinal and renal calbindin-D28K gene expression, further suggesting that the regulation of calbindin is more complex than previously thought [139,140]. In other tissues where calbindin-D28K is present in significant amounts, for example, in parts of the brain, the regulation of calbindin-D28K appears to be very different from that in the intestine and kidney. In the central nervous system (CNS), 1,25(OH)2D3 has no apparent effect on the levels of calbindin-D28K [9]. Instead, a variety of different factors have been reported to be involved in regulating neuronal calbindin-D28K. Using rat hippocampal cultures, evidence has indicated that neurotropin 3 (NT-3) [141,142], brain-derived neurotropic factor (BDNF) [141,142], fibroblast growth factor (FGF) [141], and tumor necrosis factors (TNFs) [143,144] all can induce calbindin. It has been reported that neurotropic factors may protect against excitotoxic neuronal damage [145]. The induction of calbindin by those factors suggests a role for calbindin-D28K in the process of protection against cytotoxicity. In addition, corticosterone administration in vivo has been reported to increase calbindin-D28K expression in rat hippocampus [146,147]. The glucocorticoid response is specifically localized to the CA1 region [147]. Retinoic acid has also been reported to induce calbindin-D28K protein and mRNA in medulloblastoma cells, which express a neuronal phenotype [148]. Furthermore, the content of calbindin-D28K in cultured Purkinje cells can be increased by insulin-like growth factor I (IGF-I) [149]. Similarly IGF-I, as well as insulin, promoted the expression of calbindin-D28K protein in cultured rat embryonic neuronal cells [150]. Thus, neuronal calbindin-D28K can be regulated by steroids as well as by


REGULATION OF CALBINDIN GENE EXPRESSION

factors that affect signal transduction pathways. These different modes of activation may be important for cell-specific effects of calbindin-D28K. The molecular basis for tissue-specific calbindin-D28K regulation is still not understood, but in vivo experiments using transgenic mice suggest that tissue specificity of calbindin-D28K expression to some degree is controlled by separate elements on the promoter [151]. The transgenic mouse study demonstrated the importance of an in vivo system to investigate the role of sequence elements needed for tissue-specific gene expression and regulation [151]. Gender-specific hormones have also been suggested to modulate calbindin-D28K expression. As discussed previously, calbindin-D28K is also present in the avian egg-shell gland and in the reproductive tissues of female mice. In the avian egg-shell gland, estradiol-17b induces calbindin-D28K in in vivo experiments [93]. In the mouse, calbindin-D28K gene expression was found to be downregulated by estradiol in the uterus and oviduct [92,152] but up-regulated in the ovaries [152]. Multiple imperfect half-palindromic estrogen-responsive elements, which are likely to mediate the estrogen responsiveness of the calbindin-D28K gene by estradiol-17b, were present in two regions (e1075/e702 and e175/e78) of the promoter [152]. The contrasting responses to estradiol in chick and mouse suggest species-specific regulation of calbindin-D28K by estradiol. It has also been shown that differences in gender influence Ca2þ handling in the kidney as male mice display greater urinary Ca2þ loss than female mice [153]. Significantly decreased levels of TRPV5 and calbindin-D28K are also observed in male mice compared to female mice [154]. Furthermore, levels of renal TRPV5 and calbindin-D28K mRNA and protein are increased in androgen-deficient male mice and are unaccompanied by significant differences in serum estrogen, parathyroid hormone, or 1,25 (OH)2D3 levels [154]. Testosterone treatment suppresses the elevation of renal TRPV5 and calbindin-D28K in these mice [154]. Thus, it has been suggested that gender differences in renal Ca2þ reabsorption may, in part, be explained by the inhibitory actions of androgens on renal TRPV5 and calbindin-D28K. Though calbindin-D28K was one of the first identified targets of 1,25(OH)2D3 action, the complex regulation of calbindin-D28K illustrates that the utility of calciumbinding proteins extends beyond the traditional paradigm of the vitamin D endocrine system.

Calbindin-D9K Genomic Organization of the Calbindin-D9K Gene The size of the calbindin-D9K gene is 2.5 kb, and the gene consists of three exons and two introns [155]. The first exon contains the 50 untranslated region. The second

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exon codes for the first calcium-binding site. The third exon codes for the second calcium-binding site and the 30 untranslated region. It is related to the S100 family of calcium-binding proteins. The human calbindin-D9k gene (symbol: CALB3) spans 5.5 kb and consists of three exons. The chromosomal localization of the human calbindin-D9k gene is assigned to Xp22.2. [156]. Regulation of Calbindin-D9K by 1,25(OH)2D3 Similar to the regulation of avian intestinal calbindinD28K and mammalian renal calbindin-D28K, in vivo findings have indicated that intestinal calbindin-D9K is regulated by 1,25(OH)2D3 by a small, rapid transcriptional stimulation followed by a post-transcriptional effect accounting for a sustained accumulation of mRNA long after cessation of 1,25(OH)2D3 treatment [157]. Unlike renal calbindin-D28K, there is a marked decrease of basal as well as 1,25(OH)2D3 induced levels of intestinal calbindin-D9K mRNA in VDR knockout mice, suggesting that intestinal calbindin-D9K is more sensitive to control by VDR-mediated mechanisms than calbindin-D28K [50]. Studies using transgenic mice have shown that the proximal promoter of the calbindin-D9K gene from e117 to þ365 and distal element at e3731 to e2894 together but not separately confer the 1,25(OH)2D3-induced transcriptional response [158]. Since this region does not contain a classical VDRE, these findings suggest that the 1,25(OH)2D3 regulation of calbindin-D9K may involve a nonconventional activation pathway. Regulation of Calbindin-D9K by other Steroids and Factors In vitro footprinting and gel shift assays suggested that several transacting factors other than the VDR, including a ubiquitous factor (NF1), liver-enriched factors (HNF1, C/EBP alpha and beta, and HNF4), and the intestine-specific transcription factor caudal homeobox-2 (Cdx-2), may be important for intestinespecific calbindin-D9K gene expression [159]. Further studies using transgenic mice showed that a mutation in the distal Cdx2-binding site of calbindin-D9K promoter dramatically decreased intestinal expression of the calbindin-D9K gene, directly demonstrating the crucial role of Cdx2 for the transcription of this gene in the intestine [160]. With regard to other steroids, besides 1,25(OH)2D3, the expression of calbindin-D9K in the intestine is also regulated by glucocorticoids. Glucocorticoids have been reported to inhibit intestinal calbindin-D9K expression [161]. It has been suggested that this decrease may be involved in the reported decrease by glucocorticoids in intestinal calcium absorption. Whether the effect on intestinal calbindin-D9K expression is a primary or a secondary action of glucocorticoids is not yet known.


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In the uterus, calbindin-D9K is under the control of estrogen but is unaffected by 1,25(OH)2D3. An imperfect estrogen response element (ERE) that binds the estrogen receptor (ER) has been identified at the border of the first exon and the first intron [162,163]. In vivo experiments using transgenic mice suggest the functionality of this imperfect ERE [164].

EPITHELIAL CALCIUM CHANNELS Transient Receptor Potential Vanilloid 5 (TRPV5) General Considerations and Genomic Organization of the TRPV5 Gene Transient receptor potential vanilloid (TRPV) cation channels make up one of four main classes of the transient receptor potential (TRP) superfamily [165]. In 1999, TRPV5 (ECac/CaT2) was cloned from distal convoluted tubules in rabbit kidney using Xenopus oocytes [44]. Of all TRP channels, TRPV6 shares the highest sequence homology with TRPV5 (75%) [166]. The human TRPV5 gene has been mapped to chromosome 7q31.1-q31.2, just upstream of TRPV6, and contains 15 exons coding for a protein of 729 amino acids with a molecular mass of 83 kDa [167]. The TRPV5 protein consists of six transmembrane domains with a putative pore-forming region between domains 5 and 6 as well as cytosolic N- and C-terminal domains and exists functionally as a tetramer forming a single Ca2þ channel [167]. TRPV5 Ca2þ-binding properties are lost upon disruption of the aspartate-542 residue located within the pore formed between domains 5 and 6 [168]. Immunohistochemical studies have localized TRPV5 to the apical membrane of the distal convoluted tubule and connecting tubule of the kidney and have shown colocalization of renal TRPV5 with calbindin-D28K [169]. Regulation of TRPV5 by 1,25(OH)2D3 Putative vitamin D response elements have been identified in the TRPV5 promoter [170]. Studies in mice and rats have shown that renal TRPV5 mRNA is induced by 1,25(OH)2D3 [62e64]. However the expression of renal TRPV5 mRNA is unchanged in VDR KO mice (unlike intestinal TRPV6 mRNA which is markedly reduced in VDR KO mice compared to WT mice), suggesting that factors in addition to 1,25(OH)2D3 are involved in the regulation of TRPV5 in the kidney [171,172]. Proposed Functional Significance The generation of mice lacking TRPV5 has provided an understanding of the functional significance of this

epithelial calcium channel. Renal calcium reabsorption is compromised in TRPV5 KO mice, resulting in hypercalciuria and significant changes in bone morphology, including reduced trabecular and cortical bone thickness as well as impaired bone resorption [173,174]. Furthermore, these mice show significantly elevated levels of serum 1,25(OH)2D3 and a compensatory increase in intestinal TRPV6 mRNA, intestinal calbindin-D9K mRNA, and intestinal calcium absorption [173]. Micropuncture studies localize the site of defective calcium reabsorption to the distal convoluted tubule, the site of localization of TRPV5, thereby suggesting that hypercalciuria in the KO mice results primarily from the absence of TRPV5 [173]. In TRPV5/ 1a-hydroxylase double KO mice up-regulation of intestinal TRPV6 and calbindin-D9k mRNA is not observed, indicating that the elevated serum 1,25(OH)2D3 levels are responsible for the compensatory increase in intestinal calcium transporters and intestinal calcium absorption in the TRPV5 KO mice [175]. Together, these findings demonstrate a role for TRPV5 in active calcium reabsorption in the distal nephron. Studies have also been done comparing TRPV5 KO mice, calbindin-D28K KO mice, and TRPV5/calbindinD28K double KO mice [176]. Double KO mice display compensatory up-regulation of intestinal calcium absorption (similar to that described previously in TRPV5 KO mice), increased renal calbindin-D9K expression, and up-regulation of intestinal calbindin-D9K and TRPV6 [176]. Intestinal calcium absorption and expression of calbindin-D9K and TRPV6 remain unchanged in calbindin-D28K KO mice compared to WT mice [176]. The similarities between TRPV5 KO mice and TRPV5/ calbindin-D28K double KO mice suggest that loss of calbindin-D28K may be compensated by up-regulation of renal calbindin-D9K in the double KO mice and provide further evidence of the importance of TRPV5 in renal calcium reabsorption in the distal part of the nephron.

Transient Receptor Potential Vanilloid 6 (TRPV6) General Considerations and Genomic Organization of the TRPV6 Gene Transient receptor potential vanilloid (TRPV) cation channel 6 (TRPV6) was first cloned from rat duodenum [45]. TRPV6 is expressed in highest concentrations in duodenum and cecum and in lower concentrations in proximal jejunum [177,178]. Very low levels are also expressed in the colon [177,178]. TRPV6 is expresssed in villi tips and not in villi crypts [177,178]. TRPV6 is also expressed in placenta, acinar cells of the pancreas, ductal epithelial cells of mammary glands and in skin [177,178]. The human TRPV6 gene has been mapped to chromosome 7q33-q34 downstream of TRPV5 and


373

CONCLUSION

TRPV6 (epithelial calcium channel cloned from rat duodenum): predicated membrane topology and domain structure. TRPV6 contains six transmembrane domains. The putative N-linked glycosylation site (branched chain) is marked. Reprinted with permission from Peng et al., 1999 [45].

FIGURE 20.4

consists of 15 exons coding for a protein of 725 amino acids [179]. Similar to TRPV5, TRPV6 contains six transmembrane (TM) segments and cytosolic N- and Cterminal domains [178] (see Fig. 20.4). TRPV6 as well as TRPV5 channels contain ankyrin repeats at their aminoterminal which are part of functional tetrameric channels [180]. Regulation of TRPV6 by 1,25(OH)2D3 and Functional Significance In the human TRPV6 promoter putative vitamin D response elements (VDREs) were identified at e1.2, e2.1, e3.5, e4.3, and e5.5 kb. The VDREs at e4.3 and e2.1 were found to mediate the 1,25(OH)2D3 response in intestinal cells [181]. TRPV6 is colocalized with calbindin-D9k in duodenum and jejunum. A similar regulation of intestinal TRPV6 and calbindin-D9k has been observed [64]. Both TRPV6 and calbindin-D9k are markedly induced at weaning, the time of onset of active intestinal calcium transport and intestinal responsiveness to 1,25(OH)2D3 and both are strongly induced under low calcium conditions and after 1,25(OH)2D3 injection of vitamin-D-deficient mice [64]. After a single injection of 1,25(OH)2D3 intestinal TRPV6 mRNA and calbindin-D9k mRNA are induced prior to the peak of induction of intestinal calcium absorption [64]. Since TRPV6 mRNA was found to be more markedly decreased in the intestine than calbindin-D9k in VDR KO mice, it was suggested that the expression of TRPV6 may be a rate-limiting step in the process of vitamin-D-dependent intestinal calcium absorption [172]. However, when fed a standard rodent chow diet TRPV6 KO mice were found to have serum calcium levels similar to those of WT mice [54,182,183]. Serum PTH levels have been reported to be significantly increased in TRPV6 KO mice, consistent with an observed 9.6% decrease in femoral bone density [182].

Thus, TRPV6 may have an indirect role in regulation of bone formation and/or mineralization. 1,25(OH)2D3 administration to vitamin-D-deficient TRPV6 KO mice results in a significant increase in active intestinal calcium transport, indicating that in the KO mice there is compensation by another channel or protein [54,183]. Recent studies have found that TRPV6 can interact with other proteins that modulate its function including calmodulin which facilitates rapid inactivation of TRPV6, Rab11a which has been shown to play a role in recycling of TRPV6 to the plasma membrane, and S100A10-annexin 2 protein complex which may be involved in the constitutive trafficking of TRPV6 to the plasma membrane [184e186]. These TRPV6-associated proteins may represent novel components of 1,25 (OH)2D3-regulated intestinal calcium transport that specifically influence calcium entry mechanisms.

CONCLUSION We once viewed calbindin-D28K and calbindin-D9K as exclusively vitamin-D-dependent proteins. It is now evident that the calbindins are not under the exclusive regulatory control of 1,25(OH)2D3. Calbindin-D28K and calbindin-D9K are present in many different tissues (see Table 20.1) and may serve many different functions. Accordingly, the regulation of these calcium-binding proteins is varied and quite complex. The calciumselective epithelial calcium channels, TRPV5 and TRPV6, are colocalized with calbindin in kidney and TABLE 20.1

Distribution of Calbindin

Calbindin-D9K

Calbindin-D28K

Mammalian intestine [13]

Avian intestine [3,34,37]

Mouse and neonatal rat kidney [23]

Avian, reptilian, amphibian, and mammalian kidney [3,55e57]

Rat and mouse yolk sac [14,17]

Hen egg-shell gland (uterus) [93]

Rat uterus [15,16]

Mouse reproductive tissues (uterus, oviduct, ovary) [92]

Rat and mouse placenta [14]

Avian and mammalian beta cells of the pancreas [81,85] Alpha cells of the rat pancreas [83]

Rat growth cartilage [22]

Rat and chick growth cartilage [22,73,74]

Ameloblasts and osteoblasts of rodent teeth [20,21]

Ameloblasts and osteoblasts of rodent teeth; mouse osteoblasts [20,21,76]

Rat lung [18]

Brain (avian, reptilian, amphibian, molluskan, fish, and mammalian brain) [9,94,95]


374


intestine respectively. Studies in TRPV5 KO mice have indicated that TRPV5 is important for renal calcium reabsorption. Calbindin-D28k interacts with TRPV5 and regulates its activity, suggesting a role for calbindinD28k as a modulator of calcium influx. Studies using calbindin-D9k and TRPV6 KO mice suggest that in the KO mice there is compensation by another calcium channel or protein and that other factors involved in 1,25 (OH)2D3 mediated intestinal calcium absorption remain to be identified.

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[179] J.B. Peng, X.Z. Chen, U.V. Berger, S. Weremowicz, C.C. Morton, P.M. Vassilev, et al., Human calcium transport protein CaT1, Biochem. Biophys. Res. Commun. 278 (2000) 326e332. [180] I. Erler, D. Hirnet, U. Wissenbach, V. Flockerzi, B.A. Niemeyer, Ca2þ-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat, J. Biol. Chem. 279 (2004) 34456e34463. [181] M.B. Meyer, M. Watanuki, S. Kim, N.K. Shevde, J.W. Pike, The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells, Mol. Endocrinol. 20 (2006) 1447e1461. [182] S.D. Bianco, J.B. Peng, H. Takanaga, Y. Suzuki, A. Crescenzi, C.H. Kos, et al., Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene, J. Bone Miner. Res. 22 (2007) 274e285. [183] G.D. Kutuzova, F. Sundersingh, J. Vaughan, B.P. Tadi, S.E. Ansay, S. Christakos, et al., TRPV6 is not required for 1alpha,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo, Proc. Natl. Acad. Sci. USA 105 (2008) 19655e19659. [184] I. Derler, M. Hofbauer, H. Kahr, R. Fritsch, M. Muik, K. Kepplinger, et al., Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2þ-dependent inactivation, J. Physiol. 577 (2006) 31e44. [185] S.F. van de Graaf, J.G. Hoenderop, D. Gkika, D. Lamers, J. Prenen, U. Rescher, et al., Functional expression of the epithelial Ca(2þ) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex, EMBO J. 22 (2003) 1478e1487. [186] S.F. van de Graaf, Q. Chang, A.R. Mensenkamp, J.G. Hoenderop, R.J. Bindels, Direct interaction with Rab11a targets the epithelial Ca2þ channels TRPV5 and TRPV6 to the plasma membrane, Mol. Cell Biol. 26 (2006) 303e312.



C H A P T E R

21 Mineralization Eve Donnelly, Adele L. Boskey Hospital for Special Surgery, 535 E 70th Street, New York, NY 10021; affiliated with Weil College of Cornell Medical School, New York, NY 10021, USA

INTRODUCTION Definitions “Biologic mineralization” is the physicochemical process leading to deposition of inorganic crystals (minerals) on an organic matrix within the cell or outside it. This term is more specific than “mineralization,” as biologic mineralization implies a relation between the cells, the organic matrix, and the mineral. The cell-mediated biomineralization process comprises oriented deposition of mineral within or adjacent to cells or upon an extracellular matrix. Examples of the former include iron oxides and sulfides in magnetotactic bacteria [1] and silicates in diatoms [2]; examples of the latter include calcium carbonates in shells [3] and exoskeletons [4] and calcium phosphates in bones and teeth [5]. The mineral in physiologically calcified vertebrate tissues is an analog of the geologic mineral hydroxyapatite (Fig. 21.1). The physiologic hydroxyapatite crystals are 10e60 nm in their largest dimension [6], much smaller than those found in geologic deposits, and have stoichiometries different from the predicted 10Ca:6P04:20H of the geologic mineral. For that reason biologic vertebrate mineral is often referred to as

FIGURE 21.1 The hydroxyapatite unit cell showing the major substituents occurring in biologic apatites and the ions for which they substitute.


“apatite” or “apatitic” meaning “like hydroxyapatite.” This chapter will focus on physiologic and dystrophic apatite formation in situ and in culture and on the effects of vitamin D on mineral formation. Crystalline deposits may form by several different mechanisms. De novo crystal deposition occurs when the solution supersaturation exceeds the solubility of the precipitating phase. Supersaturation refers to the ratio of the solution ion product to the solubility product of the phase in question. The process starts with “nucleation” in which several ions or ion clusters come together in solution with the orientation they will have in the final crystal. This first step requires a great deal of energy, and is facilitated by increasing the ion product or reducing the diffusion of ions in solution [5]. Most biomineralization is epitaxial or heterogeneous e occurring on the surfaces of pre-existing crystals or on protein and lipid templates, which resemble the surface of the crystal [7]. These processes use much less energy than de novo mineralization, and require less supersaturation. Crystal growth occurs as ions and ion clusters add on to the surface of the initial nuclei or other pre-existing crystals. Crystal growth requires less energy than nucleation, and is limited in the case of biologic mineralization by the template upon which the crystals are deposited. Crystals can grow in all dimensions by the addition of ions [7]; by agglomeration [8] in which crystals accumulate, not always in an oriented fashion; or by secondary nucleation. Secondary nucleation, as it is seen with hydroxyapatite crystals maturing in solutions in the presence of the dentin protein, phosphophoryn [9], is a branching process in which new nuclei form on the surfaces of existing crystals thus resulting in a new population of immature crystals.

381


382

21. MINERALIZATION

Among the vertebrate mineralized tissues, enamel contains the largest apatite crystals, but these crystals do contain carbonate [10] and other environmental contaminants (e.g., strontium, fluoride, lead, etc. [11]). Bone and tendon-bone insertions contain the smallest apatite mineral crystals [12], 6e10% carbonate [13], as well as adsorbed and incorporated citrate, fluoride, and other trace impurities. Bone apatite crystals are also hydroxide-deficient [14], but are not totally devoid of hydroxyl groups [15,16]. Cementum and dentin mineral crystals have intermediate sizes and intermediate accumulation of foreign ions. In each of these tissues the crystals that are formed initially are smaller than those in the mature tissues due both to the growth of existing crystals and the removal of the smaller crystals during osteoclast remodeling [5]. In humans, mineral also deposits at abnormal (unexpected/pathologic) locations [17]. This pathologic mineral is frequently apatitic, but calcite (calcium carbonate) is found in pancreatic stones [18]; monosodium urate, sodium pyrophosphate, as well as apatite [17] are found in cartilage; and brushite, oxalates, and uric acid occur in kidney and salivary stones (Table 21.1). The mineral in pathologic calcifications may be formed by physiologic processes or may be associated with dying cells (dystrophic calcification). An example of dystrophic calcification induced by vitamin D toxicity is seen in arteries [19]. In some cases the tissue becomes bone-like, and genes associated with osteogenesis are activated [20e22]; however it is not clear whether the calcification or the osteogenic gene expression comes first [23]. Dystrophic calcifications are distinct from TABLE 21.1

Pathologic Mineral Deposits

Mineral phase

Found in

Affected by vitamin D

Apatite

Blood vessels Kidney and bladder stones Salivary stones Pulp stones Muscle Skin Hyaline and articular cartilage

þ þ þ þ þ þ þ

Calcium carbonate (aragonite) (calcite)

Pancreatic stones

0

Oxalates

Kidney and salivary stones Soft tissues

þ

Pyrophosphates

Hyaline and articular cartilage

0

Urates

Hyaline and articular cartilage Kidney stones

0 þ

Calcified intrauterine devices

þ Indicates that deposition in these tissues is accelerated in hypervitaminosis D.

physiologic mineralization as in the latter viable cells are required, while in the former cells die, releasing calcium and phosphate and degradative enzymes [24].

Direct and Indirect Effects of Vitamin D and Vitamin D Metabolites on Mineralization Physical Chemistry of Mineralization De novo apatite formation requires Caþ2, PO-3 4 , and OHe ions to come together in the correct orientation with sufficient energy and in sufficient numbers to form the first stable apatite crystal (nucleus). After this nucleus is formed, additional ions can add on to these small crystals (nuclei) causing crystal growth. As crystals become larger, new nuclei can branch off the surface (secondary nucleation), in a fashion analogous to glycogen formation. These new nuclei grow and exponentially form additional secondary nuclei. During cell-mediated biologic apatite formation, matrix molecules provide sites for accumulation of ions and templates to orient mineral growth. Some molecules may act as heterogeneous nucleators, facilitating the deposition of mineral. Others may bind to the crystals and regulate their shape and size [25]. Nucleation occurs at multiple sites along these templates, and crystal habit (shape) and size is regulated by the template and by other matrix proteins that bind to the surface of the apatite crystals. In cartilage, bone, dentin, tendon, and ligaments, mineral crystals form on a collagenous matrix. This collagen is the “template” [26] for initial mineral formation and crystal growth; mineralization of the collagen matrix gives it increased strength [27]. Differences in the distribution and post-translational modification [28] of extracellular matrix molecules associated with the fibrillar collagen influence the crystal size and the site of initial crystal deposition. In enamel, which does not have a collagen component, there is some debate as to which of the proteins initially present in the enamel matrix are the nucleators. Recent solution data showed that amelogenin, a large hydrophobic molecule that associates into nanospheres, can induce initial calcification [29,30], facilitate the ordering and agglomeration of crystals, and hence also regulate the size to which the crystals grow [30]. (There are several other proteins in enamel whose functions are not yet established, and it must be noted that results from solution studies do not always agree with cell culture or in vivo studies, and mice lacking amelogenin can form enamel [31].) One of the ways in which vitamin D is believed to influence mineralization is by stimulating the formation of these proteins and the enzymes responsible for their post-translational modification. The genes for many of these proteins, as well as many of the enzymes that


383

INTRODUCTION

TABLE 21.2

Extracellular Matrix Proteins and Cellular Enzymes Regulated by Vitamin D and Associated with Mineralization: Solution Data

Protein

Effect on mineralization

Ref.

Regulated by vitamin D*

Ref.

Amelogenin

Regulator of crystal growth

a

þ

b

Aggrecan

Inhibitor

25

þ

c

Biglycan

Nucleator

25

?

133

Bone sialoprotein (BSP)

Nucleator

109

Expression inhibited

110, d

Collagen I

Template

25

þ

106,107

Dentin matrix protein-1 (DMP1)

Nucleator/inhibitor

e

þ

32

Dentin phosphophoryn

Nucleator

8

þ

b

Dentin sialoprotein

Weak nucleator/inhibitor

25

0

f

Enamelin

Regulator of crystal shape

30

þ

b

Fetuin

Inhibitor

g

þ

g

Matrix gla protein (MGP)

Inhibitor

25

þ

32

Matrix extracellular phosphoglycoprotein (MEPE)

Nucleator/Inhibitor

127,128

Osteopontin

Inhibitor

104,115

þ

115

Osteocalcin

Inhibitor

112

þ

111

Proteolipid

Nucleator

h

þ

117e119

ATPase

Hydrolyzes ATP, facilitates cellular Ca transport

i

þ

i

Matrix metallo-proteinases Stromolysin MMP3 Gelatinase MMP2

Degrade matrix molecules that inhibit mineralization

25

þ

32,j

EXTRACELLULAR MATRIX PROTEINS

32

ENZYMES

* Regulation by vitamin D depends on concentration, cell type and cell maturity ; hence + indicates that there is an effect and/or that the gene contains a vitamin D responsive element (VDRE). Readers are referred to other chapters to see precise effects. 0 ¼ No effect ? ¼ unknown. a. E. Beniash, J.P. Simmer, H.C. Margolis. The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro. J. Struct. Biol. 149 (2005) 182e190. b. P. Papagerakis, M. MacDougall, D. Hotton, I. Bailleul-Forestier, M. Oboeuf, A. Berdal. Expression of amelogenin in odontoblasts. Bone 32 (2003) 228e40. 156. A. Farzaneh-Far, P.L. Weissberg, D. Proudfoot, C.M. Shanahan. Transcriptional regulation of matrix gla protein. Z. Kardiol. 90 (2001) s38es42. c. D.J. Rickard, I. Kazhdan, P.S. Leboy Importance of 1,25-dihydroxyvitamin D3 and the nonadherent cells of marrow for osteoblast differentiation from rat marrow stromal cells. Bone 16 (1995) 671e678. d. J.J. Chen, H. Jin, D.M. Ranly, J. Sodek, B.D. Boyan. Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. J. Bone Miner. Res. 14 (1999) 221e229. e. A. Gericke, C. Qin, Y. Sun, R. Redfern, D. Redfern, Y. Fujimoto, et al. Different forms of DMP1 play distinct roles in mineralization. J. Dent. Res. 89 (2010) 355e359. f. H.H. Ritchie, H. Park, J. Liu, T.J. Bervoets, A.L. Bronckers. Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture. Biochim. Biophys. Acta. 1679 (2004) 263e271. g. T. Schinke, C. Amendt, A. Trindl, O. Poschke, W. Muller-Esterl, W. Jahnen-Dechent. The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J. Biol. Chem. 271 (1996) 20789e20796. h. B.R. Genge, L.N. Wu, R.E. Wuthier. Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens. J. Biol. Chem. 283 (2008) 9737e48. i. W.E. Horton Jr., R. Balakir, P. Precht, C.T. Liang. 1,25-Dihydroxyvitamin D3 down-regulates aggrecan proteoglycan expression in immortalized rat chondrocytes through a post-transcriptional mechanism. J. Biol. Chem. 266 (1991) 24804e24808. j. Y. Nakano, W.N. Addison, M.T. Kaartinen. ATP-mediated mineralization of MC3T3-E1 osteoblast cultures. Bone 2007 41 (2007) 549e561.

post-translationally modify them have vitamin-Dresponsive elements (VDRE) [32]. Extracellular matrix proteins that in solution or in culture affect the formation of apatitic mineral are listed in Table 21.2.

Cells control the mineralization process, both by regulating local calcium and phosphate concentrations and pH [33,34] as well as by the production and post-translational modification of collagen and


384

21. MINERALIZATION

noncollagenous proteins [28], which guide and direct mineral deposition. There is also a physicochemical process by which supersaturated solutions lead sometimes to mineral deposition in unwanted sites. One example of this physicochemical effect is the hypervitaminosis D syndrome [19], where injections of vitamin D into animals cause an elevation of circulating calcium and result in arterial and kidney calcification. The Nature of Vertebrate Mineral The mineral that forms in physiologically mineralized tissues e calcified cartilage, bone, dentin, and enamel, is an analog of the geologic mineral, hydroxyapatite. The chemical formula for this mineral is Ca10(PO4)6(OH)2. With the exception of enamel, physiologic mineral is deposited in an oriented fashion on a collagen template (Fig. 21.1). Unlike geologic hydroxyapatite crystals, which are quite large and visible to the naked eye, the physiologic mineral crystals are microscopic in size, 95%) so that the CDP labeling value usually reflects type I collagen synthesis. If desired, the production of different collagen types can be distinguished by ion-exchange chromatography and polyacrylamide gel electrophoresis of radiolabeled extracts of cell or organ cultures [44,45]. Type I collagen expression in human cell cultures has also been assessed by measuring secretion of the procollagen I C-terminal propeptide [46,47]. Finally, specific cDNA probes in Northern blotting and allele-specific primers in reverse transcriptase-polymerase chain reaction assays have been used to assess collagen mRNA expression in bone models. The measurement of the effect of 1,25(OH)2D3 on collagen synthesis and mRNA

levels has given comparable results using these different assays. 1,25(OH)2D3 inhibits collagen synthesis in organ cultures of 21-day fetal rat calvariae [20] and neonatal mouse calvariae [48] with little or no effect on noncollagen protein synthesis. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 in rat calvariae (about 50%) occurs at 10 nM [20]. 1,24R,25-(OH)3D3 also inhibits collagen synthesis but is less potent than 1,25(OH)2D3 [20]. 25-(OH)D3 and 24R,25(OH)2D3 do not alter collagen synthesis below 100 nM [20,48]. Vitamin D metabolites inhibit collagen synthesis and stimulate resorption of fetal rat long bones with similar relative potencies that correlate with the affinity of the metabolites for the skeletal VDRs [49]. To determine the cell selectivity of the 1,25(OH)2D3 inhibition of collagen synthesis, organ cultures of fetal rat calvariae were treated with 1,25(OH)2D3 for 22 h and then incubated with tritiated proline for the final 2 h of culture. The central bone (mature osteoblasts) was dissected free of the periosteum (less mature osteoprogenitors and fibroblasts) and both compartments were analyzed separately for the incorporation of tritiated proline. 1,25 (OH)2D3 decreases collagen synthesis in the central bone but not the periosteum, indicating selectivity of the 1,25(OH)2D3 effect for mature osteoblasts [50,51]. Using an in vivo protocol in which neonatal rats were given multiple injections of tritiated proline to radiolabel newly synthesized bone matrix, 25 ng of 1,25 (OH)2D3 given on days 1, 3, and 5 inhibited bone matrix synthesis as assessed by histomorphometry of autoradiographs of tibia and calvariae [52]. 1,25(OH)2D3 also inhibits collagen production in rat osteoblastic osteosarcoma ROS 17/2.8 cells [53], primary rat [54,55] and mouse osteoblastic cells [56], and an immortalized murine osteoblast cell line (MMB-1) [57]. 1,25(OH)2D3 has a greater inhibitory effect on type I collagen synthesis during log phase growth of primary murine osteoblastic cells than at confluence, perhaps because proliferating cells contained more VDRs [58]. Likewise, 1,25(OH)2D3 inhibition of collagen synthesis is greater in sparse cultures of MMB-1 cells that have higher VDR levels than confluent MMB-1 cells [59]. 1,25(OH)2D3 inhibition of collagen synthesis is equivalent in sparse and confluent rat primary osteoblastic cells [54], but VDR number did not change during growth of the cells [60]. Taken together, these data show that the extent of inhibition of collagen synthesis by 1,25(OH)2D3 is largely determined by the cellular quantity of VDRs. 1,25(OH)2D3 inhibits collagen mRNA levels during the proliferative phase of longterm cultures of rat primary osteoblastic cells [61] and prevents the formation of mineralized bone nodules by these cultures [61,62]. These studies show that 1,25 (OH)2D3 inhibits the differentiation of osteoprogenitors


MOLECULAR MECHANISMS OF REGULATION

that form mineralized nodules in primary rat osteoblastic cell cultures [62]. However, the inhibition of nodule formation by 1,25(OH)2D3 may be secondary to the suppression of type I collagen synthesis in the cultures. In contrast to the inhibitory effects described above, 1,25(OH)2D3 transiently stimulates collagen and noncollagen protein synthesis (about twofold), which peaks between 12 and 24 h, in the immortalized murine osteoblastic cell line MC3T3-E1 [63]. In this study, the percent collagen synthesized by the cultures (collagen relative to total protein synthesis) was not reported; as a result, it was not possible to determine the selectivity of the 1,25(OH)2D3 effect for collagen synthesis. 1,25(OH)2D3 also increases collagen expression in the human osteoblastic osteosarcoma cell line MG-63 [46,64,65] and primary cultures of human osteoblastic cells [66]. Interestingly, the increase in collagen synthesis by 1,25 (OH)2D3 in MG63 cells is blocked by insulin-like growth factor binding protein 5, which interacts directly to the VDR and prevents heterodimerization with the retinoid X receptor RXR [65]. However, in other studies, 1,25 (OH)2D3 has been shown to decrease the percent collagen synthesis in MC3T3-E1 cells [67,68]. MC3T3-E1 and MG-63 represent preosteoblastic cells that undergo in vitro osteogenic differentiation with ascorbic acid; 1,25(OH)2D3 inhibits cell growth and increases osteocalcin expression and alkaline phosphatase activity in both cell lines. MC3T3E1 cells, like most immortalized osteoblastic cell lines, display significant phenotypic variation [69]. Therefore some of these discrepant results may be due to variations in the cells used for the experiments. Collectively, these data suggest that 1,25(OH)2D3 may act as a differentiating hormone in early cells of the osteoblast lineage, which results in increased type I collagen expression. In contrast, 1,25(OH)2D3 inhibits type I collagen expression in mature osteoblasts.

MOLECULAR MECHANISMS OF REGULATION Some early studies showed that 1,25(OH)2D3 represses collagen synthesis in mature osteoblasts at a pretranslational level [50]. Measurements of procollagen mRNA activity by translation of total RNA in a reticulocyte lysate first showed that 1,25(OH)2D3 inhibited collagen mRNA in the osteoblast-rich central bone but not the periosteum of 21-day fetal rat calvariae [50]. 1,25(OH)2D3 at 10 nM inhibited procollagen mRNA activity at 6 h; maximal inhibition of about 50% occurred at 24 h [50]. A single subcutaneous injection of 1,25 (OH)2D3 (1.6 ng/g body weight) also decreased procollagen mRNA activity in calvariae [50]. Subsequently, specific cDNA probes were used to show that 1,25

405

(OH)2D3 inhibited Col1a1 mRNA levels in ROS 17/2.8 cells [53], primary rat [55], and chick calvarial osteoblastic cells [70,71]. Nuclear run-on assays in ROS 17/2.8 cells showed that 1,25(OH)2D3 represses Col1a1 and Col1a2 mRNA levels by a transcriptional mechanism [72]. 1,25(OH)2D3 at 1 and 10 nM decreased the rate of Col1a1 and Col1a2 transcription by about 50%, similar to its effect on collagen synthesis and type I collagen mRNA levels, while actin and tubulin transcription were unaffected. 1,25(OH)2D3 repressed Col1a1 and Col1a2 transcription as early as 4 h with maximal inhibition at 24 h [72]. DNA motifs that mediate stimulatory effects of 1,25 (OH)2D3 on gene expression have been well characterized for several genes [6,73,74]. Vitamin-D-responsive elements (VDREs) that mediate 1,25(OH)2D3 induction of target genes such as human [75] and rat [76] osteocalcin, mouse osteopontin [77], rat 24-hydroxylase [78], and rat calbindin D-9K [79] contain two perfect or imperfect direct hexameric repeats of the consensus AGGTCA motif separated by three spacer nucleotides [6,73,74]. The consensus VDRE binds a heterodimer of the VDR and the retinoic acid X receptor (RXR) [80]. Negative promoter elements have also been identified. The negative VDRE in the avian PTH promoter is analogous to the consensus VDRE, since it contains two imperfect direct repeats separated by three spacer nucleotides and binds VDR and RXR [81]. In contrast, the negative VDRE in the human PTH gene contains a single AGGTTC motif, and binding of the VDR to this site does not require RXR [82,83]. The negative VDRE of the parathyroid-hormone-related protein (PTHrP) gene contains two potential VDREs, one similar to the negative VDRE in the human PTH gene and another identical to the stimulatory VDRE; both motifs bind the VDR [84]. To characterize the regions of the Col1a1 gene that are involved in its repression by 1,25(OH)2D3, we produced a chimeric gene containing a fragment of the rat Col1a1 gene extending from e3518 to þ116 bp fused to the chloramphenicol acetyl transferase (CAT) reporter gene termed ColCAT3.6 [85]. 1,25(OH)2D3 inhibited ColCAT3.6 activity in transiently transfected ROS 17/2.8 cells by 50%, similar to its effect on the endogenous Col1a1 gene [85]. We then generated a series of ColCAT constructs containing progressive 50 promoter deletions of the Col1a1 promoter to map 1,25(OH)2D3 response elements [86,87]. In stably transfected cells, 1,25 (OH)2D3 inhibited a Col1a1 promoter fragment deleted to e2295 bp (ColCAT2.3) but did not affect a promoter fragment deleted to e1670 bp [87]. These experiments localized an inhibitory 1,25(OH)2D3 element to a region of the Col1a1 promoter from e2295 to e1670 bp. Sequence analysis of the Col1a1 promoter revealed a site between e2240 and e2234 bp that had high homology to both the human and rat osteocalcin VDREs.


406

22. VITAMIN D REGULATION OF TYPE I COLLAGEN EXPRESSION IN BONE

We hypothesized that the VDR binding to this motif would inhibit Col1a1 transcription. Electrophoretic mobility shift assays using VDR expressed in COS cells or by an adenovirus vector demonstrated that the VDR bound to this sequence in vitro [87]. However, deletion of the sequence between e2256 and e2216 bp from the ColCAT3.6 or ColCAT2.3 constructs did not affect the inhibitory effect of 1,25(OH)2D3 on promoter activity [87]. Therefore, 1,25(OH)2D3 does not inhibit Col1a1 transcription in ROS 17/2.8 cells solely via the e2240/ e2234 bp site. To determine the effect of 1,25(OH)2D3 on Col1a1 promoter activity in vivo, we previously produced a series of transgenic mouse lines carrying ColCAT constructs [88,89]. 1,25(OH)2D3 inhibited ColCAT3.6 activity in organ cultures of 6e8-day-old transgenic mouse calvariae [90]. 1,25(OH)2D3 inhibited CAT mRNA as early as 3 h, and maximal inhibition of CAT mRNA (50%) was seen at 24 h. The inhibition of CAT mRNA by 1,25(OH)2D3 was not affected by cycloheximide, suggesting that new protein synthesis is not required for the effect. A series of Col1a1 promoter fragments deleted to e1719 bp were fully inhibited by 1,25(OH)2D3. However, a Col1a1 promoter construct deleted to e1670 could not be analyzed because it did not have detectable basal activity in transgenic calvariae [91]. Subsequently, we showed that the rat Col1a1 promoter contains a homeodomain protein motif immediately downstream from e1683 bp that is required for high levels of promoter expression in osteoblasts in vivo [89]. A similar element is also present in the rat Col1a1 promoter [92]. In organ cultures of transgenic mouse calvariae carrying ColCAT constructs, we showed that 1,25(OH)2D3 inhibited CAT activity when the promoter was further deleted to e1683 bp. Moveover, in a transgene having the e1719 bp promoter with a large internal deletion extending from e1284 to e318 bp, the inhibitory action of 1,25(OH)2D3 promoter activity was maintained (A. Ivkovic, A. C. Lichtler and B. E. Kream, unpublished). Taken together, studies in ROS 17/2.8 cells and transgenic calvariae suggest that down-regulation of the Col1a1 promoter by 1,25(OH)2D3 involves sites located between e1683/e1284 bp or in the proximal promoter downstream from e318 bp. There are no good matches to consensus VDREs within these regions, suggesting several possible mechanisms. For one, 1,25 (OH)2D3 repression of Col1a1 could involve binding of the VDR to a novel negative VDRE. Another possibility is that 1,25(OH)2D3 inhibition of Col1a1 expression involves displacement of a stimulatory transcription factor(s) from its cognate DNA-binding site, similar to the mechanism by which 1,25(OH)2D3 inhibits the interleukin-2 gene [93]. It is also possible that 1,25(OH)2D3 inhibition of Col1a1 involves interaction of the VDR with other transcription factors rather than binding of

the VDR to DNA. Such a mechanism has been described for the inhibition of collagenase expression by glucocorticoids [94,95]. Finally, 1,25(OH)2D3 repression of Col1a1 expression could be mediated by alternative signal transduction pathways. It has been suggested that some biological effects of 1,25(OH)2D3 may be mediated by the protein kinase C (PKC) signaling pathway [74]. We have shown that stimulation of PKC with phorbol myristate acetate inhibits collagen synthesis in fetal rat calvariae [96] and ColCAT3.6 expression in transgenic mouse calvariae [97]. Therefore, 1,25(OH)2D3 activation of the PKC pathway might inhibit Col1a1 expression. This could be mediated by a putative 1,25(OH)2D3 membrane receptor, which activates intracellular signal transduction pathways leading to alteration of gene transcription. Future experiments to identify 1,25(OH)2D3 response elements in the Col1a1 gene will involve the analysis of additional constructs having selected sitedirected mutations and internal promoter deletions in cultured osteoblastic cells and transgenic mice. The previous data provide evidence of a direct action of vitamin D on type I collagen expression in osteoblasts. In other systems, the effects of vitamin D may be indirect. For example, vitamin D blocks the fibrotic effects of TGFb in lung fibroblasts and epithelial cells, and although the precise mechanism is not clear, it may involve vitamin D inhibition of TGFb transcriptional activation [98]. Vitamin D has also been shown to inhibit 5-azacytodine induction of TGFb and type I collagen expression in C3H10T1/2 multipotent mesenchymal cells [99].

CONCLUSIONS AND PERSPECTIVES The effect of 1,25(OH)2D3 on collagen expression, either inhibitory or stimulatory, may depend in part on in vitro culture conditions such as cell density, the timing and concentration of 1,25(OH)2D3 addition, the presence of ascorbic acid, and the state of maturation of the model. A model has been proposed based on the premise that cells of the osteoblast lineage differ in their response to 1,25(OH)2D3 depending on their state of maturation [100]. 1,25(OH)2D3 stimulates osteoblast markers in immature osteoprogenitor cells (MC3T3-E1 and MG-63 cells) but inhibits these markers in mature osteoblasts such as rodent calvarial organ cultures, primary rodent osteoblastic cell cultures, and ROS 17/2.8 cells [100]. Such a model is consistent with the effects of 1,25(OH)2D3 on bone remodeling during periods of calcium and phosphate deficiency. When serum calcium and phosphate are low, PTH increases the synthesis of 1,25(OH)2D3. Both hormones increase bone resorption to increase the supply of calcium and phosphate for soft tissues. During periods of mineral


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domains within the a1(I) collagen promoter in osseous and fibroblastic cells, J. Cell Biol. 116 (1992) 227e236. D. Pavlin, A. Bedalov, M.S. Kronenberg, B.E. Kream, D.W. Rowe, C.L. Smith, et al., Analysis of regulatory domains in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells, J. Cell Biochem. 56 (1994) 490e501. A. Bedalov, R. Salvatori, M. Dodig, M.S. Kronenberg, B. Kapural, Z. Bogdanovic, et al., Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice, J. Bone Miner. Res. 10 (1995) 1443e1451. M. Dodig, M.S. Kronenberg, A. Bedalov, B.E. Kream, G. Gronowicz, S. Clark, et al., Identification of a TAATcontaining motif required for high level expression of a COL1A1 promoter in differentiated osteoblasts of transgenic mice, J. Biol. Chem. 271 (1996) 16422e16429. A. Bedalov, R. Salvatori, M. Dodig, B. Kapural, D. Pavlin, B.E. Kream, et al., 1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice, Biochim. Biophys. Acta. 1398 (1998) 285e293. Z. Bogdanovic, A. Bedalov, P.H. Krebsbach, C.O. Woody, S.H. Clark, H.F. Thomas, et al., Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice, J. Bone Miner. Res. 9 (1994) 285e292. J.A. Rossert, S.S. Chen, H. Eberspaecher, C.N. Smith, B. De Crombrugghe, Identification of a minimal sequence of the mouse pro-alpha1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts, Proc. Natl. Acad. Sci. USA 93 (1996) 1027e1031. I. Alroy, T.L. Tower, L.P. Freedman, Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor, Mol. Cell Biol. 15 (1995) 5789e5799. R. Schule, P. Rangarajan, S. Kliewer, L.J. Ransone, J. Bolado, N. Yang, et al., Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor, Cell 62 (1990) 1217e1226. C. Jonat, H.J. Rahmsdorg, K.-K. Park, A.B.C. Cato, S. Bebel, H. Ponta, et al., Antitumor promotion and antiinflammation: downregulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone, Cell 62 (1990) 1189e1204. J.H.M. Feyen, D.N. Petersen, B.E. Kream, Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate, J. Bone Miner. Res. 3 (1988) 173e179. Z. Bogdanovic, Y.F. Huang, M. Dodig, S.H. Clark, A.C. Lichtler, B.E. Kream, Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMPmediated pathway in mouse calvariae, J. Cell Biochem. 77 (2000) 149e158. A.M. Ramirez, C. Wongtrakool, T. Welch, A. Steinmeyer, U. Zugel, J. Roman, Vitamin D inhibition of pro-fibrotic effects of transforming growth factor beta1 in lung fibroblasts and epithelial cells, J. Steroid. Biochem. Mol. Biol. 118 (2010) 142e150. J.N. Artaza, K.C. Norris, Vitamin D reduces the expression of collagen and key profibrotic factors by inducing an antifibrotic phenotype in mesenchymal multipotent cells, J. Endocrinol. 200 (2009) 207e221. R.T. Franceschi, J. Young, Regulation of alkaline phosphatase by 1,25-dihydroxyvitamin D3 and ascorbic acid in bonederived cells, J. Bone Miner. Res. 5 (1990) 1157e1167.



C H A P T E R

23 Target Genes: Bone Proteins Gerald J. Atkins 1, David M. Findlay 1, Paul H. Anderson 2, Howard A. Morris 2 1

Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia, 2 Chemical Pathology, SA Pathology, Adelaide, South Australia, Australia; School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia

VITAMIN D AND SKELETAL HOMEOSTASIS Genome-wide analyses indicate vitamin D, through its active metabolite 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) and the vitamin D receptor (VDR), has a potential to regulate the expression of some 3000 genes [1]. However, current evidence indicates that the strongest phenotype exhibited by vitamin-D-deficient humans or animals relates to impaired skeletal health. The first scientific reports in the early 1900s that rickets was due to vitamin D3 deficiency placed vitamin D as central to the regulation of calcium and phosphate homeostasis. In this context, it appears that the most critical and most widely studied endocrine role of vitamin D is its contribution to the maintenance of plasma calcium and phosphate at physiological levels through regulation of intestinal absorption of dietary calcium and phosphate. A central question, however, is whether vitamin D acts directly on bone tissue to modulate bone mineral homeostasis and bone strength. This question has been difficult to answer conclusively due in part to the direct actions of vitamin D on plasma calcium and phosphate levels, which indirectly affect bone mineralization and structure. One direct action that has been clearly demonstrated is the ability of plasma 1,25(OH)2D3, at least at supraphysiological levels, to stimulate bone resorption by the activation of osteoclasts [2]. The effects of vitamin D deficiency on bone in vivo can apparently be largely corrected by increasing dietary calcium and phosphate [3,4], suggesting that vitamin D is not an absolute requirement for optimal bone health. Indeed, the fact that the osteomalacic

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10023-X

phenotype observed in the vitamin D receptor gene knockout (VdrKO) mouse can be rescued by feeding a diet containing high levels of calcium and phosphate has led some to conclude that VDR-mediated activity in bone is essentially redundant [5,6]. Others have suggested that actions of VDR in bone may in fact impair mineralization [7,8]. These conclusions are, however, difficult to reconcile against an accumulating large body of evidence indicating that vitamin D activity in bone is critical for bone cell differentiation and optimal mineral status [9e12]. In this chapter, we examine the evidence for direct effects of vitamin D on the bone as a tissue, and its actions on the constituent cells of bone, in particular bone-matrix-forming osteoblasts, osteocytes, and bone-resorbing osteoclasts. There is now evidence that each of the major bone cells is capable of producing 1,25(OH)2D3 from the 25-hydroxyvitamin D3 (25(OH)D3) precursor, and that this activity is likely to account for the skeletal effects of circulating 25(OH)D3 (see Fig. 23.1). On the weight of this evidence, we have proposed that bone is an intracrine organ of vitamin D metabolism [13]. The actions of 1,25(OH)2D3 are mediated ultimately by direct effects on individual vitamin-D-responsive genes. However, the effects of vitamin D on bone tissue as a whole are not yet fully understood but are likely due to a combination of direct effects via VDREs, downstream effects of the induced gene expression and effects at specific stages of bone cell proliferation and differentiation. The effects of 1,25(OH)2D3 on important transcription factors, which in turn govern a downstream program of gene expression, will also be considered.

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FIGURE 23.1 A major source of active 1,25(OH)2D3 to regulate

bone processes of turnover and mineral homeostasis is now considered to be the bone itself. The action of the 1a-hydroxylase enzyme (CYP27B1) in bone cells including osteoblasts, osteoclasts, and osteocytes, gives rise to the active form of vitamin D, 1,25(OH)2D3. 1,25 (OH)2D3 binds to the VDR in the nucleus of target cells whereby it can alter gene transcription of vitamin-D-responsive genes, including those involved in essential bone cell activities, such as bone formation and resorption.

The 1a-hydroxylation of 25(OH)D3 to 1,25(OH)2D3 in bone cells was reported almost three decades ago [23e25] and yet only recently has evidence arisen to suggest that locally produced 1,25(OH)2D3 in osteoblasts plays a role in osteoblast differentiation and mineralization and in the regulation of osteoclastogenesis and osteoclast activity [9e12,26,27]. We and others have characterized Cyp27b1 promoter activity and Cyp27b1 mRNA expression in bone tissue associated with trabecular bone and growth plate [13,16,28] (see Fig. 23.2). Cyp27b1 mRNA levels were significantly higher in rat fetal bone than in adult bone and were shown by in situ hybridization to be present largely in growth plate chondrocytes and osteoblasts [29]. We have demonstrated the age-related regulation of Cyp27b1 mRNA expression in rat bone, which is also increased in the presence of high dietary calcium levels and is positively associated with bone mineralization [20,30]. Similar findings of a relationship between circulating 25(OH)2D levels and osteoid thickness have been reported in two separate clinical studies [31,32]. Furthermore, the expression of bone Cyp27B1 mRNA is distinct from the regulation of renal Cyp27B1 mRNA [33]. The levels of Cyp24 mRNA in bone, a gene exquisitely regulated by 1,25(OH)2D3, are coupled with the levels of bone Cyp27b21 mRNA expression and independent of changes to circulating levels of 1,25(OH)2D3, suggesting that the local bone production of 1,25(OH)2D3 is responsible for regulating CYP24 activity [20,34].

EXOGENOUS AND ENDOGENOUS SOURCES OF 1,25(OH)2D3 Circulating 1,25(OH)2D3, under nonpathological conditions, is derived by the actions of the renal 25 hydroxyvitamin D 1a-hydroxylase (CYP27B1) enzyme, which is highly expressed under certain conditions in the proximal tubular epithelial cells of the kidney [14e16]. CYP27B1 is also expressed in a wide range of extrarenal tissues including bone. Except in the cases of sarcoidosis [17] and placentation [18], extrarenal production of 1,25(OH)2D3 does not appear to contribute significantly to circulating levels [19,20]. Thus, local or cell-specific production of 1,25(OH)2D3 in bone and other tissues has been generally postulated to act in an autocrine or paracrine manner to regulate parameters of cell growth and differentiation [21,22].

Bioluminescence grayscale heat-map showing regions of Cyp27b1 promoter activity in a femur (A) of the 1501 bp Cyp27b1 promoter-Luciferase reporter transgenic mouse. The darker intensities of gray (B) represent greater activity of the CYP27B1 promoter. The overlaid grayscale heat-map with the femur image (C) demonstrates greater Cyp27b1 promoter activity in proximal and distal regions of the bone consistent with growth plate and trabecular bone structures.

FIGURE 23.2


EXOGENOUS AND ENDOGENOUS SOURCES OF 1,25(OH)2D3

In human osteoblasts, CYP27B1 mRNA expression and conversion of 25(OH)D3 into 1,25(OH)2D3 in transformed osteoblasts were demonstrated by Van Driel and colleagues. By using the pan inhibitor ketoconazole, they showed the reliance for this effect on cytochrome P450 activity [9]. We have also shown the expression of CYP27B1 mRNA in human primary osteoblasts isolated from adult femoral bone samples, and human osteosarcoma cell lines, and that 1,25 (OH)2D3 can be produced from 25(OH)D3 at physiological concentrations in these cells [12,35]. Using RNAi gene silencing, the synthesis of 1,25(OH)2D3 and the expression of osteocalcin, osteopontin, RANKL, and CYP24 mRNA in response to 25(OH)D3, were all dependent on CYP27B1 activity [12,35]. Thus, autocrine 1,25(OH)2D3 synthesis and activity is a common feature of human osteoblastic cells. We also showed that treatment with physiological levels of 25(OH)D3 inhibited cell proliferation and stimulated osteoblast differentiation, increasing the degree of matrix mineralization by human osteoblasts [12]. 25 (OH)D3 mainly circulates in vivo bound to the vitamin-D-binding protein, DBP, and this interaction is thought to exclude 25(OH)D3 from entering cells by passive diffusion [36]. The DBP-25(OH)D3 complex is reabsorbed specifically by renal tubules via the expression of two receptors for DBP, cubilin and megalin [37,38]. In further support of 25(OH)D3 metabolism representing a physiologically important pathway in osteoblasts, human osteoblastic cells have been shown to express both cubilin and megalin [9,35]. Many studies have shown an effect of 1,25(OH)2D3 on osteoclast formation, focusing on indirect effects via the osteoblast. Certainly, these effects are important, as indicated by studies using Vdr-null osteoblasts [39]. However, we have recently demonstrated that human peripheral blood mononuclear-cell-derived osteoclasts convert 25(OH)D3 into 1,25(OH)2D3 [27]. As will be discussed further below, conversion of 25 (OH)D3 to 1,25(OH)2D3 in osteoclasts dose-dependently inhibited the resorptive activity, with a maximal effect (~30% inhibition) seen at 25(OH)D3 levels >50 nmol/l. These data suggest that 25(OH)D3 metabolism in cells of the osteoclast lineage optimizes osteoclastogenesis and regulates the resorptive behavior of mature osteoclasts. Cells of the monocyte/macrophage lineage have been shown to express Cyp27b1 mRNA and convert 25(OH)D3 into 1,25(OH)2D3 [40,41]. Bone marrow macrophages also convert 25(OH)D3 into either 1,25 (OH)2D3 or 24,25(OH)2D3. Reichel and coworkers [42] demonstrated that, upon exposure to recombinant human interferon-gamma (IFN-g), bone-marrow-derived macrophages initially synthesized 1,25(OH)2D3 from 25(OH)D3.

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Growth plate chondrocytes also express Cyp27b1 mRNA and display 1a-hydroxylase activity [43,44]. Evidence suggests that 1,25(OH)2D3-VDR signaling is an important pathway for chondrocyte support of osteoclastogenesis during bone remodeling at the growth plate [45]. More recently, gene deletion and transgenic mouse models of CYP27B1 activity in chondrocytes demonstrated that locally produced 1,25(OH)2D3 in these cells regulated RANKL-mediated osteoclastogenesis, endochondral ossification, and chondrocyte development in vivo [46]. Intriguing preliminary data have been obtained suggesting that the locally produced 1,25(OH)2D3 in bone tissue exerts a differential response from that derived from the circulation. Thus in a rodent model, when circulating levels of 25(OH)D3 are below 80 nmol/L, bone loss is detectable as a result of increased bone resorption, increased osteoclastogenesis, and increased Rankl expression, driven by elevated circulating 1,25 (OH)2D3 levels [47]. On the other hand, when 25(OH) D3 levels are adequate and metabolized by bone cells to 1,25(OH)2D3 then Rankl expression is minimized. Furthermore there is clearly a differential response to induction of Cyp24 when osteoblastic cells are incubated with 1,25(OH)2D3 or 25(OH)D3 in vitro. Cells incubated with 25(OH)D3 at levels of approximately 100 nmol/L induce vitamin-D-responsive genes such as osteocalcin and osteopontin in a CYP27B1-dependent manner. Cyp24 is not significantly induced until levels of 25 (OH)D3 reach 400 nmol/L. However, when 1,25 (OH)2D3 is incubated with osteoblastic cells, Cyp24 is the first gene to be induced at the lowest levels of 1,25 (OH)2D3 [11]. The mechanism of such differential effects is unknown at this time; however it could be related to the ability of vitamin D, especially when locally metabolized, to induce osteoblast maturation. It has been well established that early or pre-osteoblast-like cells respond to vitamin D in a different manner from mature osteoblast or pre-osteocytic-like cells, as discussed in more detail later in this chapter. In summary, bone cells express the vitamin D metabolic enzyme CYP27B1 and have the capability of converting 25(OH)D3 into 1,25(OH)2D3, to elicit various effects central to bone remodeling, including bone resorption, bone formation, and mineralization. The potential and known effects of 25(OH)D3 metabolism during bone remodeling are depicted in Figure 23.3. Further innovative studies are required to identify and characterize these autocrine/paracrine networks of vitamin D metabolism and activity in the in vivo bone microenvironment to establish the relative roles of endogenous and exogenous sources of 1,25(OH)2D3. Such studies have the potential to establish a new paradigm for nutritional requirements for vitamin D for optimal skeletal health.


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Time / Bone Remodeling FIGURE 23.3 The potential effects of 25(OH)D3 metabolism by bone cells. This cartoon depicts the sequence of cellular events in bone remodeling and the potential role of metabolism of 25(OH)D3 into 1,25(OH)2D3 in osteoblasts (OB) and osteocytes (OCy), as well as in osteoclast (OC) lineage cells. Stromal osteoblasts support osteoclast differentiation from immature OC precursors (preOC), which form bone-resorbing, mature OC. The resorbed site is then populated by immature OB, which proliferate and differentiate into mature OB. These synthesize an unmineralized bone matrix, termed osteoid. Certain OBs become entrapped in osteoid (osteoid osteocytes) and these differentiate further into mature OCy, a process concomitant with bone mineralization. The effects of 25(OH)D3 to 1,25(OH)2D3 conversion, both those reported in the text, and those inferred from the known effects of exogenous 1,25(OH)2D3, at each stage are listed below each target cell type.

DIRECT ACTIONS OF VITAMIN D IN BONE Osteoblast The osteoblast regulates bone matrix synthesis and contributes to the coordination of bone resorption during remodeling in response to a large number of regulatory signals of which 1,25(OH)2D3 appears to be an important and pleiotropic member. A primary function of the osteoblast is to secrete a specialized organic extracellular matrix, which consists mainly of type I collagen but also a number of noncollagenous proteins, such as osteocalcin, osteopontin, osteonectin, bone sialoprotein-1, proteoglycans such as versican, and the small chondroitin sulfate proteoglycans, decorin and biglycan. This organic matrix is ultimately mineralized at discrete sites by the incorporation of calcium and phosphate, to form a mature bone matrix (for an excellent review of this topic, see [48]). In vitro studies have shown that 1,25(OH)2D3 is capable of regulating osteoblast gene transcription, proliferation, differentiation, and mineralization [49e51]. The genes of matrix proteins, such as osteopontin and

osteocalcin, possess vitamin-D-responsive elements (VDRE) within their promoter regions, suggesting a direct action for 1,25(OH)2D3 on their expression. Other important bone-matrix-associated genes, such as type I collagen and osteonectin, may have nonclassical VDREs in their promoters or be indirectly regulated by 1,25(OH)2D3 [52]. As mentioned above, in vitro evidence suggests that 1,25(OH)2D3 exerts effects during both the resorptive and synthetic phases of bone remodeling. In association with other factors including PTH, 1,25(OH)2D3 can also indirectly induce osteoclastogenesis by stimulating the differentiation of bone-marrow-derived promyelocytes and monocytes to active osteoclasts [53,54]. The tumor necrosis factor (TNF) ligand member, RANKL, itself a 1,25(OH)2D3-inducible protein, has been shown to be a critical mediator of 1,25(OH)2D3, PTH or inflammatory cytokine-induced osteoclastogenesis [55,56]. Thus, a paradox exists in that 1,25(OH)2D3 can potentially induce a proresorptive expression pattern, by increasing expression of RANKL, or a pro-osteogenic pattern in osteoblast lineage cells. One possibility is that the support of osteoclastogenesis and osteogenesis are performed by different types of specialized


DIRECT ACTIONS OF VITAMIN D IN BONE

osteoblasts. A second possibility is that these two diverse osteoblast functions are performed at different stages of osteoblast differentiation, as depicted in Figure 23.3. Our previous study [51] showed that in primary human osteoblasts, 1,25(OH)2D3 induced the expression of RANKL in phenotypically immature osteoblast precursors, identified by their expression of the marker STRO-1 [57]. However, in phenotypically mature osteoblasts, negative for STRO-1 expression, an osteocalcin response predominated [51]. This differential response was not related to levels of VDR expression, nor to the overall ability of the cells to respond to 1,25 (OH)2D3, evidenced by the expression of other “synthetic phase” 1,25(OH)2D3-responsive genes such as type I collagen and bone sialoprotein-1, which were found to be expressed independently of differentiation stage. Similar results were obtained in mineralizing cultures of primary mouse osteoblasts, where the 1,25 (OH)2D3 induction of RANKL expression decreased with increasing maturation of the osteoblast [58]. A third and combinatorial model is possible, with the sum of signals received by the osteoblast within a permissive window of its differentiation program determining either an osteoclastogenic or osteogenic response. The above studies imply that the particular cohort of genes expressed in response to 1,25(OH)2D3 in the osteoblast is regulated according to their stage of differentiation. Differential responses of osteoblasts to 1,25(OH)2D3 may result from different VDR signaling complexes. As detailed elsewhere in this volume, upon ligation of 1,25 (OH)2D3 with VDR and translocation to the nucleus, the complex forms a heterodimer with the retinoid X receptor (RXR). This induces a VDR conformation that is essential for effective binding to the VDRE. This association serves to recruit nuclear proteins as coactivators or corepressors, necessary for VDR-mediated transcriptional regulation. In short, the interaction of the 1,25 (OH)2D3-bound VDReRXR complex with nuclear proteins forms a so-called “pre-initiation complex,” which regulates the rate of transcription of the target gene [59]. The constitution, and therefore the promoter specificity, of this complex may change with differentiation stage of the cell. It remains to be seen whether other mechanisms of modifying the 1,25(OH)2D3 response, such as CpG methylation and inactivation of the VDRE, as has been shown for the RANKL promoter [60], occur for other bone protein genes. Osteoblast Proliferation and Matrix Synthesis In general, 1,25(OH)2D3 inhibits the proliferation of osteoblasts. This antiproliferative activity is associated with the ability of 1,25(OH)2D3 to induce osteoblast differentiation [61,62]. Numerous studies have shown the inhibition by 1,25(OH)2D3 of osteoblast proliferation in the human [51,63,64], rat [65,66], and mouse [67e69].

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The effects of 1,25(OH)2D3 on osteoblast proliferation are, however, dependent on species and maturity of the cell. For example, van den Bemd and coworkers [64] found that in human MG-63 cells, 1,25(OH)2D3 could suppress proliferation, whereas in rat osteosarcoma (ROS 17/2.8) cells, 1,25(OH)2D3 stimulated growth. Murray and coworkers [66] found that 1,25 (OH)2D3 inhibited proliferation of the rat osteoblast cell line, G2, and stimulated proliferation in rat osteoblast cell line, C12. The differences in the effects of 1,25 (OH)2D3 on osteoblast proliferation are unclear but may be due to the differentiation state of the osteoblast-like cell line tested. A further complication of the above studies is that immortalized cell lines, in general, proliferate in an uncontrolled fashion, making interpretation of the data difficult. Using carboxy-fluorescein succinimidyl ester (CFSE), a fluorescent dye that enables the number of cell divisions to be tracked with respect to other fluorescently tagged proteins [70], Atkins et al. [51] showed that while growth was inhibited overall in a heterogeneous population of human primary osteoblast-like cells in the presence of 1,25(OH)2D3, immature cells proliferated more than phenotypically mature cells. This implies that the degree of growth inhibition by 1,25 (OH)2D3 relates to the inherent growth potential of a particular cell type. Moreover, the effects of 1,25 (OH)2D3 on osteoblast proliferation appear to be dosedependent. For example, treatment of human osteoblasts with a low dose of 1,25(OH)2D3 (5 10e12 M) increased proliferation, whereas a pharmacological dose of 1,25(OH)2D3 (5 10e6 M) showed decreased proliferation [63]. Type I collagen is expressed in the proliferative stage of osteoblast development and is essential for the tensile strength of bone. Stein and coworkers [71] suggested that the inhibition of type I collagen gene expression prevents subsequent extracellular matrix development. The effect of 1,25(OH)2D3 on type I collagen expression during osteoblast proliferation, however, is dependent on the cell model of osteoblast studied. In human MG-63 osteosarcoma cells, 1,25(OH)2D3 stimulated the synthesis of type I collagen [64,72]. In rats and chickens, treatment of osteoblasts with 1,25(OH)2D3 reduced type I collagen mRNA transcription and protein synthesis [65,73e75]. The effects of 1,25(OH)2D3 on type I collagen synthesis in osteoblasts also appears to be conditional on the differentiation and proliferation state of the cells. In proliferating rat osteoblasts, acute 1,25(OH)2D3 treatment inhibited the high levels of type I collagen expression found at this stage of osteoblast development. During mineralization, however, low basal levels of type I collagen mRNA were stimulated by acute 1,25(OH)2D3 treatment and were unaltered by chronic 1,25(OH)2D3 treatment [65]. In the mouse, while 1,25 (OH)2D3 treatment was shown to promote type I


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collagen breakdown in calvarial osteoblasts [74], it has also been shown to stimulate type I collagen synthesis in early-phase MC3T3-E1 cells and have no effect in late-phase MC3T3-E1 cells [49]. Alkaline Phosphatase Alkaline phosphatase activity is important for the mineralization of bone and represents a useful biochemical marker of bone formation [76,77]. Osteoblasts express the bone- or tissue-non-specific isoform of alkaline phosphatase (TNAP), which is a glycosylphosphatidylinositol (GPI) anchored cell surface protein [78]. Treatment of rat osteoblast-like cells with 1,25(OH)2D3 promoted mineralization, which was associated with high alkaline phosphatase activity [79e81]. While the alkaline phosphatase gene promoter has no classical VDRE, 1,25(OH)2D3 was also shown to have a stimulatory effect on alkaline phosphatase mRNA levels, protein synthesis, and activity in human osteoblasts [82,83]. The stage of differentiation of osteoblasts has been shown to determine the response of alkaline phosphatase expression to 1,25(OH)2D3. During the proliferative period of osteoblast development, 1,25 (OH)2D3 inhibited the expression of alkaline phosphatase, whereas during mineralization, 1,25(OH)2D3 stimulated alkaline phosphatase mRNA expression [65]. In the mouse, however, 1,25(OH)2D3 stimulated alkaline phosphatase activity only in the early phase of osteoblast differentiation and not in the mineralization phase [49]. Matrix Gla Protein Matrix Gla protein (MGP), like osteocalcin, requires vitamin-K-dependent gamma-carboxylation for its function. MGP has been identified as a calcification inhibitor in cartilage and vasculature since MGP-null mice die soon after birth due to aberrant cartilage and arterial calcification [84]. In both rat UMR 106-01 and ROS17/ 2.8 cells, 1,25(OH)2D3 treatment markedly increased MGP mRNA and protein levels [85,86]. The stimulation of MGP mRNA by 1,25(OH)2D3 was shown to be less in the late stages of rat osteoblast differentiation [87] than in earlier stages of osteoblast growth [87].

Osteoid/Pre-osteocytes and Bone Mineralization Following cessation of proliferation and production of the collagenous matrix, certain osteoblasts become embedded in the matrix, signaling their differentiation into osteoid- or pre-osteocytes, sometimes referred to as mineralizing osteocytes. Matrix mineralization is an active process and evidence suggests that it is tightly regulated by protein products of the osteoid-osteocyte and mature osteocyte. It has been demonstrated that vitamin D augments matrix mineralization in vivo

and in vitro [11]. This may be due to effects of 1,25 (OH)2D3 on the expression of proteins known to be nucleators and regulators of mineralization, and possibly others, as will be discussed in the ensuing sections. Osteopontin Osteopontin (OPN), an extracellular glycosylated bone phosphoprotein, is one such gene that, in bone, is secreted by late-stage osteoblasts at the mineralization front [65,88]. In human bone marrow cultures, and in MG-63 cells, 1,25(OH)2D3 administration was associated with increased levels of OPN mRNA [89]. Cultured rat bone cells and ROS17/2.8 cells were both shown to increase OPN mRNA expression and protein secretion in response to 1,25(OH)2D3 administration [90]. Low basal levels of OPN mRNA were seen in rat calvarial cultures of intermediate maturity, which were markedly up-regulated by 1,25(OH)2D3 [91]. However, 1,25(OH)2D3-mediated stimulation of OPN mRNA in rat calvarial osteoblasts was shown to be far greater in premineralization cells than in mature mineralizing cells, where levels of OPN mRNA were already high [65]. OPN is a member of the small integrinbinding N-linked glycoprotein (SIBLING) family since it contains an ASARM (acidic serine- and aspartaterich motif) [92]. The OPN ASARM peptide with three phosphoserines can inhibit in vitro mineralization and is a substrate for PHEX which can rescue mineralization. It has been found that the helix-loop-helix-type transcription factor (HES-1) is expressed in osteoblastic cells and is suppressed by 1,25(OH)2D3. Overexpression of HES-1 in ROS17/2.8 cells suppressed the vitamin-Ddependent up-regulation of osteopontin gene expression in these cells [90]. TGF-b and PTH were also shown to abrogate 1,25(OH)2D3-mediated induction of OPN in ROS17/2.8 cells [93,94], suggesting that multiple transcription factors and hormones may be involved in regulating OPN activity. Bone Sialoprotein Bone sialoprotein (BSP) is largely specific for mineralized tissues and is highly expressed during the initial formation of bone and cementum [95]. The expression of BSP is suppressed by 1,25(OH)2D3 treatment in rat calvaria and ROS 17/2.8 cells [96]. A VDRE that is integrated with an inverted TATA box in the rat BSP promoter mediates the suppression of BSP transcription [97e99]. In human bone marrow stromal cells, 1,25 (OH)2D3 treatment alone did not significantly affect the expression of BSP mRNA [89]. However, data from our laboratory demonstrate a positive induction of BSP-1 mRNA by 1,25(OH)2D3 in normal human osteoblast-like cells (G.J. Atkins et al., unpublished


DIRECT ACTIONS OF VITAMIN D IN BONE

data), suggesting further differences between human and rodent responses to 1,25(OH)2D3. Osteocalcin In the mature osteoblast, 1,25(OH)2D3 downregulates the expression of BSP and type I collagen [96,100] and increases the expression of OPN and osteocalcin (OCN) [101,102]. OCN has a high affinity for calcium ions of hydroxyapatite and is the most abundant noncollagenous protein in bone [103]. OCN is expressed in postproliferative osteoblasts as well as in osteocytes. Ablation of the OCN gene in mice increases both bone formation and bone mass, although the mechanism for this remains unclear [104]. While the precise function of this protein is not well defined, it has been shown to be a chemotactic factor for osteoclasts and their precursors [105]. OCN has been widely used as a marker of bone formation [77]. It has recently been reported to be released from the matrix in the intact form rather than as fragments during remodeling as a result of osteoclast activity [106]. A number of studies have reported the induction of both OCN mRNA and protein synthesis by 1,25 (OH)2D3 in human and rat bone cells [63,65,82,83, 91,107e111], although the pattern of 1,25(OH)2D3induced expression of the OCN gene seems to depend on the culture system and the stage of maturity of the cells. For example, in human MG-63 cells, 1,25(OH)2D3 induction of OCN was highest in subconfluent cultures and decreased in confluent cultures [112]. Similarly, OCN gene expression was found to have a decreased responsiveness to 1,25(OH)2D3 in mineralizing human osteoblasts, which was suggested to be due to an accumulation of OCN in the extracellular matrix [113]. There are, however, reports of 1,25(OH)2D3 down-regulating OCN expression both in chicken embryonic osteoblasts [75] and in mouse osteoblast cultures [114,115]. Recent studies in our laboratories (H.A. Morris & G.J. Atkins et al., unpublished data), using mouse primary osteoblasts derived from adult mouse cortical bone, indicate that 1,25(OH)2D3 may stimulate OCN expression in this species, in contrast to previously published findings, and the direction of OCN expression in response to 1,25 (OH)2D3 may depend on the differentiation stage of the cells in question. The characterization of the OCN gene has identified a number of factors that are potentially involved in the development of the osteoblast phenotype. Besides the identification of a VDRE in the distal promoter of the OCN gene, several other promoter sites have been shown to be critical in the expression of the OCN and in osteoblastic differentiation. For example, the requirement for the osteoblast transcription factor, core binding factor alpha (CBFA)-1/AML-3, is best demonstrated in the CBFA-1/AML-3-null mutant mouse. These mice

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died at birth and had major skeletal deformations characterized by disrupted mineralization of osteoblasts [116,117]. Mutations of the three CBFA-1 motifs identified on the osteocalcin promoter were found to lead to abrogation of responsiveness to 1,25(OH)2D3 [118]. Another important site identified in the promoter of the OCN gene is the AP-1 site juxtaposed to the VDRE of the OCN gene. While this AP-1 site is essential for 1,25(OH)2D3 induction of the OCN gene [119], the suppression of OCN gene expression at the onset of mineralization appears to be related to the interaction of specific transcription factors at this AP-1 site. In proliferating osteoblasts, the repression of OCN gene expression is partly due to the expression of c-fos/ c-jun, which binds as a heterodimer to the AP-1 site and blocks the binding of the VDR/RXR complex [120,121]. In contrast, in postproliferative osteoblasts approaching mineralization, the expression of fra-2 results in binding to the AP-1 site, which facilitates VDR/RXR binding to the VDRE and 1,25(OH)2D3mediated expression of the OCN gene [121,122].

Osteocytes Osteocytes are the most long-lived and numerous cell type in bone tissue and have emerged over recent years as key controllers of osteoblast behavior, bone mineralization, and potentially of osteoclast activity [123,124]. While it has not been studied in detail, it is likely that osteocytes are responsible for the majority of osteocalcin synthesis, which as discussed above is under the control of 1,25(OH)2D3. Other key osteocyte derived proteins appear also to respond to 1,25(OH)2D3. Fibroblast Growth Factor (FGF) 23 FGF23 is a bone-derived hormone with known endocrine activities in regulating the renal expression of CYP27B1, having a negative effect on this enzyme and thus inhibiting the renal synthesis of 1,25(OH)2D3. The osteocyte is a major source of FGF23 although its expression has also been linked to osteoblasts. Deletion of the DMP1 gene in mice has revealed a complex relationship with FGF23, with DMP1-null cells resembling immature osteocytes and expressing excessive levels of FGF23 [125]. It is not yet known whether immature osteocytes predominantly express FGF23 in wild-type mice or in human bone. Importantly, FGF23 has been demonstrated to be 1,25(OH)2D3-responsive [126,127]. Additionally, in a recent study, Tang and coworkers demonstrated that 25(OH)D3 metabolism in cultured neonatal rat calvarial osteoblasts also resulted in the up-regulation of FGF23 expression [127]. Interestingly, the action of FGF23 on kidney tubule cells is to decrease their expression of Cyp27b1 and inhibit their synthesis of 1,25(OH)2D3 [128]. However, FGF23 has been shown to


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increase Cyp27b1 expression in bovine parathyroid cells [129]. It remains to be seen whether FGF23 also regulates osteoblast/osteocyte synthesis of 1,25(OH)2D3. Overexpression of FGF23 has been linked to rickets in X-linked hypophosphatemia (XLH) and in mouse models of this disease, such as the Hyp mouse and DMP1 mutant mice [130e133]. The pathology in these cases appears due to the increase in renal phosphate wasting and the decrease in 1,25(OH)2D3 levels. Because of the reciprocal relationship between FGF23 and circulating 1,25 (OH)2D3 levels, it might seem attractive to treat patients with XLH with 1,25(OH)2D3. A recent study, however, showed that treatment of these patients with calcitriol and phosphate resulted in increased FGF23 levels indicating that such a treatment may be counter-productive [134]. Dentin Matrix Protein 1 (DMP1) DMP1 is an acidic phosphorylated extracellular matrix protein, and like OPN, is a member of the SIBLING family [135]. First described as a product of odontoblasts [136], it is now recognized to be highly expressed in osteocytes and important for the differentiation of these cells [125]. Roles for DMP1 and its proteolytic cleavage products, the N-terminal 37 kDa and C-terminal 57 kDa fragments, are not fully understood but include nucleation of mineralization, cell attachment, and possibly as a transcriptional regulator [137]. As discussed above, DMP1, FGF23, and the CYP27B1 have a complex interplay made more complex perhaps by the findings that DMP1 is itself 1,25(OH)2D3responsive [138]. The in vivo significance of this observation has yet to be determined.

Osteoclasts Osteoclasts are multinucleated cells of the monocyte/ macrophage lineage whose primary function is to resorb bone during bone remodeling. They accomplish this by attaching to the mineral surface forming a tight sealing zone, and creating a basolateral membrane termed the ruffled border, from which bone-matrix-degrading enzymes, such as cathepsin K, and protons are secreted via proton pumps such as the vacuolar ATP-ase complex (V-ATPase) and carbonic anhydrase II, to break down the collagenous matrix and release calcium and phosphate ions, respectively. Osteoclasts may also be involved in the active coupling of bone resorption to bone formation by inducing osteoblast proliferation. Osteoclasts are generated by the proliferation and fusion of mononuclear precursors. These complex processes include several key proteins whose genes are known to be 1,25(OH)2D3-responsive. More recent data indicate that osteoclasts, like macrophages, are a site of extrarenal 1,25(OH)2D3 synthesis by virtue of their expression

of CYP27B1, and that the prevailing level of blood 25 (OH)D3 may govern aspects of both osteoclast formation and their resulting activity. Effects of Exogenous Vitamin D Evidence suggests that 1,25(OH)2D3 has direct effects on osteoclast precursors, increasing the expression of the key adhesion molecule, aVb3 integrin, in both avian osteoclast precursor cells [139e141] and in the human myelomonocytic cell line, HL-60 [142], thus potentially promoting osteoclast adhesion and the formation of the sealing zone. 1,25(OH)2D3 has been shown to facilitate adhesion of osteoclast precursors to stromal osteoblasts by increasing the expression of the intercellular adhesion molecule, ICAM-1 [143]. 1,25(OH)2D3 has also been shown to increase the expression of the receptor for RANKL, RANK, in HL-60 cells [144]. We have recently demonstrated a direct effect of 1,25(OH)2D3 on RANKL-induced osteoclast formation from the mouse preosteoclast cell line, RAW 264.7, where 1,25(OH)2D3 in the copresence of RANKL, increased the resulting numbers of multinucleated TRAP-positive osteoclasts and significantly increased osteoclast multinucleation [145]. Effects of Endogenous Vitamin D It was recently described that, similar to other macrophage cell lines and primary cells, the RAW 264.7 cell line expresses CYP27B1 and also that CYP27B1 mRNA levels increased during their differentiation into osteoclast-like cells [9]. We recently confirmed that human PBMC-derived osteoclasts possess the molecular machinery to both respond to and metabolize 25(OH)D3, as they express cytoplasmic CYP27B1 and nuclear VDR proteins [26]. Furthermore, CYP27B1 mRNA expression increased in response to M-CSF/RANKL-induced differentiation of PBMC, suggesting that 25(OH)D3 metabolism plays a role in osteoclast differentiation. In a subsequent study, we confirmed that the capacity of osteoclasts to synthesize 1,25(OH)2D3 increases substantially with their differentiation [27]. Metabolism of 25 (OH)D3 into 1,25(OH)2D3 in human PBMC-derived osteoclasts resulted in the increased expression of a number of genes, principal among these being the osteoclast transcription factor NFATc1 [27]. This may or may not be responsible for the observed concomitant increase in expression of a number of osteoclastic genes, including calcitonin receptor, tartrate-resistant acid phosphatase (TRAcP), cathepsin K, carbonic anhydrase, and V-ATPase. Notably, 1,25(OH)2D3 treatment also up-regulated the expression of these genes but generally did so to a lesser extent. It remains to be determined if some or all of these genes are vitamin-D-responsive in the classical sense or whether their expression was as a consequence of cell


REFERENCES

differentiation and the expression of transcription factors, such as NFATc1, that created a permissive environment for their expression. In the case of VATPase, which is a complex multisubunit enzyme [146], Lee et al. [147] have demonstrated that its activity is up-regulated by exogenous 1,25(OH)2D3 by an unidentified post-translational mechanism, suggesting that 1,25(OH)2D3 effects on osteoclast behavior may not be simply transcriptional. In terms of the overall effect of 1,25(OH)2D3 on the osteoclast, studies published to date [26,27] suggest that intracellular accumulation of 1,25(OH)2D3 promotes osteoclast formation but inhibits the resorptive capacity of these cells.

CONCLUDING REMARKS Overwhelming clinical evidence suggests that vitamin D is important for calcium/phosphate and skeletal homeostasis. Numerous direct and indirect effects of 1,25(OH)2D3 have been demonstrated on a range of critical bone proteins and 1,25(OH)2D3 appears to be involved in their regulation at all stages of osteoblast differentiation and, indeed, bone remodeling. Future studies will need to unravel the complexities surrounding the involvement of 1,25(OH)2D3 in the coordination of these processes. However, the evidence for a fundamental and nonredundant role for 1,25 (OH)2D3 in skeletal biology in vivo, namely, geneablated mouse models, is currently lacking. As suggested in earlier, this is in part due to an incomplete analysis of these potentially informative models, rather than a convincing lack of a biological effect. Additionally, some of the apparently contradictory data surrounding this question arise from the plethora of model systems (immortalized cell lines versus primary cells, the use of different mammalian and other vertebrate species) which have been utilized to investigate this issue, many of which may have significant and confounding limitations. Recent studies using 1,25(OH)2D3 analogs that show potent anabolic effects, for example, the compound 2-methylene-19-nor-(20S)-1-a,25dihydroxyvitamin D3 (2-MD) when administered to sham-operated or ovariectomized mice under otherwise normal dietary conditions [148], and other examples discussed elsewhere in this volume, must provide additional evidence for the bone-specific effects and importance of the natural hormone. The combination of clinical [149,150] and preclinical [47] studies provides strong evidence that adequate circulating 25(OH)D3 levels and not 1,25(OH)2D3 are required to optimize skeletal health. Such findings support the concepts described above that the local metabolism of vitamin D and the subsequent biological activity of autocrine/ paracrine-derived 1,25(OH)2D3 enhance the maturation

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and activities of each of the major bone cell types to optimize skeletal structure and strength.

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C H A P T E R

24 Vitamin D and the Calcium-Sensing Receptor Edward M. Brown Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, EBRC 223A, 221 Longwood Ave., Boston, MA 02115, USA

INTRODUCTION Calcium and vitamin D have been inextricably linked for decades by their combined roles in promoting mineral ion homeostasis and the growth and remodeling of the skeleton. For much of the history of vitamin D and the knowledge of its link to calcium metabolism, however, calcium was thought to be a largely passive partner. That is, stimulation of active intestinal calcium absorption required the receptor-mediated action of the active form of vitamin D (1,25(OH)2D3), but the resultant active transport of calcium across the intestinal epithelium did not imply any regulatory role for calcium ions per se. Similarly, during bone growth and remodeling, the role of calcium ions was primarily conceptualized as contributing to the maintenance of an adequate calciumephosphate product to enable mineralization of the bony matrix. Some early studies had shown that calcium could interact with vitamin D directly to modulate cellular functions, e.g., their coordinate up-regulation of the expression of calbindin D-28K in primary chick kidney cells [1]. It was the cloning of the extracellular calcium (Ca2þo)-sensing receptor (CaSR) in 1993 [2], however, that provided a molecular target by which Ca2þo could serve as an extracellular, first messenger. Since that time, there has been rapid progress in elucidating the mechanisms by which the CaSR exerts its regulatory roles, including its structure, intracellular signaling pathways, tissue distribution, and the range of cellular functions that it controls [3]. There has also been increasing appreciation of the wide range of tissues in which both calcium, acting via the CaSR, and 1,25(OH)2D3, acting through its nuclear vitamin D receptor (VDR), regulate the functions not only of those tissues involved in but also those uninvolved in mineral ion homeostasis in both health and


disease [4]. This chapter will briefly review the structure and functional properties of the CaSR and how it regulates, in concert with 1,25(OH)2D3, the various cells types expressing both the CaSR and VDR that participate in the maintenance of Ca2þo homeostasis as well as bone growth and development. It will then provide selected examples of the roles of the CaSR and VDR when they are coexpressed in selected cell types uninvolved in Ca2þo homeostasis. Since the VDR, its ligands, and its actions are reviewed in great detail in other chapters in this volume, the focus of this chapter will be to compare and contrast the actions of the CaSR and VDR and, if relevant, their interactions in a number of the cell types in which they are coexpressed.

WHAT IS THE CASR? To maintain near constancy of the blood Ca2þ level, a mechanism must exist that senses small changes in Ca2þo and responds in a manner that will normalize Ca2þo [5]. The CaSR is the major Ca2þo sensor involved in maintaining Ca2þo homeostasis. It is a G-proteincoupled receptor (GPCR), whose principal physiological ligand is Ca2þo. The isolation and characterization of the CaSR from bovine parathyroid gland by molecular cloning were reported in 1993 [2]. Soon afterward, the CaSR was cloned from human parathyroid gland [6] and, subsequently, from parathyroid and/or other tissues in a variety of species, including birds and fish. The CaSR is a member of family C of the GPCRs, which also comprises the metabotropic glutamate receptors (mGluRs), the GABAB receptors, whose ligand is gamma aminobutyric acid (GABA), and receptors for taste and pheromones. An additional aminoacid- and divalent-cation-sensing receptor, GPRC6A

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24. VITAMIN D AND THE CALCIUM-SENSING RECEPTOR

[7], is also a member of the family C GPCRs. Whether the latter is principally a calcium-modulated amino acid receptor or an amino-acid-modulated calciumsensing receptor, or whether it plays a physiologically meaningful role in normal mineral ion and skeletal physiology remains controversial, and GPRC6A will not be discussed further here. The predicted protein structure of the human CaSR, which is very similar to the CaSRs from the other species studied to date, has a large, 612-amino-acid extracellular domain (ECD) [6], followed by a 250-amino-acid transmembrane domain (TMD) containing seven transmembrane helices, a signature of the GPCRs, and, lastly, a 216amino-acid carboxyterminal (C)-tail. During its biosynthesis, the CaSR is targeted to the endoplasmic reticulum by a hydrophobic signal peptide, where it dimerizes through intermolecular disulfide bonds involving cysteines 129 and 131 within each monomer [8,9]. The receptor is then extensively glycosylated in the Golgi apparatus before reaching the cell surface in its biologically active, dimeric form. The receptor-activity-modifying proteins (RAMPs), RAMP-1 and RAMP-3, facilitate translocation of the CaSR to the cell membrane in some cells [10]. The cell surface CaSR undergoes little desensitization upon repeated exposure to elevated levels of Ca2þo, at least

in parathyroid cells. Its resistance to desensitization results, in part, from its binding to the large, actinbinding scaffold protein, filamin-A [11], and is presumably important to ensure the CaSR’s persistent presence on the cell surface, thereby enabling it to continuously monitor Ca2þo. Other binding partners of the CaSR include the Kþ channels, Kir4.1 and Kir4.2, caveolin-1, and the E3 ubiquitin ligase, dorfin, which likely participates in regulating the proteasomal degradation of the receptor [12]. Molecular modeling, using the known three-dimensional structures of the extracellular domains of several mGluRs, strongly suggests that the CaSR’s ECD has a bilobed, venus flytrap (VFT)-like structure with a cleft between the two lobes [13] (Fig. 24.1). The CaSR responds over a much smaller range of Ca2þo (90% at 48 h after the dose [69]. Recent studies using mouse knockout models, however, have yielded surprising results with regard to the relative importance of the CaSR and VDR in regulating parathyroid gland function in vivo. As noted earlier, homozygous knockout of exon 5 of the CaSR in the mouse results in striking hyperparathyroidism with marked increases in both PTH levels and parathyroid gland size [59]. It should be noted that, if anything, this mouse model likely underestimates the impact of losing the CaSR on parathyroid function, because knockout of exon 5 of the CaSR results, in some tissues, in production of an alternatively spliced CaSR lacking exon 5 (which encodes part of the CaSR ECD) that can apparently still signal [80]. The VDR, therefore, seemingly has limited capacity to compensate for loss of the CaSR in this animal model. Parenthetically, while a clear phenotype as seen here establishes the importance of the gene that was knocked out, the lack of a phenotype does not mean that the gene of interest serves no function in vivo, but rather that its function is nonessential, and


THE CASR AND VDR IN TISSUES PARTICIPATING IN CA2þO HOMEOSTASIS

its absence can likely be compensated for by one or more other genes. In contrast, studies in mice deficient in 1,25(OH)2D3 [81] or in the VDR [82], or both, have shown that the CaSR can effectively compensate for loss of the nuclear vitamin D signaling pathway with respect to the control of parathyroid function. VDR (VDRe/e), 1a-hydroxylase [1a(OH)asee/e] and double knockout mice [VDRe/ e 1a(OH)asee/e] mice all develop markedly elevated levels of PTH and parathyroid enlargement [81]. Administration of a calcium-rich “rescue” diet, however, normalizes the serum calcium concentration and restores PTH levels to normal in these three genotypes [81,82]. Thus hypocalcemia rather than vitamin D deficiency per se was apparently the dominant contributor to the high PTH levels. Parathyroid gland size was markedly enlarged in all three mutant genotypes of mice when hypocalcemic. In the VDRe/e mice, the rescue diet, when administered beginning early in life, completely prevented the parathyroid enlargement, indicating that hypocalcemia rather than loss of vitamin D signaling was a key contributor to parathyroid cell growth in this setting. In the 1a(OH) asee/e mice, however, the rescue diet did not fully normalize parathyroid gland size, but administration of 1,25(OH)2D3 did so [81]. Thus in this latter model both 1,25(OH)2D3 and calcium contribute to inhibition of parathyroid cell growth in the context of an intact VDR and CaSR, confirming studies reviewed earlier that both the VDR and CaSR participate in the control of parathyroid cellular growth. The capacity of the CaSR alone to mediate nearly complete inhibition of parathyroid growth in the VDRe/e mice remains unexplained, but perhaps up-regulation of the CaSR or of CaSR signaling can compensate for loss of the VDR in this setting. In addition, it is possible, since 1,25 (OH)2D3 levels are frankly high in the VDRe/e mice, that 1,25(OH)2D3 can inhibit parathyroid cell growth in a VDR-independent manner [81]. An elegant study addressing this issue further was carried out recently by Meir et al., who deleted the VDR specifically in the parathyroid gland [83]. In this way, the effects of the VDR on the parathyroid could be isolated from systemic changes in mineral ion homeostasis, e.g., resulting from loss of the VDR in kidney and intestine. The mice with parathyroid-specific ablation of the VDR exhibited modest (~30%) increases in serum PTH but had no change in the number of proliferating parathyroid cells and had exhibited normal serum calcium concentrations. Therefore, although administration of 1,25(OH)2D3 can clearly inhibit PTH gene transcription and parathyroid cellular proliferation in vivo and in vitro, under normal physiological conditions it apparently has only a limited role in parathyroid physiology in vivo. These results do not, however, mean

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that 1,25(OH)2D3 does not have a useful role therapeutically, particularly in the secondary hyperparathyroidism of renal insufficiency (see Chapter 70). In addition, it would be of interest to assess CaSR signaling efficiency in mice with deficient signaling through the vitamin D pathway to determine to what extent compensatory change in the CaSR and its downstream signaling elements contribute to the observed phenotypes in the knockout mouse models just discussed.

The CaSR and VDR in the C-cell Studies in CaSR knockout mice have proven the receptor’s role in stimulating CT secretion by showing blunting of the high Ca2þ-induced increase in circulating CT levels in CaSRþ/e mice [84] and near total loss of Ca2þ-evoked CT secretion in CaSRe/ePTHe/e mice [41]. A plausible model for the mechanism underlying CaSR-stimulated CT secretion [85] involves CaSR-mediated activation of a nonselective cation channel, which depolarizes the cells, thereby activating voltage-sensitive calcium channels and producing the increase in cytosolic calcium concentration that activates exocytosis. In contrast to the parathyroid cell, in which the CaSR and VDR exert the same biological actions (e.g., inhibition of parathyroid cell proliferation), albeit by distinct mechanisms, the VDR in the C-cell inhibits rather than stimulates CT gene expression [86,87]. In the second of these two studies, 1,25(OH)2D3 produced about a 60% inhibition of cAMP-stimulated transcriptional activity via an nVDRE located within nucleotides e920 and e829 in the 50 flanking DNA of the CT gene. The physiological significance of this bidirectional control of CT secretion is unknown.

The CaSR and VDR in the Kidney The CaSR and Regulation of 1-Hydroxylation of 25-Hydroxyvitamin D in the Proximal Tubule Both the CaSR [88] and VDR [89] are widely expressed in the kidney. The three principal sites that will be discussed here in this regard are the proximal tubule, where the CaSR and VDR regulate 1-hydroxylation of 25-hydroxyvitamin D3, the cortical thick ascending limb of Henle’s loop (cTAL), a key site of regulated calcium reabsorption, and the distal convoluted tubule/connecting segment (DCT/CNT), where the CaSR and VDR regulate tubular reabsorption of calcium. As in the parathyroid, 1,25(OH)2D3 administration in the rat up-regulates expression of the CaSR in the kidney [21], although the site(s) where this upregulation takes place was not identified. Furthermore, high calcium concentration up-regulates the VDR in


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a proximal tubular cell line [90]. Therefore, similar to the situation in the parathyroid, there is the possibility of synergistic interactions between the CaSR and VDR, whereby activation of one receptor up-regulates the other. An important aspect of the regulation of the 1-hydroxylation of 25-vitamin D3 in the proximal tubule is the VDR-mediated inhibition of the gene that 1-hydroxylates 25-hydroxyvitamin D3, the 25-hydroxyvitamin D 1a-hydroxylase (CYP27B1). It is one of the five factors that physiologically regulate this gene in vivo: 1,25 (OH)2D3 itself, the serum calcium concentration (hypocalcemia stimulates and hypercalcemia inhibits), the serum phosphate concentration (hypophosphatemia stimulates and hyperphosphatemia inhibits), FGF-23, which inhibits, and PTH, which stimulates CYP27B1. Since hypocalcemia stimulates PTH release, it can upregulate CYPB27B1 indirectly through the associated changes in circulating PTH levels. The available evidence, however, indicates that there is also direct regulation of the 1-hydroxylase by Ca2þo and that this regulation is likely mediated by the CaSR. For some time, there has been both in vivo [36,37] and in vitro [91] evidence that changes in serum and medium calcium concentration, respectively, directly modulate the 1-hydroxylation of 25-hydroxyvitamin D3. To avoid the confounding effects of calcium-induced changes in circulating PTH levels in vivo, Treschel et al. and Weisinger et al. utilized thyroparathyroidectomized rats infused with PTH to “clamp” the level of circulating PTH. Alterations in circulating 1,25(OH)2D3 levels were then determined in response to changes in the serum calcium concentration. The steep inverse sigmoidal relationship between serum calcium and 1,25(OH)2D3 levels [37] was similar to that for the relationship of PTH to the serum ionized calcium concentration. Similar results were observed in vitro using an SV40-transformed human proximal tubule cell line, namely stimulation of 1,25(OH)2D3 production at low calcium concentration and inhibition at high calcium [91]. More recent evidence has implicated the CaSR as the mediator of the direct effects of extracellular calcium concentration on the 1-hydroxylation of 25-hydroxyvitamin D3. Maiti and Beckman used a proximal tubular cell line, HK-2G, in which they had shown that high extracellular calcium inhibits the expression of CYP27B1 [90], to demonstrate that high Ca2þo up-regulates the expression of the VDR [24]. This effect was mediated by a p38 mitogen-activated protein kinase (MAPK)dependent mechanism [92]. P38 is one of several MAPKs activated by the CaSR in various cell types. Since knocking down the CaSR with siRNA prevented the high Ca2þo-evoked increase in VDR expression, the latter was CaSR-mediated [92]. The use of siRNA to prove the CaSR’s role in the concomitant inhibition of

the expression of CYP27B1 was not reported [90]. It also remains to be determined whether the high Ca2þstimulated, CaSR-mediated increase in VDR expression in the proximal tubule is sufficient by itself to account for the accompanying suppression of the expression of CYP27B1. The CaSR and Regulation of Renal Tubular Reabsorption of Calcium Favus et al. have developed an interesting model, the genetically hypercalciuric rat, which was created by repeated inbreeding of Sprague-Dawley rats with the highest urinary calcium excretion in any given generation. The hypercalciuric rats have increased intestinal calcium absorption and normal serum 1,25 (OH)2D3 concentrations [93]. The level of the VDR in the intestine of the hypercalciuric rats is twice that in the control rats, and there is no difference in the affinity of the receptor in the two groups of rats. The level of the VDR is also elevated in the kidneys of the genetically hypercalciuric rats [94]. The increased expression of the intestinal VDR provides a likely explanation for the hyperabsorption of calcium in the intestine, e.g., via up-regulation of the vitamin-Dstimulated transcellular calcium transport system, but not necessarily for the hypercalciuria, unless the latter is simply the result of absorptive hypercalciuria. However, subsequent studies [95] revealed that the level of CaSR protein was increased fourfold in the kidneys of hypercalciuric rats compared to controls. Furthermore, the level of the CaSR protein increased further and to a higher level in the hypercalciuric rats after treatment with 1,25(OH)2D3, reflecting the known VDR-mediated up-regulation of the CaSR. While the site within the kidney where the increase in the level of CaSR in the hypercalciuric rats takes place was not identified, the highest level of expression of the CaSR in the kidney is in the cTAL, an important site where the VDR [89] and CaSR [88] are both localized and in which the CaSR inhibits the reabsorption of calcium by a CaSR-mediated mechanism when the level of calcium in the blood is elevated. However, despite the importance of this nephron segment in the renal handling of calcium by the CaSR (and PTH), there are little or no data on the functional significance of the VDR in the cTAL. The distal convoluted tubule (DCT) follows immediately after the cTAL, and the connecting segment (CNT) follows the DCT and precedes the cortical collecting duct; both DCT and CNT play important roles in regulating renal tubular reabsorption of calcium. As in the cTAL [96], PTH stimulates the reabsorption of Ca2þ in the DCT [97]. It was the cloning of the TRPV5 calciumpermeable ion channel [98], however, which is the key apical Ca2þ channel involved in the transcellular



transport of calcium in the distal tubule, that clarified the biological relevance of 1,25(OH)2D3-stimulated reabsorption of calcium, as well as its molecular mechanism. Previously, it had been difficult to show convincingly a quantitatively important action of 1,25(OH)2D3 on calcium reabsorption in the DCT in vivo. Mice with knockout of TRPV5 (originally called ECAC for Epithelial Calcium Channel) [99], however, have marked hypercalciuria, along with a compensatory increase in circulating 1,25(OH)2D3 levels and intestinal hyperabsorption of calcium. 1,25(OH)2D3 acts in the DCT to increase active reabsorption of calcium by up-regulating the expression of the key molecules participating in transcellular calcium transport. These are the apical uptake channel, TRPV5, calbindins-D9K and D28K, which facilitate transcellular diffusion of calcium, and the sodiumecalcium exchanger, NCX1, and the plasma membrane calcium pump, PMCA1B. Both NCX1 and PMCA1B pump calcium across the basolateral plasma membrane. The role of vitamin D in regulating this pathway was shown unequivocally using 1a(OH)asee/e mice, which lack any endogenous 1,25(OH)2D3 [100]. Repleting these mice with 1,25(OH)2D3 increases the expression of TRPV5, calbindin-D28K, calbindin-D9K, NCX1, and PMCA1B. The effect of dietary calcium rescue on the expression of these same genes was also examined in this study. Administration of the calcium-enriched rescue diet to 1a(OH)asee/e mice normalized their serum calcium concentration in association with upregulation of TRPV5, calbindin D28K, NCX1, and PMCA1b. These effects of calcium supplementation were presumably related to direct actions of calcium on the same cell type(s) upon which 1,25(OH)2D3 acts in the distal tubule, but indirect effects were not formally ruled out. However, Clemens et al. had shown some years before [1] that 1,25(OH)2D3 and/or elevated concentrations of calcium in the medium up-regulate expression of calbindin-D28K in primary kidney cells from the chick. It seems highly likely that calcium and 1,25(OH)2D3 were acting on the same calbindin-D28Kexpressing cell type in this study, and, because it was carried out in vitro, this effectively rules out indirect effects of 1,25(OH)2D3 and calcium mediated by other cells inside or outside of the kidney. The rescue diet, however, did not normalize renal calcium handling in the VDRe/e mice, as urinary calcium excretion in the VDRe/e mice receiving the rescue diet was twice that in normal mice receiving the same diet [101]. Apparently, therefore, the dietary calcium-induced changes in the expression of the components of the transcellular absorption pathway for calcium could not completely substitute for loss of the VDR. A recent study supports the role of the CaSR in mediating the actions of calcium on the transcellular calcium

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transport system in the DCT [102]. In this study, the CaSR and TRPV5 were shown to be coexpressed in the same cells, and activation of the CaSR on the apical membrane stimulated the activity of TRPV5, with a resultant increase in the intracellular calcium concentration, by a pathway that involves a PKC-dependent phosphorylation of amino acid residues S299 and S654 in the channel protein. This activation of TRPV5 was inhibited by a dominant negative CaSR construct, documenting the receptor’s involvement. What is the purpose of a CaSR-dependent stimulation of calcium reabsorption in DCT? This contrasts with the CaSRmediated inhibition of the paracellular reabsorption of calcium in the CTAL [103], which plays an important role in the defense against hypercalcemia. Topala et al. [102] suggested that this represents a local feedback mechanism that adjusts the reabsorption of calcium in the DCT to the prevailing urinary calcium concentration, perhaps mitigating the risk of stones when the urine reaching the DCT has an excessively high calcium concentration.

The CaSR and VDR in Cartilage and Bone Our understanding of the functions of the CaSR in bone and cartilage has lagged behind that in parathyroid and kidney, owing, at least in part, to controversy about whether the receptor actually exists in skeletal cells, to say nothing about whether it exerts biological relevant effects on those cells. While some studies found clear evidence for the receptor’s presence in cartilage or chondrocytic cell lines [104,105] and/or bone [105] as well as in osteoblastic cell lines, and/or osteoclasts and related cell lines, others did not (for review, see [106]). The following discussion summarizes the current state of this field and compares and contrasts the relative roles of the CaSR and VDR in cartilage and bone cells. The CaSR in Cartilage The chondrocytic cell line, RCJ3.1C5.18, expresses readily detectable levels of the CaSR. When incubated with elevated levels of Ca2þo, it shows suppression of the early differentiation marker, aggrecan, and increased expression of the markers of terminal differentiation, osteopontin, osteonectin, and osteocalcin, as well as increased production of matrix mineral, indicating stimulation of differentiation [107]. Several of these effects of the receptor were potentiated by overexpression of the wild-type CaSR or inhibited by transfection of the cells with a signal-defective CaSR, consistent with a mediatory role for the CaSR. Growth plate cartilage also expresses the CaSR [105]. Initial studies of cartilage in mice with homozygous knockout of exon 5 of the CaSR demonstrated rickets [108], suggesting a functional


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role for the receptor in cartilage in vivo. However, when these severely hyperparathyroid mice were “rescued” by knocking out the PTH gene (CaSRe/ePTHe/e) [109] or the key transcription factor, Gcm-2, that is required for formation of the parathyroid glands (CaSRe/e Gcme/e) [110], there was no obvious cartilage phenotype. Other studies carried out at around the same time, however, had shown that keratinocytes from the CaSRe/e mice were capable of generating a variant CaSR in which exon 5 had been spliced out [111]. This observation raised the possibility that the “exon-5-less” CaSR could have biological activity and be capable of rescuing the CaSRe/ePTHe/e and CaSRe/eGcme/e mice from any skeletal defects that might otherwise result from loss of only the full-length CaSR. Indeed, when chondrocytes were isolated from CaSRe/e mice, they still exhibited the same responses to Ca2þo that were present in wild-type chondrocytes [80], consistent with the presence of a biologically active CaSR lacking exon 5. Regardless of this observation, however, it has not been possible as yet to directly demonstrate biological activity of the exon-5-less CaSR by expressing it in heterologous cell systems. To further study the biological roles of the CaSR in the skeleton in vivo using knockout models, Chang, Shoback and Bikle and coworkers developed mice in which exon 7 of the CaSR had been “floxed” by insertion of loxP sites at either end of this exon [112]. If these mice are then mated with a strain of mice expressing the Cre recombinase only in the tissue of interest, the recombinase excises the floxed exon, recombines the ends of the remaining gene, and transcription and translation of that gene will produce a protein in which exon 7 is missing. Thus, mating the mice with exon 7 of the CaSR floxed with mice expressing the Cre recombinase in chondrocytes was utilized to examine the consequences of knocking out the CaSR in cartilage. Exon 7 encodes the entire transmembrane domain of the CaSR and the CaSR gene lacking exon 7 can only generate a receptor comprising the CaSR ECD, which would be released into the extracellular fluid as a soluble protein and presumably be incapable of signaling [112]. Presently, there is no convincing evidence that this soluble ECD protein has any biological role(s). Chondrocytespecific deletion of the CaSR resulted in death of embryos by day 13 [112], a surprisingly severe phenotype given the multiple hormonal or other factors known to regulate chondrocyte development and function. By using an inducible Cre recombinase, it was possible to delete exon 7 of the CaSR on days 16e18 of embryonic life, i.e., after the time when the embryos died in the previous model, which resulted in viable embryos that nevertheless displayed delayed growth plate development. These data supported, therefore,

a critical, nonredundant role of the CaSR in growth plate function. VDR in Chondrocytes An early, classic observation in the vitamin D field was that rickets, with its disordered structure of the growth plate and associated impaired growth, could be cured by the stimulation of the endogenous production of vitamin D or its exogenous administration (see additional chapters in this section). It was widely assumed that this represented direct actions of vitamin D on the cartilaginous growth plate. Indeed, direct effects of 1,25(OH)2D3 on chondrocytes had been shown in vitro that could potentially occur in vivo, such as stimulation [113] or inhibition [114] of growth, and promotion of differentiation [115,116]. Some of these actions may be mediated by cell surface receptors for 1,25(OH)2D3 [114] (see Chapter 28). While there is some overlap in the functions of the VDR and CaSR in chondrocytes identified in these studies (i.e., in promoting cellular differentiation), there have been limited studies to date directly assessing functional interactions between the two receptors [117]. A seminal observation was made, however, in mice with targeted disruption of the VDR (reviewed in [118]). As noted earlier, VDRe/e mice developed hypocalcemia, hypophosphatemia, and high PTH levels along with severe rickets after weaning. When administered a rescue diet, however, which included highcalcium and high-phosphate contents and lactose, the levels of serum minerals normalized and rickets was totally prevented. These findings, therefore, indicated that a major mechanism by which vitamin D cures rickets is indirect, by ensuring normal circulating mineral levels, which, in turn, promote mineralization of bone independent of direct cellular effects mediated by the VDR in chondrocytes. Nevertheless, these findings do not rule out the possibility that signaling through the VDR participates in growth plate function in VDR intact animals. For instance, up-regulation of signaling though the CaSR or some other pathway in the VDRe/e mice might compensate for loss of the VDR. Moreover, 1-hydroxylase knockout mice, unlike the VDRe/e mice, do not totally normalize their growth plates when receiving a rescue diet [117]. Thus the total lack of 1,25(OH)2D3 in these mice may have a detrimental effect on growth plate function not fully explained by absent VDR signaling. Of note the VDRe/e mice have markedly elevated 1,25(OH)2D3 levels, as noted above. Perhaps the latter contributes to normalization of their growth plates on the rescue diet, potentially through a VDR-independent mechanism, such as a membrane receptor for 1,25(OH)2D3. In addition, recent studies have shown that specific inactivation of the VDR in chondrocytes results in



reduced RANKL expression by these cells, which is accompanied by reduced osteoclastogenesis and decreased FGF-23 levels, the latter producing increased serum phosphate concentrations [119]. These studies directly demonstrate novel functions of the VDR in chondrocytes in vivo related to the regulation of bone and mineral ion homeostasis. The CaSR in Osteoblasts As noted earlier, some [105,120,121] but not all [122] studies demonstrated the presence of the CaSR in bone, primary cultured osteoblasts and osteoblastic cell lines (for review, see [106]). In osteoblastic cells expressing the CaSR, high Ca2þo, acting at least in part via the CaSR, exerts effects that would be expected to promote bone formation. These include stimulation of the proliferation of preosteoblasts [123], increased expression of the mRNAs encoding the osteoblast differentiation markers, CBFA-1, osteocalcin, osteopontin, and collagen 1, and enhanced mineralized nodule formation [124]. As was the case with studies of cartilage in mice with homozygous knockout of exon 5 of the CaSR, bone histology in these mice was dominated by severe hyperparathyroid bone disease owing to loss of the CaSR in the parathyroid, which precluded ready interpretation of the impact of loss of the CaSR on osteoblast function. Studies in the two “rescue” models described above, CaSRe/ePTHe/e [109] and CaSRe/eGcme/e [110], showed that there were little or no differences in their bone histology and histomorphometry from those of control mice with intact CaSR with or without PTH or Gcm, respectively, suggesting a minimal role for the CaSR in the formation and turnover of the skeleton. However, conditional knockout of exon 7 of the CaSR in osteoblasts using the Cre recombinase driven by the 2.3-kilobase promoter for type 1 collagen produced viable mice, which exhibited, however, poor postnatal growth and skeletal development, with small poorly mineralized skeletons [112]. Most mice developed long bone and rib fractures and died within 3 weeks of birth. The bones were osteopenic and poorly mineralized and exhibited decreased levels of both early and late markers of osteoblast differentiation, such as type 1 collagen, alkaline phosphatase, insulin-like growth factor-1 (IGF-1, a key osteoblast growth factor), and osteocalcin. There was also increased osteoblast apoptosis. These results suggest key roles for the CaSR in promoting proliferation, survival, and differentiation of osteoblasts as well as skeletal mineralization [112]. For unclear reasons, the control mice with exon 7 of the CaSR floxed, which had not been crossed with Cre mice, had mildly elevated serum calcium concentrations. The significance of this finding is unclear, and it would be important to compare their serum calcium concentrations to those

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of the strain of mice from which they were derived in order to rule out an impact of the insertion of the loxP sites on expression or function of the CaSR. The VDR and Osteoblasts There is an extensive literature addressing the roles of the VDR in osteoblasts (for review, see [125]), which will be summarized briefly here as this is covered in detail elsewhere (see additional chapters in this section). Fetal rat calvarial osteoblasts grown in culture undergo a characteristic cellular program of proliferation, followed by differentiation and then mineralization [126]. Type I collagen production is initially expressed at high levels during the proliferative phase, then decreases during differentiation, while the production of alkaline phosphatase, osteopontin, and osteocalcin increase in that order during the differentiation and mineralization phases. Several of these genes are vitamin-D-responsive, including type I collagen, osteocalcin, and osteopontin, to name just a few of the osteoblastic genes that have been shown to be regulated by vitamin D during osteoblastogenesis and subsequent mineralization of bone. In fact, the use of chromatin immunoprecipitation techniques (ChIP), which enables identification of genes associated with specific transcription factors, such as the VDR, bound to their regulatory regions, has identified several thousand binding sites for the VDR/RXR heterodimer in the DNA of the mouse osteoblastic cell line MC3T3-E1 [127]. The analysis of the phenotype of mice with knockout of the VDR [128] and/or the 1a(OH)ase [129] has had a major impact on our understanding of the role of 1,25(OH)2D3 and the VDR on osteoblast function, analogous to the impact it had on the roles of the VDR in parathyroid cell and chondrocyte biology (see Chapter 33). Knockout of the VDR alone, as noted above, revealed that a rescue diet normalizing serum calcium, phosphate, and PTH could heal the rickets of the VDRe/e mice with normalization of the growth plate in 70-dayold mice, as noted above. Histomorphometry of adjacent metaphysis (the flared bone connecting the epiphysis to the shaft of a long bone) was likewise normal, including osteoblast number and the ratio of osteoblast surface to total bone surface. In addition, femoral strength, providing an assessment of the integrity of cortical bone, was equivalent to that in control mice [128]. This study concluded that the principal function of the VDR in bone formation and modeling was indirect, e.g., ensuring adequate serum levels of calcium and phosphate, rather than by directly exerting essential actions of osteoblasts. A follow-up study by the same group, however, demonstrated that primary calvarial osteoblasts isolated from VDRe/e mice exhibited increased numbers of osteoblastic colony-forming units (a measure of the number of cells capable of


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differentiating to osteoblasts) [130]. Moreover, the osteoblastic cells in vitro manifested earlier onset and increased magnitude of alkaline phosphatase expression and earlier and sustained mineralized matrix formation. These results point out that even though loss of the VDR can clearly alter the function of osteoblastic cells in vitro, additional factors, including (and almost certainly not limited to) the CaSR when serum calcium is normalized can compensate to maintain normal or nearly normal bone formation in vivo. Panda et al. carried out a similarly designed study in older mice (120 days old), utilizing VDRe/e mice and/ or 1a(OH)asee/e mice [129]. When the mutant mice received the rescue diet, the length of the femurs of the mutants (which were markedly shorter than that in the wild-type if the rescue diet was not utilized), were nearly normalized, indicating near normal bone growth, reflecting, in part, much improved growth plate function. With regard to the properties of osteoblasts per se, the rescue diet reduced the number of osteoblasts in the three mutants below that in the wild-type mice, with an associated decrease in mineral apposition rate and bone volume [130]. Furthermore, in contrast to the observations of Amling et al., cultured osteoblastic cells from all three mutants after they had been maintained on the rescue diet, exhibited a reduced, rather than increased, capacity to form osteoblastic colonies. Finally, the expression of RANKL, the key osteoblastic protein needed to promote the osteoblast-dependent formation of osteoclasts was substantially below that in wild-type mice. These authors concluded that 1,25(OH)2D3 and the VDR serve an anabolic role in osteoblastic function in vivo [129]. The difference between these results and those of Amling et al. [128] were ascribed to differences in the ages of the mice (120 vs. 70 days, respectively). Thus, the impact of the loss of the VDR on bone in the two studies can be compensated partially [129] or nearly completely [128] in vivo by ensuring a normal milieu of mineral ions, presumably mediated by the crucial roles of the CaSR and perhaps other compensatory genes in chondrocytes and osteoblasts. The key role that VDR-mediated intestinal calcium absorption, rather than the VDR in bone cells per se, plays in maintaining normal development and growth of cartilage and bone was shown by the studies of Xue et al. [131], who showed that transgenic expression of the VDR on a VDR-null background normalized calcium transport in the intestine, serum calcium concentration, serum PTH, and somatic growth. There were changes in bone per se, however, as bone mineral density and bone volume were higher in the transgenic mice compared with the normal mice as a result of increased mineral apposition rate and osteoblast number. An increase in 1,25(OH)2D3 levels owing to loss of the VDR in kidney and a resultant decrease in degradation

by the 25-hydroxyvitamin D 24-hydroxylase and increase in synthesis of 1,25(OH)2D3 may have contributed to an anabolic effect of 1,25(OH)2D3 in bone postulated by others [129]. The CaSR in Osteoclasts While some studies have failed to detect the CaSR in osteoclasts [132], recent studies have provided strong evidence that the CaSR is expressed in cell lines thought to resemble osteoclast precursors (e.g., RAW 264.7) [133], in multinucleated osteoclasts differentiated from such precursors in vitro and in at least some mature osteoclasts in intact bone. The CaSR appears to serve a permissive role in osteoclastogenesis in vitro, but high Ca2þo concentrations also directly inhibit osteoclast activity and stimulate their apoptosis [133]. Thus assuming that the CaSR mediates similar actions in vivo, high Ca2þo, via the CaSR, inhibits bone resorption in a homeostatically appropriate manner, actions that at least theoretically could be mimicked by calcimimetics, although one in vitro study that failed to detect transcripts for the CaSR in osteoclast precursors or osteoclasts formed in vitro did not observe any functional effect of a calcimimetic on these cells [132]. The effects of extracellular calcium on osteoclast function have also been ascribed to an entirely different calcium-sensing mechanism (for review, see [134]). VDR in Osteoclasts In contrast to the extensive literature on the presence and actions of vitamin D and the VDR in chondrocytes and osteoblasts, that related to osteoclasts is much more limited. Instead, there has been a greater emphasis on indirect effects of 1,25(OH)2D3 on osteoclasts that are mediated by osteoblasts, such as up-regulation of RANKL, with subsequent stimulation of osteoclastogenesis and bone resorption by pre-existing osteoclasts [135] (for additional details see Chapters 17 and 18). Available data indicate that the VDR is expressed in osteoclasts and their precursors [136e138], but the functional implications of the VDR in these cells are not known. It should be pointed out, however, that studies on osteoclasts in intact bone carried out using mice with knockout of the VDR and/or 1-hydroxylase have provided some insights into the role of the VDR, at least in mice. As noted above, Amling et al. [128] studied 70-day-old VDRe/e mice when on a rescue diet and found little if any difference in osteoclast number or in the percent of the bone surface covered by osteoclasts. On the basis of these results, it was not possible to ascribe any function to the VDR in osteoclasts (or indirect effects of the VDR on osteoclasts mediated by osteoblasts) in the setting of the normocalcemic, normophosphatemic, and euparathyroid state. More recently, Panda et al. [129] performed similar studies in 120-day-old mice,



utilizing VDRe/e, 1a(OH)asee/e mice and double knockout mice as before. While receiving the rescue diet, osteoclasts in all three of the mutant genotypes were normal in size and number. Of note, when hypocalcemic owing to lack of the rescue diet, all three genotypes of mutant mice did not show the expected increase in osteoclast number owing to hypocalcemia and secondary hyperparathyroidism [129]. This observation is most likely not due to an intrinsic osteoclast defect, but rather to the reduced RANKL expression by the osteoblasts of the mutant mice, as noted above. Neither of these studies, therefore, supports a defect in osteoclasts per se or their precursors in the absence of the VDR and/or 1,25(OH)2D3.

The CaSR and VDR in the Intestine While the CaSR is widely expressed in the gastrointestinal tract [17], many of its functions are not directly related to Ca2þo homeostasis (e.g., regulation of gastric acid secretion) and are not covered here. Some of these locations, e.g., the colon, provide useful examples, however, of how the CaSR and VDR can interact in ways unrelated to mineral ion homeostasis, such as in the development of colon cancer, and will be discussed later. With regard to Ca2þo homeostasis, the stimulation of transcellular calcium transport in the intestine, particularly the proximal small intestine, is one of the bestknown actions of vitamin D. Although there are rapid nongenomic effects of 1,25(OH)2D3 on intestinal calcium transport in vitro [139], the best-characterized intestinal actions of vitamin D mediated by the VDR involve upregulation of the transport machinery for transcellular transport of calcium. This includes the apical uptake channel, TRPV6 (which is closely related to TRPV5 in the kidney), calbindin-D9K, and PMCA1b. The expression of all of these components is up-regulated by 1,25 (OH)2D3 in 1a(OH)asee/e mice [140], thereby providing a molecular basis for the stimulation of intestinal Ca2þ absorption by 1,25(OH)2D3 through a genomic mechanism very similar to that in the DCT/CNT. The CaSR is expressed in the small intestine [141] (presumably in the same cells expressing the VDR but this has not been proven) as well as in cell lines derived from the intestine [142], but its functions in the intestine have not been well characterized in many cases. Nevertheless, available data suggest that it can act in concert with vitamin D or partially substitute for the actions of 1,25(OH)2D3 in certain circumstances. In organ cultures of fetal rat duodenum, for example, not only 1,25 (OH)2D3 but also increases in medium Ca2þ concentration increase the expression of the mRNA for calbindin-D9K [143]. More recently, van Abel et al. showed that administration of a rescue diet to 1a(OH)asee/e mice restored normocalcemia in association with

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statistically significant, >tenfold increases in the expression of the mRNAs for TRPV6 and calbindin-D9k [140] in the intestine, similar to what was observed in the kidney. Although not formally proven, it seems likely that these actions of calcium are mediated by the CaSR. What is the significance of these actions of dietary Ca2þ on intestinal calcium absorption? On the one hand, high concentrations of Ca2þ can apparently substitute, at least in part, for a lack of vitamin D in the maintenance of the level of expression of key elements of the transcellular calcium transport system in the intestine. In the presence of vitamin D, on the other hand, there might be the possibility of a physiologically undesirable feedforward mechanism, whereby intake of increasing amounts of calcium in the vitamin-D-sufficient state might foster excessive GI calcium absorption by the mechanism just described. However, as long as the source of vitamin D was not 1,25(OH)2D3 itself or one of its analogs, inhibitory feedback regulation on renal 1-hydroxylation via decreases in PTH or increases in serum calcium or phosphorus (e.g., by directly inhibiting the 1-hydroxylase) could prevent such a scenario.

The CaSR and VDR in the Placenta A key role that the placenta plays during intrauterine life is to provide adequate quantities of calcium for the developing fetal skeleton, primarily during the third trimester in humans. This is accomplished by “pumping” calcium transcellularly using the same machinery that is used in other Ca2þ-transporting epithelia, including TRPV6, calbindin D9K, and PMCA. In the mouse, much of this transport is thought to occur in the placental yolk sac, the site of expression of this transport machinery in this species. The most cogent evidence regarding the relative importance of the CaSR and VDR for placental Ca2þ transport comes from mouse knockout models. The role of the CaSR in regulating placental Ca2þ transport was explored by Kovacs et al. [144] utilizing mice heterozygous or homozygous for knockout of exon 5 of the CaSR. There were several phenotypes of fetal CaSRe/e mice relevant to calcium homeostasis. These include hypercalcemia, markedly elevated PTH levels owing to defective Ca2þ sensing by the fetal parathyroids, elevated levels of the bone resorption marker, deoxypyridinoline, and increased urinary calcium excretion, likely as a result of PTH-induced bone resorption. Thus, as is also observed postnatally in this knockout model, loss of the full-length CaSR leads to severe hyperfunction of the fetal parathyroid glands. In addition, however, placental transport in the CaSRe/e fetuses measured in vivo using 45Ca (each fetus has its own placenta) was significantly less than


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that of both the wild-type (CaSRþ/þ) and heterozygous (CaSRþ/e) fetuses [144]. This result implicates the CaSR in promoting normal placental transport of calcium. It does so, at least in part, through a PTHrPdependent pathway, since knocking out PTHrP in vivo results in a decrease in 45Ca transport to a level similar to that found in CaSRe/e fetuses [144], which no longer varies as a function of CaSR genotype. That is, the CaSR can only stimulate placental calcium transport in the presence of PTHrP, which, therefore, is presumably “downstream” of the CaSR. Despite the presence in the murine placenta of the vitamin-D-dependent calcium transport machinery similar to that present in intestine and kidney, cogent evidence obtained using VDR knockout mice indicates that the VDR is not required for fetal mineral ion homeostasis, skeletal mineralization, or for normal placental calcium transport, at least in mice [145]. When heterozygous, VDRþ/e mothers were mated, the offspring (VDRþ/þ, VDRþ/e, and VDRe/e fetuses) were similar with regard to placental 45Ca transport, serum calcium and phosphate, and the calcium content of their skeletons. Thus the CaSR, and not the VDR, functions in the placenta to regulate key placental functions vital to mineral ion and bone homeostasis.

The CaSR and VDR in Breast Analogous to the role of the placenta in maternoefetal calcium and bone homeostasis, the breast serves as the dominant source of calcium for the newborn and growing infant. Recent studies by van Houten and colleagues have identified the CaSR as a key player in this process [146]. Expression of the CaSR increases markedly during lactation in the mouse and then returns to baseline levels following the termination of breastfeeding. The receptor is expressed basolaterally in breast epithelial cells (i.e., on the opposite side of the cell from the apical surface facing the milk), particularly in the milk-producing alveoli. The CaSR in the lactating breast plays two key roles in the breast during lactation: (1) suppressing the secretion of PTHrP, and (2) stimulating the transport of calcium into breast milk. Any decrease in maternal serum calcium concentration stimulates PTHrP secretion, both into the milk as well as into the systemic circulation. The increase in circulating PTHrP level stimulates bone resorption, providing a mechanism for increasing the serum calcium concentration in the mother and thereby providing calcium for transport into the milk. Activation of the basolateral CaSR in the breast by the increase in maternal serum calcium concentration then stimulates the transport of calcium into the milk by a mechanism involving activation of the apical calcium pump, PMCA2 [147]. In a sense, therefore, the CaSR serves as

an “accessory parathyroid gland” during lactation, showing inverse regulation of a calcium-elevating hormone (PTHrP) that acts through the same mechanisms utilized by PTH, namely stimulating bone resorption and, presumably, renal tubular calcium reabsorption. Since the PMCA is stimulated by calmodulin (CaM) following activation of the latter by increases in the cytosolic calcium concentration [147], a CaSRstimulated increase in CaM activity could underlie the activation of PMCA, although the signal transduction pathways involved have not been studied in detail. Is there a role for the VDR during lactation? Relatively little work has been carried out on the role of the VDR and vitamin-D-responsive genes participating in calcium transport on calcium transport into milk during lactation. As assessed by binding of radioactive 1,25 (OH)2D3, epithelial cells of the ductal and alveolar cells of the breast express the vitamin D receptor, and the levels increase during pregnancy and lactation [148]. It is currently unknown, however, whether key elements of the epithelial calcium transport machinery, e.g., TRPV5/6, calbindin D9K and D28K, PMCA, etc., function in transcellular calcium transport in the lactating breast. In this regard it would be of interest to study the effect of 1,25(OH)2D3 on transport of calcium into milk in vitro and in vivo during lactation using the tools used to study the role of the CaSR in this process, including mice with global or conditional knockout of the VDR (and/or CaSR) in the breast. Recent studies have addressed the role of the VDR in regulating other aspects of breast structure and function. In mice with knockout of the VDR, there was accelerated lobuloalveolar development and premature expression of casein, a milk protein, as well as delayed postlactational involution compared with control mice [149]. Thus the VDR appears to regulate mammary cell turnover during the reproductive cycle. Comparable studies have not been carried out with regard to the CaSR, for example, using mice with conditional knockout of the CaSR in the breast to examine breast development and involution during lactation as well as at other times.

The Utility of CaSR-based Therapeutics Alone or in Combination with VDRAs for Treating Parathyroid Hyperfunction Nearly simultaneous with the isolation and characterization of the CaSR was the development of the calcimimetic CaSR activators, which provided a means of pharmacologically inhibiting hyperfunctioning parathyroid glands, particularly in the setting of chronic renal insufficiency. The discussion that follows provides an overview of this area, which is relevant to another clinically useful way of treating hyperparathyroidism in this setting, i.e., the use of vitamin D



receptor activators (VDRAs) [150]. The latter share with calcimimetics the capacity to suppress PTH gene expression and parathyroid cellular proliferation, as detailed earlier. These shared actions plus the additional direct suppression of PTH secretion by the calcimimetics offer new opportunities for sequential or initial combined therapy with these drugs. Given the voluminous literature on VDR-based therapy for stage 5 CKD and a related discussion of this issue in this volume (see Chapter 70), the emphasis of the discussion that follows will be on CaSR-based therapy and our evolving understanding of its relationship to VDR-based therapy. Studies of the Utility of Calcimimetics in Animals with Uremic Hyperparathyroidism It has not yet been shown definitively that all of the actions of calcimimetics on rats with experimentally induced uremia are applicable to humans. However, these animal studies have provided proof-of-concept that administration of calcimimetics in vivo, as would have been expected, sensitize the CaSR in the parathyroid gland to Ca2þo, thereby producing the expected alterations in parathyroid function. Moreover, the results of these animal studies provide benchmarks for the subsequent and ongoing investigation of potentially beneficial actions that the drug might have in humans. Studies in normal rats demonstrated that oral administration of a single dose of the “second-generation” calcimimetic, cinacalcet (Fig. 24.7), lowered serum PTH, with a nadir at 1e2 hours [151]. Serum calcium concentration also declined with a maximal decrease at 1e4 hours, depending on the dose. Cinacalcet also increased circulating calcitonin levels, but the apparent in vivo EC50 values for suppressing PTH and elevating calcitonin were 0.5 and 16 mg/kg, respectively [151]. Therefore, doses of cinacalcet that suppress PTH release would have minimal, if any, effects on calcitonin secretion in vivo. An extensive body of evidence subsequently showed that the “first-generation” calcimimetic, NPS R-568, lowers serum PTH and calcium concentrations in rats with experimentally induced renal insufficiency, usually produced by subtotal (i.e., 5/6) nephrectomy [151]. A calcimimetic also decreased PTH mRNA [47], which likely

FIGURE 24.7 Chemical structure of the calcimimetic, CinacalcetÒ

Ca2þo (also known as SensiparÒ). From Nemeth et al. 2004 J Pharmacol 308:627e35, with permission.

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contributes to the reduction in PTH secretion, and upregulated both the CaSR and VDR in the parathyroid [152]. Administering calcimimetics to uremic rats also obviated the parathyroid hyperplasia that would otherwise take place in the setting of renal insufficiency [153], owing, in part, to up-regulation of the cyclin-dependent kinase inhibitor, p21 [154]. Some studies have also demonstrated regression of established parathyroid hyperplasia [155]. While there is little apoptosis of the long-lived parathyroid chief cells under normal circumstances, there is some evidence that calcimimetics induce apoptosis of this cell type, albeit at a high dose (10e4 M) [156]. Finally, treating uremic rats with a calcimimetic has reduced the hyperparathyroid bone disease, termed osteitis fibrosa cystica, that occurs in the setting of renal failure [157]. Further studies in rats revealed additional actions of calcimimetics in other tissues. Rats administered NPS R-568 manifested a reduction in rate of progression of renal failure that otherwise occurs in rats that have undergone subtotal nephrectomy [158]. The same study documented that the drug also decreased blood pressure and LDL cholesterol levels, which could have a favorable impact on the substantially increased risk of cardiovascular complications of uremic animals and humans [159]. Indeed, the hearts of the treated animals showed less interstitial fibrosis and had a reduction in arteriolar wall thickness compared to control animals. Subsequent studies have also shown decreases in the thickness and calcification of blood vessel walls outside of the heart, and the life span of uremic rats treated with calcimimetic was significantly longer than in controls [160]. The use of atherosclerosis-prone, apolipoproteinE-deficient mice made it feasible to demonstrate that NPS R-568 retarded uremia-induced vascular calcification and atherosclerosis in this animal model [161]. Finally, a recently developed calcimimetic, AMG 641, produced actual regression of pre-existent aortic and soft tissue calcification in uremic mice of this strain [162]. The relative contributions of alterations in PTH vs. serum mineral ions (i.e., Ca2þ, phosphate, and calciumephosphate product) vs. potentially direct actions of calcimimetics on the vasculature are topics of ongoing investigation. Active vitamin D compounds, such as 1,25(OH)2D3 and its less calcemic analog, paracalcitol, are an important component in the therapy of secondary hyperparathyroidism in humans with stage 5 CKD by virtue of their capacity to reduce PTH gene expression and parathyroid cellular proliferation [163]. Studies in uremic rats have, however, shown that vitamin D analogs, particularly 1,25(OH)2D3, while effective in lowering PTH, can decrease survival and cause extraosseous calcification and progression of renal failure [160]. These deleterious


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actions of the vitamin D analogs in rats can be significantly decreased by coadministering a calcimimetic [164]. Studies of the Utility of Calcimimetics in SHPT in Humans with Stage 5 CKD A substantial body of evidence in humans with stage 5 CKD has demonstrated actions of cinacalcet that are similar to those just described in animal models of uremia, albeit with some differences, as pointed out below (the rats are not being dialyzed for one). This field will be summarized relatively briefly, emphasizing gaps in our knowledge or areas of controversy, as this topic is covered in detail in recent reviews [165]. In randomized controlled studies, cinacalcet lowers serum PTH, calcium, phosphate, and the calciumephosphate product [28]. The decrease in serum phosphate was surprising at first. In a dialysis patient, there is little or no renal phosphate excretion, so the change in serum phosphate observed during therapy with cinacalcet must result from other, as yet poorly defined, mechanisms. The decreases in these indices of mineral metabolism have improved the attainment of targets developed for these parameters in patients with stage 5 CKD. The U.S. National Kidney Foundation (NKF) provides guidelines (so-called NKF KDOQI (kidney disease outcomes quality initiative) guidelines) for the levels of serum calcium, phosphate, PTH, and the calciumephosphate product that are to be achieved in patients with CKD. These guidelines have as their goal to reduce morbidity and mortality in this patient population (http://www.kidney.org/Professionals/Kdoqi/ guidelines_ckd/toc.htm). For stage 5 CKD, the goals for serum calcium, phosphorus, calciumephosphate product, and PTH are, respectively, 8.4e9.5 mg/dl, 3.5e5.5 mg/dl, 90% decrease in the rate of parathyroidectomy and about a 40% reduction in cardiovascular hospitalizations. The study was not sufficiently powered, however, to assess the effect of the drug on “hard” cardiovascular endpoints, such as myocardial infarction or death. Two large randomized controlled studies are currently under way to address this latter point, the ADVANCE (“A randomized study to evaluate the effects of cinacalcet plus low dose VDRA on vascular calcification in subjects with chronic kidney disease (CKD) receiving hemodialysis”) and EVOLVE studies (EValuation Of Cinacalcet HCl Therapy to Lower cardio Vascular Events) [174]. The latter has primary end-points of all-cause mortality and first nonfatal cardiovascular event in 3800 chronic dialysis patients who are being treated with a flexible regimen of traditional therapies and will additionally receive either cinacalcet or placebo [174]. In addition to the use of Cinacalcet for treating secondary hyperparathyroidism in stage 5 CKD, the drug has also been approved by the FDA for use in parathyroid cancer and the drug has been used off-label (i.e., without FDA approval) for other forms of primary hyperparathyroidism, lithium-induced hyperparathyroidism, secondary hyperparathyroidism in patients being treated for phosphate-wasting disorders, and others. The interested reader is referred to a recent review of this topic [175].

THE CASR AND VDR IN TISSUES UNINVOLVED IN CA2DO HOMEOSTASIS There has been increasing interest in the roles of calcium and dietary calcium intake, on the one hand, and vitamin D, on the other, in maintaining the normal function of various cells and tissues (e.g., keratinocytes) as well as in preventing or treating a variety of pathological conditions not directly related to Ca2þo homeostasis. These pathological states include cancer (e.g., colon,

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breast, prostate, and others), infectious disease, autoimmune diseases (viz., inflammatory bowel disease), hypertension, and other cardiovascular diseases, and obesity, metabolic syndrome, and type 2 diabetes [176,177]. In general, more data are available regarding the mechanisms by which 1,25(OH)2D3 and the VDR, as opposed to calcium and the CaSR, regulate normal and pathological tissues, owing to the relatively recent recognition that the CaSR is expressed in a multitude of tissues uninvolved in Ca2þo homeostasis [178]. Indeed, in only a minority of cases are the molecular mechanism(s) fully understood by which calcium exerts beneficial and, in some cases, detrimental effects on normal and neoplastic cells outside of Ca2þo homeostatic tissues. In only a few of these, in turn, is there convincing evidence that the CaSR serves as the molecular target for the effects of calcium, as opposed to some other mechanism, such as a calcium channel, for instance. Rather than trying to cover this rapidly growing field in detail, this discussion will focus on several examples where the CaSR and VDR both appear to contribute to modulating the function(s) of selected normal and pathological tissues outside of those participating in Ca2þo homeostasis.

CaSR, VDR, and Renin Secretion Renin plays a key role in sodium, volume, and blood pressure homeostasis: hypovolemia is sensed by the juxtaglomerular (JG) cells of the afferent arteriole of the kidney, which increase their release of renin. In turn, renin converts circulating angiotensinogen to angiotensin II, a potent endogenous pressor, which also stimulates the production of the sodium-retaining hormone, aldosterone, by the zona glomerulosa of the adrenal gland [179]. The combination of the direct elevation in blood pressure due to angiotensin II and the sodium retention caused by aldosterone will promote normalization of sodium and volume homeostasis. It has been known for several decades that raising the extracellular calcium concentration suppresses the release of renin by the JG cells [180]. Only more recently, however, has it been shown convincingly that the CaSR is expressed by JG cells [181] and that it mediates the inhibitory action of elevated calcium concentrations on renin release [182]. An unexpected observation in VDRe/e mice was that they exhibit increased production of renin and angiotensin, which leads to hypertension and cardiac hypertrophy [183,184]. The CaSR and the VDR, therefore, can modulate not only on mineral ion homeostasis but also on sodium, blood pressure, and volume homeostasis. These observations highlight unexpected roles of the CaSR and VDR that suggest previously unknown interactions between Ca2þo homeostasis, on the one hand, and sodium and volume homeostasis, on the


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other. What might the physiological significance of these observations be? In some studies, intravenous infusion of calcium produces an acute increase in blood pressure in normal individuals and especially in those with chronic renal insufficiency [185,186], who cannot efficiently excrete a sodium load. Viewed in this context, suppression of renin secretion and the resultant decrease in angiotensin II production could serve as a counterregulatory, blood-pressure-lowering mechanism. In some but not all studies, calcium administration can exert a beneficial effect on some forms of hypertension, e.g., preeclampsia [187], although the relevance of the CaSR in JG cells to this effect is unknown.

CaSR and VDR in the Growth and Differentiation of Normal and Neoplastic Tissues CaSR and VDR in Keratinocytes Both elevating Ca2þo and treatment with 1,25(OH)2D3 inhibit the proliferation and stimulate the differentiation of keratinocytes in culture in a manner that mirrors that occurring in normal skin. Keratinocytes cultured at concentrations of 1 nm 1,25(OH)2D3, is associated with reductions in the mRNA levels for the growth promoters, cyclin D1 and c-Myc, as well as increases in the cell cycle inhibitors, p21cip and p27kip (reviewed in [193]) (Fig. 24.8). In addition to 1,25 (OH)2D3 produced in the kidney, it is likely that 1,25 (OH)2D3 synthesized locally by CYP27B1 expressed by skin cells [194] also acts on the VDR present in the keratinocyte. 1,25(OH)2D3-induced changes in gene expression contributing to differentiation include increases in the expression of involucrin, loricrin, profilagrin, and transgluatminase, a key protein for the cross-linking the cornified envelope of the skin. VDRe/e mice have reduced cutaneous levels of involucrin and loricrin and loss of keratinohyalin granules, confirming the physiological importance of the VDR in the keratinocyte [118]. Thus, unlike the parathyroid gland, chondrocyte and osteoblast, where the CaSR can support normal or nearly normal cellular function in the absence of the VDR, both the CaSR and VDR appear to have similar, nonredundant roles in the keratinocyte. Therefore, there is substantial overlap in the mechanisms by which calcium and 1,25(OH)2D3, acting through two entirely different classes of receptors, produce growth arrest and differentiation of keratinocytes (Fig. 24.8). In fact, they synergistically up-regulate the expression of involucrin and transglutaminase [195], which results in part from up-regulation of the CaSR by 1,25(OH)2D3 [196]. The reciprocal experiment, examining the effects of elevated Ca2þo on the level of expression of the VDR, does not appear to have been carried out. It would be of interest to determine whether activation of the CaSR by high Ca2þo up-regulates this receptor, as in the parathyroid cell. Calcium and vitamin D also exert important biological actions on a variety of tumor cells which are potentially relevant to treatment of these tumors in vivo. This field is considerably more advanced with regard to the antiproliferative and other effects of vitamin D than with respect to similar effects mediated by the CaSR. It should also be noted that, even though there is experimental evidence, primarily in vitro, that the CaSR modulates the biology of tumor cells, it is far from certain that any effects of dietary calcium on cancer risk (for review, see [176]) are CaSR-mediated. CaSR and VDR in Colon Cancer Garland et al. carried out an early prospective study showing that reduced dietary intakes of calcium and vitamin D were associated with significantly increased risks of colon and/or rectal cancer [197]. A recent review reported that of 30 studies of colon cancer or adenomatous polyps [198], 20 studies found a significant benefit of vitamin D level or status (i.e., sunlight exposure) on


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FIGURE 24.8 Interactions between Ca2þo, acting via the CaSR, and 1,25(OH)2D3, acting via the VDR, in promoting keratinocyte differentiation. Activation of the CaSR stimulates phospholipase C in a G-protein-dependent manner, resulting in formation of diacylglycerol (DAG) and inositol tris-phosphate (IP3). The former activates protein kinase C bound to the receptor for activated PKC (RACK), while the latter releases calcium from intracellular stores in the endoplasmic reticulum (ER) and participates in activation of influx of extracellular calcium. The increase in cytosolic calcium and the active protein kinase C can activate the transcription factors (AP-1) and stimulate calcium-responsive DNA elements (CaRE). Increases in expression of keratinocyte genes, such as Involucrin and Loricrin, which are cross-linked through the activity of transglutaminase, enhance formation of the cornified envelope in the outer layer of skin. 1,25(OH)2D3 acts in concert with high Ca2þo by increasing expression of the CaSR as well as of that of phospholipase C and by increasing expression of the same keratinocyte genes. From Bikle et al. 2003 J Cell Biochem 88:290e295, with permission.

cancer risk or mortality or on the incidence of adenomatous polyps. Five other studies showed beneficial effects of borderline statistical significance and five showed no effect. Three recent, large studies in the US or Japan found statistically significant inverse relationship between calcium intake and risk of colorectal cancer with an average relative risk of about 0.7 in subjects with a higher intake of calcium [199e201]. Therefore, substantial epidemiological evidence supports the concept that increased intake or supplementation with calcium and/or vitamin D can substantially reduce the risk of colorectal cancer and the risk of recurrence of colonic polyps, thereby potentially substantially reducing the morbidity, mortality, and cost of medical care related to colorectal neoplasia in this country and elsewhere. What are the mechanisms by which calcium and vitamin D act to reduce the risk of colon cancer? Elevated levels of Ca2þo, acting via the CaSR, inhibit the proliferation of colon cancer cells. In Caco-2 cells,

high Ca2þo lowers c-Myc expression and up-regulates p21 [176]. Furthermore, in CBS colon cancer cells, in addition to increasing p21, high Ca2þo up-regulates E-cadherin expression and inhibits the nuclear transcription factor, TCF4, which influences signaling by the Wnt pathway [202,203]. Activation of the CaSR in colon cancer cells also down-regulates thymidylate synthase and survivin, which enhances cellular sensitivity to 5-FU. Kallay, Peterlik, and Cross have shown that vitamin D exerts antiproliferative actions in colon cancer cells by down-regulating cyclin D1 (176). 1,25(OH)2D3 also upregulates p21waf1 and p27 and down-regulates survivin, an inhibitor of apoptosis, and thymidylate synthase, which increases the sensitivity of colon cancer cells to the cytotoxic agent, 5-fluorouracil, as just noted. Of interest, these latter effects occurred in a CaSR-dependent manner [203]. As just detailed, a number of these actions are shared by high Ca2þo. Of note with regard to the in vitro effects of 1,25(OH)2D3, VDR knockout mice exhibit


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colonic hyperproliferation, supporting the role of 1,25 (OH)2D3 and the VDR in tonically suppressing proliferation of the colonic mucosa in vivo [204]. Therefore, high Ca2þo and 1,25(OH)2D3 each have a number of actions in colon cancer cells that would be expected to reduce proliferation and enhance differentiation (e.g., up-regulating E-cadherin, as in keratinocytes). Recent work indicates that these actions may not take place totally independently of one another, as noted above. There are also interesting and potentially important interactions between the CaSR and VDR in their actions on normal colon or colon cancer cells. Both high Ca2þo and 1,25(OH)2D3 up-regulate the CaSR [202], providing a feedforward mechanism by which high Ca2þo can amplify its own effects. In addition, high dietary calcium suppresses expression of the 24-hydroxylase in vivo, providing a potential mechanism by which elevating Ca2þo could increase the local level of 1,25(OH)2D3 by reducing its degradation, even though the colonic level of CYP27B1 did not change [176]. Future studies in this area could investigate in more detail the mechanisms, including signaling pathways and transcriptional control, by which Ca2þo exerts its actions on the colon and develop and utilize mice with conditional knockout of the CaSR in the intestine to further solidify the receptor’s role in the colon and its interaction with vitamin D and the VDR. CaSR and VDR in Prostate Cancer Thirteen of 26 studies in patients with prostate cancer found a statistically significantly reduced risk of prostate cancer with increasing vitamin D status, 11 reported no significant association and only one reported a significant inverse correlation, but the last of these only used latitude as an indirect measure of vitamin D status and prostate cancer risk [198]. Available data for the effect of calcium intake on prostate cancer risk, however, are even less clearcut than with vitamin D status. One study found a positive association with calcium intake and the risk of prostate cancer [205], while others found no such association [206]. However, a recent meta-analysis of 45 observational studies did not show a clearcut association between calcium intake and risk of prostate cancer [207]. Mechanisms by which vitamin D could exert beneficial effects on prostate cancer include cell cycle arrest through induction of p21, promotion of differentiation, and inhibition of tumor cell invasion and metastasis [208]. As with colon cancer and breast cancer cells, prostate cancer cells express the 1-hydroxylase enzyme, allowing for local production of 1,25(OH)2D3. The mixed effects of calcium intake on prostate cancer risk may relate to actions of the CaSR on prostate cancer cells that would enhance rather than reduce prostate cancer cell growth and/or metastasis, or it could exert a combination of stimulatory and inhibitory actions.

For example, the CaSR inhibits apoptosis of AT-3 prostate cancer cells [209] and activates the epidermal growth factor receptor in PC-3 prostate cancer cells, which would likely increase cellular proliferation and tumor growth [210]. The CaSR also promotes cellular proliferation and enhances metastasis in an in vivo mouse model in which the malignant and metastatic behavior of either control PC-3 cell or those with knockdown of the CaSR were compared [211]. The CaSRinduced stimulation of PTHrP production by prostate cancer cells [210], if it occurs in prostate cancer cells metastatic to bone in vivo, might aggravate malignant osteolysis owing to the stimulation of further bone resorption by the PTHrP secreted by cancer cells near sites of bone resorption (i.e., with a locally high level of Ca2þo), begetting further resorption, and so forth. Therefore, activation of the CaSR need not produce the same biological actions exerted by 1,25(OH)2D3 in cancer cells, and the effects of the CaSR and VDR in various forms of cancer must be compared on a caseby-case basis. Clearly, there is also no definitive evidence that the CaSR on prostate cancer cells serves as the molecular target for the potentially deleterious actions of calcium supplementation on the risk of prostate cancer.

POLYMORPHISMS OF THE CASR AND VDR AND CALCIUM AND BONE HOMEOSTASIS Identifying an association between polymorphisms in a gene (often single nucleotide polymorphisms (SNPs) and more recently the use of SNPs in genome-wide association studies (GWAS)) and disease states or alterations in physiological or other variables can provide clues into disease pathogenesis that can be pursued by experimental approaches that prove causality. There have been a number of studies examining the associations of CaSR polymorphisms with variables such as serum calcium concentrations in normal individuals, PTH level and severity of hyperparathyroidism, calcium excretion and renal stones, fractures and bone mineral density, and risk of colon cancer. In some studies, there have been concomitant investigations of association with polymorphisms of the VDR in order to look for interactions between polymorphisms in the CaSR and VDR genes. The following briefly describes some of these studies to provide a brief overview of the state of this field. There have been several studies of the associations between CaSR SNPs, particularly those producing changes in amino acids within the receptor’s C-tail (A986S, R990G, and Q1011E), and serum calcium concentration in normals. Most [212,213] but not all


SUMMARY AND PERSPECTIVES

[214] found modest changes in serum calcium concentration associated with specific genotypes (i.e., higher serum calcium with A986S or with haplotypes such as SRQ/ARE (a haplotype is a series of SNPs or alleles on a single strand of DNA) [212]). A recent study utilized GWAS to show the association of a different polymorphism with serum calcium concentration [215]. The studies showing a positive association are consistent with the idea that SNPs that alter the function or the expression of the CaSR may contribute to small variations in serum calcium concentration that are observed within the normal population. Two studies found an association between the presence of homozygosity for arginine at residue 990 and the severity of hyperparathyroidism in dialysis patients [216] or in primary hyperparathyroidism [217]. Another study, however, found no relationship of the three polymorphisms in the CaSR’s C-tail to persistent hyperparathyroidism after kidney transplantation [218]. Only one [219] out of about eight studies (viz., [220,221]) found an association between CaSR polymorphisms and bone mineral density, and no studies have reported an association with fracture risk. Therefore, despite its importance in chondrocyte and osteoblast biology, at least in the mouse, genetic variation in the CaSR in the general population does not appear to impact bone health. Several studies have shown an association between certain polymorphisms in the CaSR and either urinary calcium excretion [222] or renal stone disease [223e225] in normocalcemic stone formers, as well as in patients with primary hyperparathyroidism [226,227], but some have not [228]. Therefore, the presence of these associations is consistent with the known role of the CaSR in the control of renal calcium excretion. In this regard, it is also possible that a CaSR antagonist could be developed that would be of clinical utility in patients in whom altered function of the CaSR appears to be contributing to hypercalciuria by reducing the receptor’s activity. Three [229e231] out of four studies have found a positive association between risk or recurrence of colon cancer and polymorphisms in the CaSR. For instance, the A986S polymorphism was associated with a fourfold greater risk of cancer in one study [230], while a different SNP (rs1801726) was associated with a lower risk of cancer in another [229]. In one of these three studies that yielded positive results, the association was only with cancer in the proximal colon [231]. These results are consistent with those from the experimental studies described earlier showing that the CaSR may participate in the control of cell growth in the colon, and suggest that it may be possible to identify genetic variants that could predict persons at increased risk for the disease. In no study to date out of about ten that have studied both VDR and CaSR polymorphisms at the same time

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has there been any interactions between polymorphisms in the two genes and parameters such as bone mineral density [221] or disease states, e.g., renal stone disease [228] and colon cancer [231]. Surprisingly, therefore, despite the many interrelationships and interactions between the two genes in experimental studies carried out either in vivo or in vitro that were detailed earlier, polymorphic variations in the two genes do not appear to interact with one another in the studies carried out to date.

SUMMARY AND PERSPECTIVES Research carried out over the past 20 years, largely as a result of ongoing improvements in molecular techniques and the development of mouse knockout models, has dramatically altered our understanding of how calcium and vitamin D interact in bone and mineral ion homeostasis. In this regard, the summary that follows will assume that mice and men are more similar than different in calcium and bone metabolism. An important advance in this area has been the identification and characterization of the CaSR [2]. As a result, it has become possible to understand the role of extracellular calcium ions as not just serving as a passive participant in processes such as bone development and renal calcium excretion, but also acting as an extracellular first messenger. As such, calcium can actively regulate the tissues that make up the systems governing mineral ion and bone metabolism. The following discussion highlights and summarizes key areas covered earlier in which there have been dramatic shifts in our understanding of how calcium and vitamin D act and interact through their respective receptors, the CaSR and VDR. All calcium destined to serve extra- or intracellular roles originally entered the body through the GI tract. Studies in mice with global knockout of the VDR in which this receptor was expressed transgenically in the intestine support VDR-stimulated absorption of calcium as a crucial function of the receptor in mineral ion and bone homeostasis [131]. However, the rescue diet utilized in the global VDR knockout animals can overcome the defect in calcium absorption resulting from loss of the VDR in the intestine simply by ensuring sufficient dietary mineral ions. Thus the main purpose of the VDR in the intestine is to increase the efficiency of calcium absorption to the point where adequate calcium is absorbed with physiologically relevant levels of calcium intake. Until recently, this dietary rescue might have been assumed to simply reflect massaction-driven, passive absorption of calcium through the paracellular pathway. Surprisingly, however, normalization of the serum calcium concentration in mice with knockout of the 1-hydroxylase is


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accompanied by up-regulation of several of the genes participating in the active transcellular pathway of intestinal calcium absorption [232,233]. The latter observation has not been formally proven to be CaSRmediated but appears to reflect the capacity of the CaSR to serve a backup role in calcium absorption in the presence of abundant calcium in the gut lumen. How about the other elements of the calcium homeostatic mechanism? Once adequate calcium is absorbed, a key function of the CaSR is to serve as a “calciostat” that ensures adequate, stable circulating levels of calcium in bodily fluids [5]. Experiments in which the VDR was knocked out only in the parathyroid suggest that the parathyroid VDR serves only a relatively minor, secondary role [83]. That is, the loss of the well-established capacity of vitamin D to inhibit PTH gene expression and parathyroid cellular proliferation can be largely substituted for by the CaSR, with maintenance of normal or nearly normal parathyroid function even without the VDR in the parathyroid. However, the capacity of the VDR and CaSR to up-regulate both their own expression as well as that of the other receptor noted earlier raises the possibility that the two can “tune” or adjust the gain of the other receptor in the parathyroid or other tissues, although the physiological relevance of such a mechanism has not been shown as yet. What is the relative importance of the two receptors in the kidney with regard to their roles in Ca2þo homeostasis? Knockout models have not yet explored the interplay between the VDR and CaSR in regulating the 1-hydroxylase in the proximal tubule. The VDR is clearly a key feedback inhibitor of the formation of 1,25(OH)2D3, but can the CaSR exert physiologically relevant inhibition of the 1-hydroxylase in VDRe/e mice? One scenario by which the CaSR has been postulated to inhibit the 1-hydroxylase is by up-regulating the VDR, i.e., acting by a VDR-dependent mechanism [92]. The development of a proximal tubule-selective knockout of the CaSR, VDR, or both would permit this issue to be addressed experimentally. A second key site where urinary calcium excretion is adjusted to suit the homeostatic needs of the organism is in the thick ascending limb of Henle’s loop [103]. Studies in mice with knockout of the CaSR and PTH genes [41] have shown a nonredundant role of the CaSR in cTAL in up-regulating renal calcium excretion, independent of the regulation of PTH secretion by the CaSR. Does the VDR have any role in Ca2þo homeostasis in this nephron segment? The data are sparse, but up-regulation of the CaSR by the VDR in CTAL may be a mechanism by which the VDR can enhance urinary calcium excretion, perhaps contributing to the body’s defense against hypercalcemia in this setting. It would be of interest to determine whether loss of the VDR in the thick ascending limb has any functional consequences.

Knockout models have also not explored the relative importance of the VDR and CaSR in the DCT/CNT. As in the intestine, the VDR is clearly a key regulator of the components of the transcellular pathway that stimulate calcium absorption in the DCT/CNT [233]. Again, however, as in the intestine, dietary calcium rescue in mice lacking the 1-hydroxylase is capable of up-regulating some of these components, suggesting a backup role for the CaSR not only in intestine but also in kidney if vitamin D is deficient but dietary calcium abundant. However, the dietary calcium rescue of VDRe/e mice is associated with substantial hypercalciuria [101], supporting an important role of the VDR, presumably in DCT/CNT, in renal calcium reabsorption. Thus, loss of the VDR in DCT/ CNT cannot be effectively compensated for by the CaSR. The relative contributions of PTH- and 1,25(OH)2D3mediated changes in bone resorption and formation to Ca2þo homeostasis vs. that of rapid alterations in calcium fluxes into or out of bone by a largely PTH- or 1,25(OH)2D3-independent mechanism(s) [43] is uncertain. Both the CaSR and VDR can promote bone formation [112,125], which could dispose of calcium in the setting of hypercalcemia, and yet both can support osteoclastogenesis [133,234]. High concentrations of calcium, in excess of those ever encountered in the blood, but relevant to the levels measured near actively resorbing osteoclasts, exert a homeostatically appropriate inhibition of osteoclast activity and promote osteoclast apoptosis [133]. These actions are mediated, at least in part, by the CaSR [133]. The relevance of these actions of the VDR and CaSR on osteoblast and osteoclast function to homeostatically significant changes in fluxes of calcium into or out of bone is not clearly defined. Some evidence also suggests a role for the CaSR in regulating rapid fluxes of calcium into or out of bone, presumably independent of changes in bone formation and/or resorption [43]. Although experimentally challenging, further studies of how the receptor exerts such an action would be illuminating in terms of providing further understanding of the homeostatic relevance of the regulated movement of calcium ions into or out of the skeleton over a wide range of timeframes. Our understanding of how the VDR and CaSR interact has perhaps been altered most dramatically with regard to their roles in the development and maintenance of the skeleton. The dramatic phenotypes of mice with conditional knockout of exon 7 of the CaSR in chondrocytes and osteoblasts [112] came as a surprise given earlier work with mice with knockout of exon 5, which showed little, if any, cartilage or bone phenotype, apparently due to the capacity of the exon-5-less CaSR to rescue the cartilage and bone phenotypes [109]. Subsequent studies using conditional knockouts have established unexpected, essential, and nonredundant roles of the CaSR in


REFERENCES

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C H A P T E R

25 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in Osteoblast Differentiation and Morphology William R. Thompson 1, Mary C. Farach-Carson 2 1

Department of Physical Therapy, Program in Biomechanics and Movement Science; University of Delaware, Newark, Delaware, USA, 2 Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA

SYSTEMIC AND INTRACELLULAR CA2D HOMEOSTASIS Tight regulation of plasma and intracellular Ca2þ concentrations is essential to ensure proper cellular function and phenotype for essentially all cells in complex tissue. Mammalian Ca2þ homeostasis is hormonally regulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) at three major organ sites: the intestine, kidney, and bone. A decrease in serum Ca2þ concentration increases 1,25(OH)2D3 production, increasing intestinal Ca2þ absorption, decreasing excretion of Ca2þ into the urine, and increasing osteoclast activity ultimately to favor bone resorption. The first two processes occur quickly and are largely physiological responses, the latter is delayed and requires osteoclast expansion and activation. Approximately 99% of Ca2þ in the human body is held in the skeleton. During skeletal remodeling, Ca2þ released from mineralized tissue becomes freely exchangeable with the extracellular fluid, thus creating a buffer and reservoir system to help maintain circulating Ca2þ concentrations. A key function of bone-lining cells is to protect the mineral component of bone from being lost except at sites of bone remodeling. Intracellular Ca2þ levels remain in dynamic balance due to the activity of channels, pumps, and exchangers present in the plasma membrane and within organelles such as the endoplasmic reticulum, mitochondria, and nuclei. Organelles and intracellular Ca2þ binding proteins only transiently buffer cytosolic increases. The majority of intracellular Ca2þ regulation is accomplished


by extrusion of Ca2þ into the extracellular space by the concerted efforts of Ca2þ-ATPases and Naþ/Ca2þ exchangers [1,2]. Uptake of Ca2þ into the mitochondria and endoplasmic reticulum is an additional mode of buffering intracellular Ca2þ. Increases in cytosolic Ca2þ concentrations result from release of Ca2þ from internal stores in the endoplasmic reticulum through leak channels and by extracellular influx of Ca2þ through a variety of membrane channels that include voltage-sensitive calcium channels (VSCCs), voltage-insensitive calcium channels (VICCs), mechanosensitive divalent cation channels (MDCCs) and receptor-operated calcium channels (ROCs). Plasma membrane Ca2þ channel activity is modulated by various mechanisms including the influence of calcitropic hormones such as 1,25(OH)2D3, parathyroid hormone (PTH), and by mechanical stimuli. VSCCs are major regulators of Ca2þ permeability in osteoblasts and are the major Ca2þ channels expressed in the plasma membrane of these cells [3].

VOLTAGE-SENSITIVE CALCIUM CHANNELS VSCCs are ubiquitous, multimeric complexes that regulate Ca2þ influx in response to membrane depolarization. VSCCs regulate numerous intracellular processes including contraction, secretion, neurotransmission, and gene expression in excitable tissues and in most nonexcitable cell types. Several tissues involved in the vitamin D endocrine system express VSCCs,

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including the kidney [4], intestine [5], and bone [3,6]. Tight spatial and temporal regulation of Ca2þ influx [7,8] is achieved by the presence of multiple unique subtypes of VSCCs, each with differing pharmacological, kinetic, and electrophysiological properties. The highly regulated patterns of Ca2þ influx help balance the need for Ca2þ entry with the potential cytotoxic effects of high intracellular Ca2þ levels such as may occur around active sites of bone remodeling, when Ca2þ stores are being solubilized from the bone reservoir. VSCCs first were purified from skeletal muscle transverse tubules [9]. Solubilization and purification of the channel complex initially revealed three subunits, a1, b, and g with consensus sites for cAMP phosphorylation present within the a1 and b subunits [9]. Later analysis demonstrated the presence of covalently linked fourth and fifth subunits from a common precursor that associate together termed a2d [10]. A model was produced based on a detailed analysis of protein sequences, hydropathicity, and glycosylation sites of the five VSCC subunits that included a transmembrane a1 subunit associated in a complex with the intracellular b subunit, a transmembrane g subunit, and a disulfidelinked a2d dimer [11e13]. A diagram of this general structure is shown in Figure 25.1. The a1 subunit is the largest of the complex consisting of an approximate mass of 175 kDa and has been the focus of numerous functional studies because it incorporates the Ca2þ conduction pore and can generate a Ca2þ current in the absence of other subunits [14]. The a1 subunit is the putative binding site for three classes of organic Ca2þ channel blockers and is regulated by various second messengers and toxins [15,16]. Similar to the a subunit of Naþ channels, the a1 subunit of VSCCs contains four homologous domains (IeIV), with six transmembrane segments (S1eS6) in each. The fourth transmembrane segment of each domain serves as the voltage sensor and the pore loop between segments S5 and S6 determines ion conductance and selectivity. Interestingly, a change of only three amino acids in the pore loops of domains I, II, and IV converts a Naþ channel to Ca2þ selectivity [15]. Also similar to the topology of Naþ channels, the a1 subunit of VSCCs have both the short amino-terminal and long carboxyterminal segments positioned on the intracellular or cytoplasmic face where they are accessible for modification during signal transduction [17e20]. The a1 subunits of VSCCs are encoded by at least ten distinct genes [20,21] and historically there have been various names given to the corresponding gene products. These subunits first were named by a lettering system starting with a1S for the original skeletal muscle isoform and a1A through a1E for those discovered subsequently [22]. This arbitrary nomenclature frequently led to confusion; therefore, a more rational naming system

was adopted [23] based on the well-defined potassium channel nomenclature [24]. Under the new system, calcium channels were identified using the chemical symbol of the primary permeating ion (Ca) with the principal physiological regulator (voltage) indicated as a subscript (Cav). The gene subfamily of each a1 subunit is denoted with a number (currently 1 to 3) and the order of discovery of the a1 subunit within that subfamily (1 to n). Thus the VSCC subfamily responsible for mediating L-type Ca2þ currents is labeled Cav1.1eCav1.4, which includes channels formerly known as a1S, a1C, a1D, and a1F respectively. The Cav2 subfamily mediates P/Qtype, N-type, and R-type currents and are labeled Cav2.1eCav2.3 respectively. T-type Ca2þ currents are mediated by Cav3 subfamily channels (Cav3.1e3.3), which include those formerly known as a1G, a1H, and a1I, respectively [15]. Amino acid sequences of a1 subunits share greater than 70% similarity within a subfamily, but are less than 40% identical among the three subfamilies [15]. This variation on a common structure demonstrates how these Ca2þ permeating subunits may regulate a variety of specialized events in varied systems and yet also share a number of common features. The b subunit has an approximate molecular weight of 56 kDa and is highly and dynamically phosphorylated in vitro. The lack of a membrane-spanning segment suggests an intracellular orientation of this subunit and pulse chase analysis demonstrates the ability of b subunits to be palmitoylated, which aids in membrane association [25]. Additionally, b subunits generate a motif termed the beta interacting domain (BID), identified as the minimum sequence necessary to influence a1 subunit expression and binding to the a1 subunit [26,27]. Genes encoding four distinct b subunits have been identified in rabbit brain. The activation and inactivation kinetics of a1 subunits expressed in Xenopus oocytes, in the absence of b subunits, are significantly reduced compared to native cell preparations [16]. Normal channel activity is nearly fully restored following coexpression of recombinant b subunits, demonstrating the ability of these intracellular subunits to regulate channel kinetics, voltage-dependent gating properties, and channel density. The b subunit also plays a role in trafficking the a1 subunit to the membrane [28], reportedly by the masking of an ER retention sequence on the a1 subunit [29]. In osteoblasts, the b2 isoform is found in a complex with the L-type, Cav1.2 subunit, and with a large cytoplasmic protein ahnak. This association forms a stable complex that permits Ca2þ signaling independently of the cytoskeleton [30]. The ability of different b subunits to associate with the a1 subunit allows for calcium channels with diverse electrophysiological properties [16,25,31e34].


VOLTAGE-SENSITIVE CALCIUM CHANNELS

459 FIGURE 25.1 General model of voltage-

gated calcium channel structure showing subunit organization. Note that the poreforming a1 subunit is complemented by the exteriorly disposed a2d subunit and the cytoplasmic b subunit. The g subunit is not always present. The a2d subunit is positioned such that it can interact with the ECM. The b subunit is intracellularly disposed such that it can be modified during signaling such as occurs following exposure to 1,25(OH)2D3 (see text). Figure modified with permission from Dooley et al. [109].

The a2d subunits are encoded as a single gene product, which is post-translationally cleaved and subsequently joined together by a disulfide bond [35]. The reduced form of this subunit yields a 143-kDa a2 and 27-kDa d polypeptide [13,36]. The a2 subunit is extensively glycosylated [13] and is positioned extracellularly, while the d subunit, with a single transmembrane domain, resides in the plasma membrane. There are four distinct genes that encode for the a2d subunit. Osteoblastic cells express the a2d1 and a2d3 isoforms [37]. The extracellular a2 subunit promotes assembly and trafficking [38] of the a1 subunit on the plasma membrane, and the ability of a2d to modulate a1-induced current is enhanced by coexpression with the b subunit [35,39e43]. These properties demonstrate that similar to the b subunit, the a2d subunit can regulate VSCC current amplitude [16,39,40]. Additionally, the a2d1 isoform facilitates spreading, migration, and attachment of myoblasts, suggesting the ability of this

subunit to form attachments to the extracellular matrix (ECM) [44]. Similar interactions of the external face of VSCCs, in particular the a2d subunit, with ECM components likely occur in other components of connective tissue, including bone. The g subunit is the last of the peptides that forms the native high-voltage activated (HVA) calcium channel complex. This highly glycosylated subunit is approximately 35 kDa and contains several hydrophobic regions consistent with membrane-bound proteins [45]. The function of this auxiliary subunit remains largely unknown and, although purification studies have not yet revealed a g subunit within cardiac tissue, a gsubunit-like cDNA has been isolated from cardiac mRNA by PCR cloning [2,16]. In bone, our earlier studies showed that osteoblastic cells did not have a classic g subunit [37], however we cannot exclude the presence of those other rarer g subunits discovered since the time that study was published.


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Calcium currents may be generated with only the a1 subunit; however, in the absence of auxiliary subunits, these channels display lower levels of membrane expression and altered kinetics and voltage dependence compared to native channels [14]. The presence of b subunits increases membrane trafficking while shifting the voltage dependence of activation and inactivation to more negative membrane potentials and increasing the rate of inactivation [11]. Coexpression of the a2d subunit enhances expression of the channel and confers normal gating properties [28]. The g subunit creates a negative shift of the voltage dependence of inactivation resulting in altered peak currents and a reduction in channel availability [46]. This is similar to that effect of 1,25(OH)2D3 on calcium currents in osteoblasts, discussed in the following section.

1,25-DIHYDROXYVITAMIN D3 ACTIONS ON VOLTAGE-SENSITIVE CA2D CHANNELS Calcium homeostasis is modulated by 1,25(OH)2D3, altering bone remodeling through a primary effect on regulation of osteoblast function. Resorptive signals generated by osteoblasts in response to 1,25(OH)2D3 alter osteoclast activity to ultimately favor bone resorption. Feedback between these two processes through paracrine regulation at sites of bone remodeling allows 1,25(OH)2D3 to function as a metabolic “switch” that maintains a balance of cellular activities and preserves bone mass in normal bone. Biological responses are generated by 1,25(OH)2D3 through two mechanisms, regulation of gene transcription and by rapid, membrane-associated events. Altered gene transcription occurs following long-term (hours to days) treatment with 1,25(OH)2D3 by binding the nuclear vitamin D receptor (nuclear VDR) [47e52]. Production of several noncollagenous matrix proteins, including osteopontin is stimulated by 1,25(OH)2D3 in mice [49]. Additionally, 1,25(OH)2D3 down-regulates osteocalcin (OCN) [50] and parathyroid hormone (PTH) production [51,52]. The most well-characterized cellular response to 1,25(OH)2D3 is the binding of 1,25(OH)2D3 to the nuclear VDR, translocation to the nucleus, interaction with coactivators, and modulation of gene expression, processes discussed in great detail in Chapters 7e13 of this volume. However, the rapidly initiated actions of 1,25(OH)2D3 have become more greatly appreciated and commonly accepted to occur in many cells and tissues. Membrane-initiated actions have been observed among other steroid hormones including estrogen [53,54], glucocorticoids [55], androgens [56,57], and progesterone [58], thus the role of 1,25(OH)2D3 in membraneinitiated events has warranted study in bone. Rapid

responses of 1,25(OH)2D3 have been associated with the induction of protein kinase C [59], phospholipase C [60], activation of the phosphatidylinositide-30 kinase/AKT (PI3K/AKT) pathway [61], exocytosis [62], and ATP secretion [63] in osteoblasts, phosphorylation of matrix proteins including osteopontin (OPN) [64], and modulation of intracellular Ca2þ levels [65]. Previous studies using microarray analysis demonstrated altered gene expression through nuclear VDRdependent and independent pathways in osteoblasts following treatment with 1,25(OH)2D3 [66]. Many genes lacking vitamin D response elements in their proximal promoters including stress response proteins, transcription factors, and various matrix proteins demonstrated changes in expression as early as 3 h following treatment with 1,25(OH)2D3. While it is possible that some of these responded owing to the presence of more distant response elements, these observations, along with 1,25(OH)2D3 responsiveness in nuclear VDR-free membranes, suggest the presence of separate nuclear and membrane receptors for 1,25(OH)2D3, both of which can alter gene transcription [66]. A potential membrane receptor for 1,25(OH)2D3 has been identified called 1,25D3-MARRS (membrane-associated rapid response to steroids) [67e70]. An antibody generated against the N-terminal sequence of a component of the membrane vitamin D response system in other tissues to functionally block this 64.5-kDa receptor, inhibited intracellular Ca2þ signaling mediated by 1,25(OH)2D3 [71e73]. Additionally, Wali and colleagues demonstrate that the nuclear VDR is not required for the rapid actions of 1,25(OH)2D3 in mouse osteoblasts [74]. These data indicate that 1,25D3-MARRS is one player that is involved in initiating rapid signals in response to 1,25(OH)2D3, perhaps by virtue of its ability to alter the activities of signaling molecules such as protein kinase C, which also modulate VSCC activities. Recently, Nemere and colleagues demonstrated that knockout (KO) mice lacking the 1,25D3-MARRS receptor exhibited blunted or completely absent Ca2þ uptake in response to 1,25(OH)2D3 in intestinal epithelial cells. Additionally, intestinal cells isolated from 1,25D3-MARRS KO mice show significantly diminished PKA activity in response to 1,25(OH)2D3 treatment [75]. Interestingly, Ca2þ responses following activation of the plasma membrane vitamin D receptor have been associated with altered gene expression, similar to that seen with prostaglandin treatment [59,66,76]. The rapid transcriptional effects of 1,25(OH)2D3 are related to Ca2þ influx rather than activation of the nuclear VDR, because changes in gene transcription are observed within 3 h following treatment with 1,25(OH)2D3 or with Ca2þ mobilizing vitamin D3 analogs [66]. Many of the rapid actions of 1,25(OH)2D3 are negated with the addition of Ca2þ channel blockers, including the ability to modify


1,25-DIHYDROXYVITAMIN D3 ACTIONS ON VOLTAGE-SENSITIVE CA2þ CHANNELS

the phosphorylation state of OPN [64], demonstrating that the activity of plasma membrane Ca2þ channels is necessary for membrane-initiated actions of 1,25(OH)2D3. A rapid and transient increase in intracellular Ca2þ is observed in some cell types following addition of 1,25(OH)2D3 [77e79]. Intracellular Ca2þ elevation is dependent on the influx of Ca2þ through the plasma membrane and by release from internal stores [79]. A transient local elevation of intracellular Ca2þ can be elicited by low nanomolar concentrations of 1,25(OH)2D3; however, supraphysiological levels are necessary to promote release of Ca2þ from intracellular stores and yield measurable Ca2þ transients [77]. Several signaling pathways are activated upon binding of 1,25(OH)2D3 to the membrane vitamin D response system. In rat osteoblastic cells, 1,25(OH)2D3 stimulates VSCC activity resulting in activation of the PI3K/Akt pathway [62]. Electrophysiological studies have shown that the primary mechanism of Ca2þ influx in osteoblastic cells is through the L-type VSCC, which demonstrates a prolonged open time in response to 1,25(OH)2D3 treatment [3]. Addition of the L-type agonist Bay K 8644 or the 1,25(OH)2D3 analog AT (25-hydroxy-16-ene-23-yne-D3), which does not bind the nuclear VDR but does result in increases in Ca2þ influx [80], increases Ca2þ influx in osteoblasts and shifts the threshold of activation towards the resting potential, an event termed “left shift” [3,81]. A “left shift” of the resting membrane potential predicts that osteoblastic plasma membrane VSCCs are more susceptible to opening or are “primed” for the action of other hormones acting through ROCs or in response to small membrane depolarizations such as by activation of VICCs or MDCCs. This has been shown to be the case

461

for interactions between 1,25(OH)2D3 and PTH in terms of generating calcium signals in osteoblastic cells and cultured bony calvaria [81]. If one considers the osteoblast to be a polarized cell as has been well demonstrated [82], then these hormonal interactions with VSCCs occur on the cellular face distal from the face attached to the mineralized matrix. This places the Ca2þ signaling face of the osteoblast near the blood supply, in an ideal location to sense physiological levels of circulating Ca2þ. The physiological outcomes in response to a “left shift” are apparent in the coordinated actions of 1,25(OH)2D3 and PTH. PTH stimulates Ca2þ influx in osteoblastic cells through gadolinium-sensitive Ca2þ channels and the resulting local depolarization can influence neighboring VSCCs [83]. Osteoblastic cell cultures pretreated with low nanomolar concentrations of 1,25(OH)2D3 for 10 min results in enhanced PTHinduced Ca2þ influx associated with increased bone resorption rates [81,84]. This indicates that 1,25(OH)2D3 serves a priming function to augment PTH-induced Ca2þ influx at the plasma membrane. Addition of the L-type VSCC inhibitor nitrendipine or removal of extracellular Ca2þ, inhibits the elevation of 1,25(OH)2D3 and PTH-induced Ca2þ influx [81], demonstrating that the enhanced PTH-induced Ca2þ influx is dependent on the presence and influx of Ca2þ through the L-type VSCC [84]. A diagram illustrating the concerted actions of calcitropic hormones in modifying the calcium permeability of the osteoblast plasma membrane is shown in Figure 25.2, where it is shown that the overall conductance of the channel is increased in the presence of 1,25(OH)2D3, which then allows for a greater degree of Ca2þ entry in the presence of a second calcitropic signal such as provided by PTH.

Calcitropic regulation of voltage-sensitive (VSCC) and voltage-insensitive (VICC) calcium channels in the plasma membrane of the osteoblast. The 1,25(OH)2D3 first serves to sensitize the VSCC channel by moving the threshold of activation toward the resting potential, a process referred to as “left shift.” Small amounts of Ca2þ enter the cell during this phase, insufficient to trigger Ca2þ release from stores. Subsequent activation of neighboring VICCs by PTH triggers a local depolarization that is sensed by the left-shifted VSCC to allow Ca2þ entry to occur. This Ca2þ entry (dark arrow) then is able to trigger release of Ca2þ from internal stores, resulting in full-fledged Ca2þ transient and physiological response. See text for details and references.

FIGURE 25.2


462


1,25-DIHYDROXYVITAMIN D3REGULATED OSTEOBLAST DIFFERENTIATION Osteoblast differentiation is influenced by interactions with the ECM [85] and is regulated by mechanical [86] and hormonal signals [87]. Differentiation of mesenchymal precursors to osteoblasts is accompanied by increases in collagen type I, alkaline phosphatase (ALP) [88], and fibronectin (FN) [89,90]. Addition of 1,25(OH)2D3 to human osteoblastic cells increased expression of FN and enhanced cell-associated ALP activity, followed by altered morphology with cells containing more cytoplasmic processes [87], demonstrating the ability of 1,25(OH)2D3 to influence osteoblast differentiation. Previous studies have definitively determined that the L-type VSCC Cav1.2 is the primary subunit responsible for Ca2þ entry into the proliferating osteoblast [3,91] and that treatment with 1,25(OH)2D3 increases the mean open time of the L-type VSCC and enhances

permeability by shifting the threshold of activation toward the resting potential [3,84]. Blockage of the Ltype VSCC using benidipine promotes differentiation in murine preosteoblast cells [92], consistent with the idea that markers of osteoblast differentiation are enhanced following a loss of L-type membrane currents. Interestingly application of 1,25(OH)2D3 for 24e48 h to rat osteoblastic cells reduced L-type VSCC mRNA levels and increased markers of differentiation including OPN and OCN [93]. Previous studies also have demonstrated that terminally differentiated osteocytes express the T-type Cav3.2, and little or none of the L-type Cav1.2 VSCC subunit [94]. Figure 25.3 shows the distribution of expression of Cav1.2 and Cav3.2 in developing prenatal murine bone (Shao and Farach-Carson, unpublished). While Cav1.2 is present in rapidly growing cells present in both bone and cartilage at this stage of development, Cav3.2 is largely absent from cartilage, but is richly expressed by well-differentiated osteoblasts and by entrapped osteocytes [94]. Collagen X staining is strong in the hypertrophic zone of cartilage, separating FIGURE 25.3 Expression of calcium channels in developing bone. (A) Expression of L-type VSCC Cav1.2 in developing long bone (red). Note expression in cartilaginous growth plate, in bone marrow cells amongst trabecular bone spicules, and in both cortical and trabecular bone. (B) Expression of T-type VSCC Cav3.2 (red). Note more restricted expression with absence in the growth plate where only nuclei (green) can be seen. Also note absence in bone marrow. Staining (red) is seen at sites of osteocyte entrapment. (C) Staining of collagen X (red), specific to the hypertrophic zone separating the growth plate and the marrow cavity. Note that the yellow color results from overlap of the VSCC stain and the nuclea stain. Please see color plate section.


INFLUENCE OF 1,25(OH)2D3 ON OSTEOBLAST CELL SURVIVAL

the chondrocytic compartment of the growth plate from trabecular bone and bone marrow. Note down-regulation of staining for Cav1.2 in the hypertrophic zone where cells are not dividing. This is consistent for a role for Cav1.2 in growth and cell division, and for Cav3.2 in post-proliferative cells. Strong data suggest that 1,25(OH)2D3 plays a key role in the dynamic changes in VSCC expression during bone development [3,93]. Treatment of murine preosteoblasts for 24 hours with 1,25(OH)2D3 coordinately down-regulates L-type Cav1.2 and uniquely up-regulates T-type Cav3.2 VSCC expression at both the mRNA and protein levels [95]. Of the ten VSCC a1 subunits assayed, only the T-type Cav3.2 subunit was up-regulated. Application of 1,25(OH)2D3 also alters the Ca2þ permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity [95]. The physiological roles of T-type VSCCs are well characterized in neuronal [96] and cardiac cells [97], where T-type currents result in low-threshold Ca2þ spikes. These Ca2þ spikes are associated with burst firing and oscillatory behavior [98], leading to rapid neuronal depolarization and generation of pacemaker currents. Mice null for the T-type, Cav3.2 subunit are viable, but demonstrate focal coronary and arteriole defects as well as skeletal abnormalities [99] (Kronsberg A, Duncan RL, and Farach-Carson MC, in preparation). Up-regulation of the T-type VSCC Cav3.2 subunit following 1,25(OH)2D3 treatment suggests the need to compensate for the loss of L-type VSCC-mediated Ca2þ influx. If a subsequent up-regulation of another VSCC a1 subunit did not occur following the 1,25(OH)2D3-mediated down-regulation of the L-type Cav1.2, the ability of the cell to maintain Ca2þ-dependent cellular processes would be compromised. Alterations in Ca2þ permeability following 1,25(OH)2D3 application in osteoblasts demonstrate a drastic shift from L-type sensitivity to T-type sensitivity. One likely explanation for this change is that the high Ca2þ permeability in the preosteoblast supports cell proliferation and growth, whereas the depletion of Ca2þ permeability during 1,25(OH)2D3-stimulated differentiation attenuates Ca2þ-dependent proliferation signals and related gene expression. These alterations likely occur in parallel with the down-regulation of L-type Cav1.2 expression in terminally differentiated osteocytes [94]. Interestingly, treatment of human osteoblastic cells with 1,25(OH)2D3 results in a transition to a more stellate morphology with increased cytoplasmic processes [87], a phenotype characteristic of terminally differentiated osteocytes [100]. Additionally, the lipid growth factor lysophosphatidic acid (LPA) is a potent stimulator of osteocyte dendrite outgrowth and treatment of the osteocytic cell line MLO-Y4 results in

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a more stellate morphology [101]. The LPA receptor antagonist Kil6425 and pertussin toxin blocked the LPA-induced increase in dendrite formation [101]. Further studies demonstrated that 1,25(OH)2D3-induced osteoblast differentiation was inhibited in the presence of the Kil6425 and that 1,25(OH)2D3 and LPA act synergistically to generate mature osteoblasts, which was inhibited by pertussis toxin [102]. Increases in ALP in cells cotreated with LPA and 1,25(OH)2D3 were blocked with Y-27632, an inhibitor of Rho-associated coiled kinase (ROCK). Taken together, these data suggest that the alterations in osteoblast differentiation initiated by 1,25(OH)2D3 that alter VSCC expression may be augmented by cooperative actions of LPA [102].

INFLUENCE OF 1,25(OH)2D3 ON OSTEOBLAST CELL SURVIVAL Hormonal regulation of bone cell apoptosis has been implicated as a crucial mechanism to control osteoblast to osteoclast cell ratios, and thus the state of remodeling and mineralization of bone [61]. Several studies have demonstrated the ability of 1,25(OH)2D3 to promote cell survival in osteoblasts [61,103,104]. Various pathways have been shown to be influenced by application of 1,25(OH)2D3 to osteoblasts including activation of the PI3/Akt survival pathway [61] and inhibition of the proapoptotic Fas pathways [105]. Chronic elevations in intracellular Ca2þ are well known to participate in the initiation of apoptosis [106]. Application of 1,25(OH)2D3 for seconds to minutes increases the mean open time and Ca2þ permeability of the L-type VSCC [3,107]. Treatment of primary osteoblast cultures with 1,25(OH)2D3 produces an increase in Ca2þ entry that, along with subsequent release of Ca2þ from intracellular stores, elevates cytoplasmic Ca2þ levels [77,78]. Treatment of osteoblasts with 1,25(OH)2D3 results in down-regulation of the Cav1.2 VSCC subunit mRNA transcription and protein production [95]. Transcript levels of other L-type VSCC a1 subunits remained unchanged following application of 1,25(OH)2D3, indicating that the Cav1.2 subunit is the primary L-type pore-forming subunit whose transcriptional regulation is modulated by 1,25(OH)2D3 in osteoblasts. Prolonged exposure of osteoblasts to 1,25(OH)2D3 also revealed diminished Ca2þ entry through the L-type VSCC by radioactive 45Ca2þ influx assays, most likely because of decreased Cav1.2 expression [95]. We propose that a rational role for down-regulation of the Cav1.2 subunit in response to long-term 1,25(OH)2D3 treatment is to protect the cell from chronic increases in intracellular Ca2þ levels that could result in apoptosis. Hippocampal neurons react similarly in that neuronal vulnerability to excitotoxicity is mediated by Ca2þ influx


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through the L-type VSCC, and down-regulation of these channels following long-term exposure to 1,25(OH)2D3 enhances neuroprotection [108]. These studies demonstrate that short- and long-term exposure to 1,25(OH)2D3 have distinct and sometimes opposite effects. The initial application of 1,25(OH)2D3 elicits a rapid cellular response, including activation of various protein kinases, protein lipases, and cAMP, by increasing Ca2þ influx through the L-type Cav1.2 subunit. In contrast, long-term exposure down-regulates the Cav1.2 subunit resulting in diminished Ca2þ entry, preventing Ca2þ toxicity. The previously described shift from L-type VSSC currents to T-type currents during differentiation from osteoblasts to osteocytes also may have a role in protection of these terminally differentiated bone cells. The L-type channel has a conductance of ~25 pS, whereas the T-type VSCC has a much reduced conductance of ~8 pS. The ability of osteoblasts to simultaneously decrease L-type Cav3.2 levels and increase expression of the T-type Cav3.2 subunit in the presence of 1,25(OH)2D3 [95], along with the observation that osteocytes predominantly express the T-type Cav3.2 subunit [94], together suggest that the decreased current potential in osteocytes may protect these terminally differentiated cells from toxic levels of Ca2þ. Ongoing studies in our laboratory are designed to further test these ideas. Ultimately, we believe that a complete understanding of the responses of osteoblasts to 1,25(OH)2D3 will create new avenues to design pharmacological interventions that will maintain bone mass in the elderly, perhaps while also preserving function of other excitable tissues such as nerve and muscle that express high levels of calcium channels.

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Acknowledgments The authors would like to thank our long-term collaborator, Dr. Randall L. Duncan, for many helpful discussions and for sharing unpublished data of voltage-sensitive channel knockouts. We also thank Jason M. Koons for his assistance in the preparation of the figures. We especially thank Dr. Ying Shao for contributing her images for Figure 25.3. William Thompson was funded in part by a Florence Kendall Scholarship and a Promotion of Doctoral Studies II training fellowship from the Foundation for Physical Therapy and an Adopt-A-Doc Scholarship from the American Physical Therapy Association Section on Geriatrics.

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[71] Z. Jia, I. Nemere, Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. Chick kidney and brain, Steroids 64 (8) (1999) 541e550. [72] H.A. Pedrozo, Z. Schwartz, S. Rimes, V.L. Sylvia, I. Nemere, G.H. Posner, et al., Physiological importance of the 1,25(OH) 2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3, J. Bone Miner. Res. 14 (6) (1999) 856e867. [73] I. Nemere, D. Larsson, K. Sundell, A specific binding moiety for 1,25-dihydroxyvitamin D(3) in basal lateral membranes of carp enterocytes, Am. J. Physiol. Endocrinol. Metab. 279 (3) (2000) E614eE621. [74] R.K. Wali, J. Kong, M.D. Sitrin, M. Bissonnette, Y.C. Li, Vitamin D receptor is not required for the rapid actions of 1,25dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts, J. Cell Biochem. 88 (4) (2003) 794e801. [75] I. Nemere, N. Garbi, G.J. Hammerling, R.C. Khanal, Intestinal cell calcium uptake and the targeted knockout of the 1,25D3MARRS (membrane associated, rapid response steroid binding) receptor/PDIA3/Erp57. J. Biol. Chem. [76] A.M. Choi, R.W. Tucker, S.G. Carlson, G. Weigand, N.J. Holbrook, Calcium mediates expression of stressresponse genes in prostaglandin A2-induced growth arrest, FASEB J. 8 (13) (1994) 1048e1054. [77] R. Civitelli, Y.S. Kim, S.L. Gunsten, A. Fujimori, M. Huskey, L.V. Avioli, et al., Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells, Endocrinology 127 (5) (1990) 2253e2262. [78] M. Lieberherr, Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts, J. Biol. Chem. 262 (27) (1987) 13168e13173. [79] G. Vazquez, J. Selles, A.R. de Boland, R. Boland, Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study, Br. J. Pharmacol. 126 (8) (1999) 1815e1823. [80] R. Khoury, A.L. Ridall, A.W. Norman, M.C. Farach-Carson, Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx, Endocrinology 135 (6) (1994) 2446e2453. [81] W. Li, R.L. Duncan, N.J. Karin, M.C. Farach-Carson, 1,25 (OH) 2D3 enhances PTH-induced Ca2þ transients in preosteoblasts by activating L-type Ca2þ channels, Am. J. Physiol. 273 (3 Pt 1) (1997) E599eE605. [82] C.V. Gay, V.R. Gilman, T. Sugiyama, Perspectives on osteoblast and osteoclast function, Poult. Sci. 79 (7) (2000) 1005e1008. [83] R.L. Duncan, K.A. Hruska, S. Misler, Parathyroid hormone activation of stretch-activated cation channels in osteosarcoma cells (UMR-106.01), FEBS Lett. 307 (2) (1992) 219e223. [84] W. Li, M.C. Farach-Carson, Parathyroid hormone-stimulated resorption in calvaria cultured in serum-free medium is enhanced by the calcium-mobilizing activity of 1,25dihydroxyvitamin D(3), Bone 29 (3) (2001) 231e235. [85] A.M. Moursi, C.H. Damsky, J. Lull, D. Zimmerman, S.B. Doty, S. Aota, et al., Fibronectin regulates calvarial osteoblast differentiation, J. Cell Sci. 109 (Pt 6) (1996) 1369e1380. [86] S. Kapur, D.J. Baylink, K.H. Lau, Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways, Bone 32 (3) (2003) 241e251. [87] R.T. Franceschi, W.M. James, G. Zerlauth, 1 Alpha, 25dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line, J. Cell Physiol. 123 (3) (1985) 401e409.


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[88] B.U. Steinmann, A.H. Reddi, Changes in synthesis of types-I and -III collagen during matrix-induced endochondral bone differentiation in rat, Biochem. J. 186 (3) (1980) 919e924. [89] R.E. Weiss, A.H. Reddi, Synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentiation of cartilage and bone in vivo, Proc. Natl. Acad. Sci. USA 77 (4) (1980) 2074e2078. [90] R.E. Weiss, A.H. Reddi, Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow, J. Cell. Biol. 88 (3) (1981) 630e636. [91] R. Liu, W. Li, N.J. Karin, J.J. Bergh, K. Adler-Storthz, M.C. Farach-Carson, Ribozyme ablation demonstrates that the cardiac subtype of the voltage-sensitive calcium channel is the molecular transducer of 1, 25-dihydroxyvitamin D(3)stimulated calcium influx in osteoblastic cells, J. Biol. Chem. 275 (12) (2000) 8711e8718. [92] Y. Nishiya, N. Kosaka, M. Uchii, S. Sugimoto, A potent 1,4dihydropyridine L-type calcium channel blocker, benidipine, promotes osteoblast differentiation, Calcif. Tissue Int. 70 (1) (2002) 30e39. [93] J.G. Meszaros, N.J. Karin, K. Akanbi, M.C. Farach-Carson, Down-regulation of L-type Ca2þ channel transcript levels by 1,25-dihyroxyvitamin D3. Osteoblastic cells express L-type alpha1C Ca2þ channel isoforms, J. Biol. Chem. 271 (51) (1996) 32981e32985. [94] Y. Shao, M. Alicknavitch, M.C. Farach-Carson, Expression of voltage sensitive calcium channel (VSCC) L-type Cav1.2 (alpha1C) and T-type Cav3.2 (alpha1H) subunits during mouse bone development, Dev. Dyn. 234 (1) (2005) 54e62. [95] J.J. Bergh, Y. Shao, E. Puente, R.L. Duncan, M.C. Farach-Carson, Osteoblast Ca(2þ) permeability and voltage-sensitive Ca(2þ) channel expression is temporally regulated by 1,25-dihydroxyvitamin D(3), Am. J. Physiol. Cell Physiol. 290 (3) (2006) C822eC831. [96] R. Llinas, Y. Yarom, Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro, J. Physiol. 315 (1981) 569e584. [97] J.P. Benitah, A.M. Gomez, J. Fauconnier, B.G. Kerfant, E. Perrier, G. Vassort, et al., Voltage-gated Ca2þ currents in the human pathophysiologic heart: a review, Basic. Res. Cardiol. 97 (Suppl. 1) (2002) I11eI18. [98] J.R. Huguenard, Low-threshold calcium currents in central nervous system neurons, Annu. Rev. Physiol. 58 (1996) 329e348.

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[99] C.C. Chen, K.G. Lamping, D.W. Nuno, R. Barresi, S.J. Prouty, J.L. Lavoie, et al., Abnormal coronary function in mice deficient in alpha1H T-type Ca2þ channels, Science 302 (5649) (2003) 1416e1418. [100] L.F. Bonewald, Establishment and characterization of an osteocyte-like cell line, MLO-Y4, J. Bone Miner. Metab. 17 (1) (1999) 61e65. [101] S.A. Karagiosis, N.J. Karin, Lysophosphatidic acid induces osteocyte dendrite outgrowth, Biochem. Biophys. Res. Commun. 357 (1) (2007) 194e199. [102] J. Gidley, S. Openshaw, E.T. Pring, S. Sale, J.P. Mansell, Lysophosphatidic acid cooperates with 1alpha,25(OH)2D3 in stimulating human MG63 osteoblast maturation, Prostaglandins Other Lipid Mediat. 80 (1-2) (2006) 46e61. [103] A.M. Vertino, C.M. Bula, J.R. Chen, M. Almeida, L. Han, T. Bellido, et al., Nongenotropic, anti-apoptotic signaling of 1alpha,25(OH)2-vitamin D3 and analogs through the ligand binding domain of the vitamin D receptor in osteoblasts and osteocytes. Mediation by Src, phosphatidylinositol 3-, and JNK kinases, J. Biol. Chem. 280 (14) (2005) 14130e14137. [104] O. Morales, M.K. Samuelsson, U. Lindgren, L.A. Haldosen, Effects of 1alpha,25-dihydroxyvitamin D3 and growth hormone on apoptosis and proliferation in UMR 106 osteoblast-like cells, Endocrinology 145 (1) (2004) 87e94. [105] G. Duque, K. El Abdaimi, J.E. Henderson, A. Lomri, R. Kremer, Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways, Bone 35 (1) (2004) 57e64. [106] A. Verkhratsky, Calcium and cell death, Subcell Biochem. 45 (2007) 465e480. [107] X.T. Wang, S. Nagaba, Y. Nagaba, S.W. Leung, J. Wang, W. Qiu, et al., Cardiac L-type calcium channel alpha 1-subunit is increased by cyclic adenosine monophosphate: messenger RNA and protein expression in intact bone, J. Bone Miner Res. 15 (7) (2000) 1275e1285. [108] L.D. Brewer, V. Thibault, K.C. Chen, M.C. Langub, P.W. Landfield, N.M. Porter, Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons, J. Neurosci. 21 (1) (2001) 98e108. [109] D.J. Dooley, C.P. Taylor, S. Donevan, D. Feltner, Ca2þ channel alpha2delta ligands: novel modulators of neurotransmission, Trends Pharmacol. Sci. 28 (2) (2007) 75e82.



S E C T I O N I V

TARGETS


C H A P T E R

26 Vitamin D and the Kidney Peter Tebben, Rajiv Kumar Departments of Medicine, Biochemistry and Molecular Biology, Divisions of Nephrology, and Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic and Foundation, Rochester, Minnesota, 55905, USA

INTRODUCTION The kidney has a unique function in mineral homeostasis and plays a vital role in the control of plasma calcium and phosphorus. While examining how the kidney controls calcium and phosphate homeostasis it is worthwhile to keep the following facts in mind. 1. In humans, in a 24-hour period, about 8 grams of calcium are filtered at the glomerulus, and about 7.8 grams are reabsorbed in the proximal and distal tubules and the loop of Henle [1e5]. This is carried out in a manner such that, under normal circumstances, i.e. in states of neutral calcium balance, the amount of calcium in the urine closely approximates that absorbed in the intestine. The mechanisms by which calcium is reabsorbed in the kidney are complex and yield several insights into the cellular regulation of calcium transport. 2. The reabsorption of calcium in the kidney is controlled by several factors such as the filtered load of sodium, urine flow, and the activity of several hormones, most notably, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D) and calcitonin, in addition to others [1e5]. 3. The kidney is the major site of synthesis of 1,25 (OH)2D, the active, hormonal form of vitamin D [6,7]. 4. The kidney expresses several 1,25(OH)2D-dependent proteins such as the plasma membrane calcium pump (PMCa), the epithelial calcium channel (ECaC), the sodium calcium exchanger, and the calbindins, some of which play a vital role in calcium transport. 5. The kidney expresses the vitamin D receptor (VDR). 6. The kidney expresses 25-hydroxyvitamin D3 1ahydroxylase (1a-hydroxylase or CYP27B1 gene) and 1,25-dihydroxyvitamin D3-24-hydroxylase (24-hydroxylase or CYP24A1 gene), and other


1,25-dihydroxyvitamin D3 and vitamin D analog metabolizing enzymes [6,7]. 7. Finally, the kidney has an equally important role in the control of plasma phosphate, and the filtration and reabsorption of phosphate. As in the case of calcium, the reabsorption and secretion of phosphate are under hormonal control and many of the same hormones and factors involved in calcium regulation, play a significant role in the regulation of phosphate reabsorption [8e13]. Given the complex role of the kidney in calcium and phosphate homeostasis, a brief overview of calcium and phosphate reabsorption in the kidney is in order.

Calcium Handling by the Kidney About 55% of total plasma calcium is ultrafilterable [14]. The ultrafilterable calcium concentration is about 1.35 mM (5.4 mg/dl) and closely approximates the concentrations of calcium present in glomerular fluid [15e17]. The total amount of calcium filtered at the glomerulus in a 24-hour period is about 8000 mg. Approximately 98% of the filtered load of calcium is reabsorbed in the tubules. Thus, the amount of calcium excreted in the urine in a 24-hour period is about 150e200 mg [1,14]. In the proximal tubule, about 50e60% of the filtered load of calcium is reabsorbed [2,3,18]. The reabsorption of calcium is thought to occur as a result of solvent drag by a paracellular route and is sodium-dependent. Volume expansion and a reduction of tubular sodium reabsorption inhibits calcium reabsorption, while volume contraction and an increase in sodium reabsorption enhance calcium reabsorption [2,19]. Inhibition of sodiumepotassium ATPase activity and sodium reabsorption by ouabain reduce the amount of calcium

471


472

26. VITAMIN D AND THE KIDNEY

reabsorbed as does the substitution of sodium with lithium [19]. The concentration of calcium at the end of the proximal tubule is similar to that in the glomerular fluid. Importantly, calcium reabsorption in the proximal tubule is not influenced by thiazide diuretics, hormones such as PTH or 1,25(OH)2D3, or by hydrogen ions [2,3,18,19]. As we shall discuss later, some vitaminD-dependent proteins such as the calbindins, ECaC, and PMCa pump are either not expressed in the proximal tubule or are expressed in low amounts when compared with the amounts expressed in the distal tubule. Calcium reabsorption in the descending loop and the thin ascending limb of the loop of Henle is minimal. In the thick ascending limb of the loop of Henle about 20% of the filtered load of calcium is reabsorbed while another 10e15% is reabsorbed in the distal tubule with the remaining 5% being reabsorbed in the collecting ducts [2,3,18,19]. There are important distinctions between the factors influencing calcium reabsorption in the proximal tubule and the mechanisms of calcium reabsorption in the distal segments of the nephron. Firstly, the movement of calcium in the distal nephron occurs against a concentration gradient (lumen relative to extracellular fluid). Secondly, the lumen of the tubule is electronegative and becomes progressively more so towards the end of the distal tubule. Thirdly, calcium reabsorption can be dissociated from sodium reabsorption by thiazide diuretics which inhibit sodium reabsorption but enhance calcium reabsorption. Fourthly, hydrogen ions inhibit calcium reabsorption in the distal tubule whereas they have no effect on calcium reabsorption in the proximal tubule.

Phosphate Handling by the Kidney Virtually all inorganic phosphate in the serum is filtered by the glomerulus [8e10]. About 80% of filtered phosphorus is reabsorbed in the kidney, mostly in the proximal tubule. The amount of phosphorus reabsorbed in the proximal tubule is greatest in the first half of the proximal tubule and exceeds that of sodium. There is evidence for further phosphorus reabsorption in the pars recta. Little or no phosphorus reabsorption occurs in the loop of Henle or the distal tubule although there is some debate about whether there is phosphorus reabsorption in the distal tubule. The reabsorption of phosphate is sodium-dependent and is mediated by sodiumephosphate cotransporters (Na-Pi IIa,SLC34A1; Na-Pi IIc, SLC34A3, and Pit-2, SLC20A2) [20,21]. Na-Pi IIa activity is increased by a low-phosphate diet and decreased by PTH [22e25]. The recently described phosphatonins, fibroblast growth factor-23 (FGF-23) and secreted frizzled related protein-4 (sFRP-4) are also able to inhibit sodium-dependent phosphate transport [13,26]. In opossum kidney (OK) cells, NaPi II is

TABLE 26.1 Factors that Alter Renal Phosphate Excretion Increase

Decrease

1. High-phosphate diet 2. Parathyroid hormone 3. Calcitonin 4. Chronic vitamin D 5. Glucagon 6. Glucocorticoids 7. Volume expansion 8. Increased pCO2 9. Chronic acidosis 10. Starvation 11. Diuretics 12. “Phosphatonin” FGF-23 sFRP-4

1. 2. 3. 4. 5. 6. 7. 8.

Low-phosphate diet Thyro-parathyroidectomy Thyroxine Acute vitamin D Insulin Growth hormone Volume contraction Decreased pCO2

internalized from the cell membrane in response to FGF-23 and sFRP-4, similar to the effects of PTH [27,28]. Additional factors involved in phosphorus reabsorption are noted in Table 26.1.

ROLE OF THE KIDNEY IN THE METABOLISM OF 25(OH)D Formation of 1,25(OH)2D3 25-Hydroxyvitamin D3-1a-hydroxylase is a multicomponent, cytochrome P-450-containing enzyme in the mitochondria of renal proximal tubular cells [29e35]. The central role of the kidney in the formation of 1,25(OH)2D3 was first noted by Fraser and Kodicek, who demonstrated that nephrectomy abolished the formation of 1,25(OH)2D3 [36,37]. This was subsequently confirmed by others [38,39]. Nephrectomy greatly decreases circulating 1,25(OH)2D3 concentration in vivo except during pregnancy, granuloma-forming diseases, and lymphomas associated with the ectopic production of 1,25(OH)2D3 [40e43]. While the kidney is the major site of 1,25(OH)2D3 production, 25-hydroxyvitamin D3-1a-hydroxylase activity has been found in several other cell types throughout the body [44e49]. In vitro, chick renal epithelial cells in culture, mammalian nephron segments and homogenates derived from avian and mammalian (mostly rodent) renal cells all appear to metabolize 25(OH)D3 to 1,25(OH)2D3 [50e54]. For some time the proximal tubule was felt to be the only site of 1,25(OH)2D3 synthesis in the kidney. However, there is evidence that other tubular segments synthesize 1,25(OH)2D3 [55,56]. Using immunohistochemistry and in situ hybridization techniques, Zehnder et al. have demonstrated 1a-hydroxylase mRNA and

IV. TARGETS

EFFECTS OF VITAMIN D, 25(OH)D3 AND 1,25(OH)2D3 ON THE RENAL HANDLING OF CALCIUM AND PHOSPHORUS

protein in the distal convoluted tubule, cortical collecting duct, thick ascending limb of the loop of Henle, and Bowman’s capsule [56]. Recent experiments in which the 25-hydroxyvitamin D 1a-hydroxylase cytochrome P450 gene (CYP27B1) was deleted in mice point to the central role of this enzyme in vitamin D metabolism [57]. Table 26.2 summarizes some of the key factors known to regulate the activity of this enzyme in vivo and in vitro. The major regulators appear to be PTH, inorganic phosphorus, and 1,25(OH)2D3 itself. Regulators such as 1,25-dihydroxyvitamin D3 which alter the expression of the CYP27B1 gene have reciprocal effects on the expression of the CYP24A1 gene [6] that are mediated via vitamin-D-responsive elements in the promoters of the respective genes [58,59].

24-Hydroxylase Activity in the Kidney The 24-hydroxylase enzyme is a multicomponent cytochrome P450 enzyme expressed in the kidney as well as many other tissues [60e78]. In the kidney, 24-hydroxylase is expressed primarily in the proximal TABLE 26.2 Effect of Increased Level or Activity of Various Factors on 1,25(OH)2D3 Concentration or 1ahydroxylase Activity Factor

Animals

Humans

Ref.

Parathyroid hormone

[

[

[17,52,257e265]

Serum inorganic phosphorus

Y

Y

[255,266e268]

1,25(OH)2D3

Y

Y

[257,269]

Calcium (direct)

?

Y

[270,271]

Calcitonin

[,Y,0

[

[14,52,257,258,272,273]

Hydrogen ion

Y

0

[259,274,275]

Sex steroids

[

[

[254,276]

Prolactin

[

0

[277e279]

Growth hormone and insulin-like growth factor-1

[

[,Y,0

[184,271,280e285]

Glucocorticoids

Y,0

[,Y,0

[136,286e289]

Thyroid hormone

?

Y*

[290e292]

Fibroblast growth factor 23

Y

?

[293,294]

Frizzled related protein 4

Y

?

[13]

Pregnancy

[

[*

[295,296]

* Effects may be secondary to changes in calcium, phosphorus or parathyroid hormone. (With permission, modified from Kumar R [6].) [, Stimulation or increase; Y, suppression or decrease; 0, no effect; ?, effect not known.

TABLE 26.3

25(OH)D3

473

The Metabolism of 25(OH)D3 by the Kidney 24R,25(OH)2D3

24-keto-25(OH)D3

25S,26(OH)2D3

25(OH)D3-lactone

23S,25(OH)2D3

23-keto-25(OH)D3

tubule but is also present in more distal segments [79,80]. It is responsible for the conversion of 25hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3, respectively. There is conflicting evidence whether 25(OH)D3 or 1,25(OH)2D3 is the preferred substrate for 24-hydroxylase [81]. Some have reported 1,25(OH)2D3 is the preferred substrate with a Km approximately tenfold lower than that for 25(OH)D3 [82,83] while others have found Km values substantially lower for 25(OH)D3 [84]. It has been suggested that these metabolites have certain unique properties and actions [85e88], however, others have not confirmed these observations [89e95]. The 24-hydroxylase enzyme activity and mRNA expression in the kidney is up-regulated by 1,25 (OH)2D3 [96e102]. The effect of 1,25(OH)2D3 is blunted in vitro and in vivo by parathyroid hormone [96e99]. The kidney is also capable of transforming 25(OH)D3 to several other compounds listed in Table 26.3 [103e123]. The specific physiological roles of these various metabolites are not known with certainty. Several polar metabolites of 1,25(OH)2D3 are formed in the liver, including calcitroic acid and glucuronide and sulfate conjugates of the hormone; these and small amounts of unchanged dihydroxylated and trihydroxylated metabolites of vitamin D are excreted in the urine [124e138]. Many of the tranformations that occur with 25(OH)D3 also occur in the case of 1,25(OH)2D3.

EFFECTS OF VITAMIN D, 25(OH)D3 AND 1,25(OH)2D3 ON THE RENAL HANDLING OF CALCIUM AND PHOSPHORUS Clinical studies have shown that vitamin D deficiency is associated with low urine calcium concentration whereas vitamin D excess or intoxication is associated with hypercalciuria [1]. The concentrations of calcium and phosphorus in the urine in these in vivo situations, however, reflect the decreased or increased calcium absorption in the intestine, the presence of hypo- or hypercalcemia, and the presence of diminished or elevated concentrations of circulating PTH. Puschett et al. examined the effect of vitamin D3 and 25(OH)D3

IV. TARGETS

474


on the renal transport of phosphate, sodium, and calcium in parathyroidectomized dogs [139e141]. They showed that short-term infusions of vitamin D3 and 25 (OH)D3 were associated with decreases in the clearances of phosphate, calcium, and sodium relative to the clearance of inulin. These studies were interpreted as showing that vitamin D3 and 25(OH)D3 enhance phosphate, calcium, and sodium reabsorption. A follow-up study by Puschett et al. showed that 1,25(OH)2D3 had similar effects on the excretion of phosphate, calcium, and sodium in thyroparathyroidectomized (TPTX) dogs [142]. We performed similar studies examining the effects of 25(OH)D3 on renal bicarbonate and phosphate reabsorption [143]. Unlike the studies of Puschett et al., we observed that phosphate and bicarbonate reabsorption increased only in intact animals and not in parathyroidectomized animals, suggesting the need for PTH. Our observations are similar to those of Popovtzer et al. [144]. Yamamoto et al. carried out perhaps the most comprehensive examination of the effects of 1,25 (OH)2D3 on the reabsorption of calcium [145]. VitaminD-deficient rats, vitamin-D-deficient rats supplemented with dietary calcium to normalize plasma calcium and PTH levels, and vitamin-D-replete rats were examined following TPTX and the infusion of graded amounts of calcium. Figure 26.1 shows the relationship between the amount of calcium excreted in the urine and serum calcium concentrations in the three groups of animals. Urinary calcium excretion was lower in vitamin-Dreplete rats than in vitamin-D-deficient rats, suggesting that vitamin D administration increased the efficiency of renal calcium reabsorption in the absence of PTH. In a second group of experiments, rats treated in the manner noted above were TPTX and infused with PTH. The results of this experiment show that a lower dose of PTH is needed to exhibit a comparable effect on renal calcium reabsorption in vitamin-D-replete rats when compared to vitamin-D-deficient rats (Fig. 26.2). This finding may be explained by in vitro studies of distal convoluted tubule cells in which 1,25(OH)2D3 increased PTH/PTHrp receptor mRNA levels [146]. Winaver et al. used micropuncture to examine the sites along the nephron at which 25(OH)D3 exerted its antiphosphaturic and hypocalciuric effects [147]. They observed that the effects of 25(OH)D3 were not mediated at the level of the superficial proximal tubule but were likely to have an effect on other segments of the nephron, most likely the distal tubule. These studies are similar to those of Sutton et al. and others who observed distal tubular effects of 25(OH)D3 in the dog [148]. These effects occurred shortly after the administration of 25(OH)D3 and were likely to have been independent of conversion to 1,25(OH)2D3 although this cannot be entirely ruled out since amounts of 25(OH) D3 and 1,25(OH)2D3 were not measured. Harris and

FIGURE 26.1 Relationships between urinary calcium excretion and serum calcium concentration in three groups of thyroparathyroidectomized (TPTX) rats. Serum concentration and urinary excretion of calcium were determined 16e19 h after continuous infusion of an electrolyte solution containing 0e30 mM of CaCl2. Each point represents the data pooled according to a continuous series of 0.25mM changes in serum calcium concentration. Horizontal bars indicate standard error of mean serum calcium concentration, and vertical bars indicate standard error of mean urinary calcium excretion. The lines were derived from the regression analysis of the linear portion of data. (:): Group A rats fed vitamin-D-deficient standard diet. (B): Group B rats fed vitamin-D-deficient diet containing high calcium and lactose. (•): Group C rats fed vitamin-D-replete standard diet. For any given serum calcium level, the urinary calcium excretion was significantly lower in vitamin-D-replete rats (group C) than in vitamin-Ddeficient rats (groups A and B). Thus, the apparent serum calcium threshold determined as an intercept of the regression line on the serum calcium axis was higher in vitamin-D-replete rats (~1.5 mM) than in vitamin-D-deficient rats (~1.0 mM). There was no significant difference in the calcium threshold between group A and group B. (With permission from Yamamoto M et al. [145].)

Seely examined the effects of 1,25(OH)2D3 on tubular calcium handling and concluded that the enhanced reabsorption of calcium occurred in distal tubular segments of the nephron [149]. All of these studies do not exclude an effect of the vitamin D analogs on a segment of the proximal tubule that is not accessible to micropuncture but are consistent with the localization of many vitamin-D-responsive proteins exclusively in the distal nephron. In vitro studies have shown that vitamin D deficiency is associated with decreased calcium uptake in membranes derived from the distal segments of the

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DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY

475

FIGURE 26.2 The effects of PTH infusion on the renal handling of calcium among three groups of TPTX rats. The data are presented as described in the legend to Fig. 26.1. PTH was delivered at 2.5 U/h (•) on groups A and B rats, and 0.75 U/h (B) on group C rats. A, B, and C illustrate the results in group A, group B, and group C, respectively. The enhancement of calcium reabsorption by PTH is shown as the shifts of the lines to the right in each group. The striking difference exists between vitamin-D-deficient (groups A and B) and vitamin-D-replete (group C) rats in the doses of PTH required to induce a comparable shift in the calcium threshold. , data of TPTX rats in each group (from Fig. 26.1). (With permission from Yamamoto M et al. [145].)

nephron [150]. This occurs in membranes of both luminal and basolateral origin. In addition, Bindels et al. have demonstrated that cultured rabbit connecting tubule cells show an increase in the transport of calcium when exposed to 1,25(OH)2D3 [151]. Protein and mRNA expression of the recently described epithelial calcium channels (ECaC1 and ECaC2 or TRPV5 and TRPV6) are diminished in rats fed a diet deficient in vitamin D [152e154]. TRPV5 protein is present in the apical membrane of the distal convoluted tubule and is responsible for uptake of calcium from the tubule fluid into the cell [154,155]. TRPV6 is localized to the principal cells of the cortical and medullary collecting ducts and is also regulated by 1,25(OH)2D3 [153,154]. 1,25 (OH)2D3 increases mRNA and protein expression of the PMCa pump which is involved with active transport of calcium through the basolateral membrane of distal tubule cells [156]. A synthesis of the experimental results suggest that vitamin D3 metabolites have effects on the distal tubular reabsorption of calcium through several mechanisms. Expression of proteins responsible for distal tubule fluid uptake, intracellular trafficking, and basolateral transport of calcium are responsive to vitamin D3. Figure 26.3 depicts the transcellular transport of calcium through a distal tubule cell.

DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY Several vitamin-D-dependent proteins are expressed in the kidney and many of these play a role in calcium

and phosphate transport. The VDR, calbindins, PMCa pump, and the ECaCs (TRPV5 and TRPV6) are all found in renal tubule cells and act coordinately in the regulation of calcium transport in the nephron [5,18,146,152,154,157e163]. We will discuss pertinent information regarding these vitamin-D-dependent proteins. Additional vitamin-D-responsive proteins involved in renal calcium and phosphate transport are listed in Table 26.4.

1,25-Dihydroxyvitamin D3 Receptor (VDR) in the Kidney The VDR mediates many, if not all, of the effects of 1,25(OH)2D3 in diverse organs [164e172]. The distribution of the VDR in the kidney has been assessed using a variety of techniques including ligand-binding assays with protein obtained from specific microdissected nephron segments, the localization of radiolabeled 1,25 (OH)2D3 by autoradiography, and by the use of various antibody techniques [79,173e177]. Using protein from microdissected nephron segments, the VDR was found in proximal and distal tubules [79,175]. With autoradiographic methods, following the administration of labeled ligand in vivo, silver grains were localized over distal tubule segments [79,174]. We have used sensitive polyclonal antibodies to localize the receptor in human and rat kidneys [161]. These antibodies were raised against highly purified antigen that was expressed in bacteria. The specificity of these antibodies was determined by absorption with purified antigen and by Western analysis which showed that they detected a protein band with a molecular mass of approximately 50 000 [161,178]. With these antibodies,

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476


FIGURE 26.3 Integrated model of active Ca2þ reabsorption in the distal part of the nephron. Apical entry of Ca2þ is facilitated by ECaC, Ca2þ

then binds to calbindin-D28K, and this complex diffuses through the cytosol to the basolateral membrane, where Ca2þ is extruded by an Naþ/Ca2þ exchanger and a plasma membrane Ca2þ-ATPase. The individually controlled steps in the activation process of the rate-limiting Ca2þ entry channel include 1,25(OH)2D3-mediated transcriptional and translational activation, shuttling to the apical membrane, and subsequent activation of apically located channels by ambient Ca2þ concentration, direct phosphorylation and/or accessory proteins. (With permission from Hoenderop et al. [250].)

we found that the VDR was present abundantly in the distal tubule and to a lesser extent in the proximal tubule (Fig. 26.4). Cells expressing calbindin-D28K also express the calcium pump and the epithelial calcium channel [152,179]. Interestingly, not all cells in the distal tubule expressed the receptor. Acid-secreting cells do not express the VDR in significant amounts. Taken together, the results are consistent with the notion that the VDR is present in significant amounts in the distal tubule where it regulates the amount and the activity of several vitamin-D-dependent proteins such as the plasma membrane calcium pump, epithelial calcium channel, and calbindin D28K. Although in lesser amounts, the

TABLE 26.4

Vitamin D Responsive Proteins in the Kidney

Vitamin D receptor 24-Hydroxylase Plasma membrane calcium pump Epithelial calcium channels, TRPV5 and TRPV6 Calbindin D28K Calbindin D9K Calcium sensing receptor PTH/PTHrp receptor Sodiumephosphate cotransporter type 2

proximal tubule also expresses the VDR where it regulates the activity of 1a-hydroxylase and 24-hydroxylase. We have shown that the VDR is present in cells of the developing rodent kidney [180] (Figs 26.5 and 26.6) and in the cultured metanephros (Fig. 26.7). The VDR was detected as early as day 15 post-coitum (p.c.) in the developing rat kidney in vivo. Significant amounts of the receptor were found in the metanephric mesenchyme as well as in the ureteric bud. As the kidney matured, the receptor was observed in the S-shaped and comma-shaped bodies and in the developing glomerulus, specifically in the parietal and visceral endothelial cells. The VDR staining in the latter cells persists in the adult kidney as well. Despite slightly different gestational periods, similar patterns of VDR were found in the developing mouse kidney in vivo. Of great interest is the observation that calbindin D28K appears in the distal tubule only around day 18 p.c. This is when urine flow begins in the kidney. The VDR is also present in mouse metanephric cultures in the same distribution pattern as is found in vivo. By day 3 of culture, the pattern of expression of the VDR was similar to that seen in the mouse kidney in vivo at day 15. Calbindin D28K does not appear in the cultured metanephros. These results showing that the VDR appears well before the appearance of calbindin D28K suggest that it may play a role in fetal renal development. Additional evidence for the regulation of calbindin D28K and calbindin D9K expression in the kidney can be found in

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(A)

(D)

(G)

(B)

(E)

(H)

(C)

(F)

(I)

477

FIGURE 26.4 (AeC) Immunohistochemical detection of VDR in normal human kidney tissue with polyclonal anti-hVDR antibody 2-152 (A, 200; B, 400; C, 400). (DeF) Immunohistochemical detection of 25(OH)D3 24-hydroxylase cytochrome P-450 in human kidney (D 200; E, 400; F, 400). (G) Immunohistochemical detection of calbindin D28k in human kidney (G, 400). (H and I) Control panels showing kidney tissue stained with preimmune serum (H, 200; I, 400). (With permission from Kumar R et al. [161].) Please see color plate section.

VDR knockout mice where calbindin expression is reduced [181]. The VDR is regulated by several factors in diverse tissues [182]. Concentrations of the VDR in the kidney and parathyroid glands are mainly regulated by 1,25 (OH)2D3, PTH, and dietary calcium. Studies have shown that somewhat different results are obtained in vivo with respect to VDR abundance when 1,25(OH)2D3 concentrations are altered by dietary manipulations when compared to the effects of the intravenous administration of the hormone [183,184]. Exogenous administration of 1,25(OH)2D3 in rats increases VDR levels in duodenal and renal tissues [183,184]. When endogenous 1,25(OH)2D3 concentrations are increased by adapting an animal to a low-calcium diet (Table 26.5), VDR concentrations in the duodenum and kidney do not increase. The difference appears to be due to increases in the levels of PTH elicted by the low-calcium diet and decreased PTH following 1,25(OH)2D3 administration. Differences in CYP24A1 expression (decreased in the presence of a low-calcium diet, and increased following the administration of 1,25(OH)2D3) might also contribute to this difference in VDR expression. PTH has been shown to down-regulate VDR in osteosarcoma cells as well as block up-regulation of VDR in rats infused with both PTH and 1,25(OH)2D3 [185]. These

results demonstrate the opposing effects of PTH and 1,25(OH)2D3 on VDR expression in the kidney. The results obtained from in vivo studies are consistent with those obtained following the administration of 1,25(OH)2D3 to yeast cells transfected with a VDR construct [186]. There is evidence that in certain cells such as fibroblasts, 1,25(OH)2D3 may have its predominant effect to increase VDR abundance by ligandinduced stabilization of the protein [187]. Wiese et al. showed that the addition of 1,25(OH)2D3 to fibroblasts did not result in a significant increase in VDR mRNA concentrations but did result in increases in the amount of VDR protein suggesting that the predominant effect of 1,25(OH)2D3 in fibroblasts is the stabilization of preexisting VDR protein or the stabilization of preexisting vitamin D receptor mRNA. In addition to the VDR, several vitamin-D-dependent proteins are expressed in the kidney. The pattern of regulation of these proteins is of great interest, in as much as it casts light on the different mechanisms by which calcium is transported in the kidney. Those proteins involved in calcium transport include: calbindin D28K and calbindin D9K, the plasma membrane calcium pump, the epithelial calcium channel, and the calcium-sensing receptor. Additionally, although not involved in the transport of calcium, the 24-hydroxylase

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478


(A)

(A)

(B)

(B)

(A) Vitamin D receptor (VDR) immunostaining in metanephros of rat fetus on gestational day 15. (B) Higher-power view of boxed area in (A). Branching ureteric buds (arrows) and mesenchyme (M) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.

FIGURE 26.5

and the sodiumephosphate type 2 cotransporter (NaPi 2) are also expressed in the kidney. All of these proteins appear to be regulated by 1,25(OH)2D3.

Calbindins-D The calbindins-D are widely distributed in many tissues of the body and in a variety of species. Except in the brain, their synthesis is dependent on vitamin D but not calcium or phosphorus. There are two forms of calbindin-D, namely, calbindin-D28K and calbindinD9K that are variably distributed in different tissues of the body [188e205]. The apparent molecular weight of the larger protein is about 30 000 daltons whereas that of the smaller protein is 9000 daltons [188]. The proteins are classical EF hand proteins, the calbindinD28K having six EF hand structures and the calbindinD9K having two such motifs [206e225]. The proteins bind calcium with high affinity and in different molar amounts. Calbindin D28K binds 3e4 moles of calcium per mole of protein and calbindin D9K two moles of

FIGURE 26.6 VDR (A) and calbindin D28k (B) immunostaining in metanephros of rat fetus on gestational day 17. Parietal epithelial cells (small arrows), visceral epithelial cells (open arrows), and tubule portion of developing comma-shaped body (T) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.

calcium per mole of protein [188]. In the mouse kidney, both forms are present and are regulated by vitamin D. In other species, only calbindin-D28K is expressed in the kidney. Both proteins undergo conformational change upon binding to calcium. This phenomenon has been studied extensively by us in the case of calbindinD28K. We have shown that the protein undergoes a two-step change in conformation that is associated with binding to a high-affinity site in EF-hand 1 and a further change upon calcium binding to sites in EF hands -4 and -5 [215,217,226]. We recently solved the structure of Ca2þ-loaded calbindin-D28K [227]. The protein is comprised of a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2þ in loops on EF1, EF3, EF4, and EF5. Ranbinding protein M, myo-inositol monophosphatase, and procaspase-3-derived peptides interact with the protein on a surface comprised of alpha5 (EF3), alpha8 (EF4), and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2þ. The

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479


(A)

(B)

FIGURE 26.7 VDR immunostaining in mouse metanephric organ culture explant following 120 h of incubation. Parietal epithelial cells (small arrow), visceral epithelial cells (large arrow), proximal tubule (P), and distal tubule (D) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.

TABLE 26.5

conformation change is probably what allows the protein to act as a modulator of the activity of Ranbinding protein M, myo-inositol monophosphatase, procaspase-3, the plasma membrane calcium pump, TRPV5, and TRPV6 channels (see below). Calbindin D28K and calbindin-D9K are regulated by 1,25(OH)2D3 in the kidney [219e225,228]. Both calbindins have lower expression in 1a-hydroxylase knockout mice [229]. Calbindin D28K and calbindinD9K expression was normalized by treatment with 1,25(OH)2D3, however, only calbindin D28K expression is increased when 1a-hydroxylase knockout mice were fed a high-calcium diet. In a mouse VDR-KO model, renal calbindin D9K expression is nearly abolished whereas calbindin D28K expression returns to control values as the animals age [230]. Cao et al. demonstrated that calbindin-D9K induction by 1,25 (OH)2D3 in vitro is absent in VDR-null cells [231]. Furthermore, calbindin D9K regulation by 1,25 (OH)2D3 was restored after transfection of the VDRnull cells with human VDR clearly showing the effect of 1,25(OH)2D3 is mediated through the VDR. In vitro, PTH has a synergistic effect with 1,25(OH)2D3 on calbindin D9K expression but has no effect alone [231]. In contrast, calbindin D28K expression is enhanced by PTH infusion without elevations in 1,25(OH)2D3 concentrations [232]. Using distal tubule membranes, preincubation with calbindin D28K increased calcium uptake in luminal membranes while calbindin D9K preincubation increased calcium uptake in basolateral membranes [233,234]. Calbindin D28K knockout mice fed a high-calcium (1%) diet have an elevated urinary

Effect of Dietary Calcium on Unoccupied VDR Content in Rat Duodenum and Kidney Day Diet

2

7

14

21

1% calcium

341 26

197 17

202 17

259 26

0.02% calcium

365 27

226 28

221 16

267 28

1% calcium

ND

163 11

165 9

124 8

0.02% calcium

ND

120 4*

131 10*

77 3*

1% calcium

9.98 0.18

9.82 0.08

9.16 0.16

9.52 0.41

0.02% calcium

9.45 0.12

9.15 0.17*

9.09 0.14

8.70 0.29*

1% calcium

8.40 0.17

8.88 0.22

8.73 0.17

7.92 0.29

0.02% calcium

8.15 0.22

8.65 0.13

8.37 0.28

8.41 0.23

1% calcium

153 11

113 13

139 9

160 32

0.02% calcium

180 20

392 45**

682 44**

829 59**

>

VDR

Duodenum

Kidney

>

Calcium (mg/dL)

>

Phosphorus (mg/dL)

1,25-(OH)2D> 3

(pg/mL)

>

Values are mean SEM. Unoccupied vitamin D receptor content expressed as fmols [3H]1,25(OH)2D3 bound per mg cytosol protein. * P < 0.05; ** P < 0.001. ND ¼ not done. Modified from Goff JP et al. [183].

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480


calcium/creatinine ratio despite no difference in serum calcium or PTH compared to wild-type littermates [235,236]. However, the elevated urinary calcium/ creatinine ratio in calbindin D28K knockout mice was not apparent after fasting [235]. This is consistent with our finding that fractional excretion of calcium is not altered in calbindin D28K knockout mice fed a normalcalcium diet [237]. The effects of calbindin D28K on renal calcium conservation in mice are modest, perhaps because of compensatory increases in calbindin D9K. The interactions between vitamin D, PTH, calcium, and the calbindins appears to be quite complex. The exact mechanisms and their interactions with other calcium transport proteins is not yet completely understood.

The Plasma Membrane Calcium Pump We raised monoclonal and polyclonal antibodies directed against the plasma membrane calcium pump and used them to examine the distribution of these proteins in the adult human kidney [18, 157e160]. We found that epitopes for the calciume magnesium ATPase (calcium pump) were expressed in the basolateral membrane of the distal tubular cells (Fig. 26.8). Similar patterns of expression were apparent in the rat [158] and rabbit [238] kidney. Interestingly, not all cells of the distal tubule stained positively for the calcium pump. Further investigation showed that cells expressing carbonic anhydrase and presumably involved in acid secretion did not express the plasma membrane calcium pump whereas the other cells of the distal tubule did so. We found that the calbindin D28K was present in the same cells of the distal tubule as the plasma membrane calcium pump. Our studies on the distribution of the plasma membrane calcium pump in the kidney and its localization, predominantly in the distal tubule of the kidney are supported by others who have shown that calcium ATPases are present mostly in the distal tubule [238]. In addition, it appears that the plasma membrane calcium pump is widely distributed in a large number of other calcium transporting tissues many of which display vitamin-D-dependent calcium transport [239e245]. Table 26.6 shows the distribution of the plasma membrane calcium pump in different tissues. In MDBK (bovine distal tubule) cells, 1,25(OH)2D3 increases PMCa pump mRNA and protein [156]. Bouthinay et al. have examined the effects of vitamin D deficiency on the activity of the plasma membrane calcium pump [233,234]. They observed that vitamin D deficiency was associated with a decrease in PMCa pump activity in the distal tubule of the kidney. These same authors also observed that calbindin D9K increased the

activity of the renal basolateral membrane calcium pump. In collaboration with Wasserman’s group at Cornell University, we have shown that the plasma membrane calcium pump is regulated in the intestine by vitamin D [240,246,247]. We showed by Western analysis using monoclonal antibodies directed against the pump that shortly after the administration of 1,25(OH)2D3 to vitamin-D-deficient chicks, there is an increase in the amount of immunoreactive plasma membrane calcium pump in the cells of the duodenum, jejunum, and ileum [240]. This is associated with an increase in the amount of mRNA for the pump in the same segments of the intestine [246]. The increase occurs within 3e6 hours following the administration of 1,25(OH)2D3 and the effect is dose-dependent. Furthermore, dietary calcium and phosphorus depletion are also associated with an increase in the amount of the pump expressed in the intestine. Thus, in the intestine, 1,25(OH)2D3 increases the synthesis of the plasma membrane calcium pump. In addition, 1,25(OH)2D3 increases the activity of the plasma membrane calcium pump in intestinal cell basolateral membranes. The mechanism by which up-regulation of the calcium pump occurs in the kidney and intestine is uncertain although several possibilities arise. It could be a direct effect of either 1,25(OH)2D3 or via stimulation of calbindin D9k or calbindin D28k [233,234]. There is also evidence that stimulation of the calcium-sensing receptor (CaSR) decreases calcium absorption by inhibiting PMCa pump activity [248].

The Epithelial Calcium Channel, VanilloidReceptor-Related Transient Receptor Potential Channels 5 and 6 Hoenderop et al. described epithelial calcium channels (ECaCs) which are expressed in the apical membrane of the distal tubule and principal cells of the collecting duct and are distinct from previously described calcium channels [153,155,179,249,250]. There are at least two members in this family of calcium channels, ECaC-1/TRPV5 and ECaC-2/TRPV6 [250]. ECaC1/TRPV5 expression is limited to the kidney while ECaC2/TRPV6 is expressed in several other tissues [155,250e252]. The ECaCs/TRPV channels have six putative transmembrane-spanning domains including a pore-forming hydrophobic region between transmembrane domains 5 and 6 [249]. Several putative vitaminD-response elements (VDRE) have been identified within the promoter region of the human TRPV5 channel. Hoenderop et al. also demonstrated that ECaC1/TRPV5 mRNA and protein levels are increased to near control levels after vitamin D rescue in rats fed a vitamin-D-deficient diet [152]. In a study using

IV. TARGETS


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

481

Immunoperoxidase localization of Caþþ-Mgþþ ATPase within human kidney distal tubules and human spleen erythrocytes. Kidney: (a and b) Monoclonal antibody JA3. (c and d) Monoclonal antibody JA8. (e and f) Negative control. (g and h) Double stain, PAS, and JA3; (arrows) PASpositive proximal tubule brush border. Spleen: (i) Monoclonal antibody JA3. (j) Negative control. (a, c, e and g) 200. (b, d, f, h, i, and j) 640. (With permission from Borke et al. [157].) Please see color plate section.

FIGURE 26.8

1a-hydroxylase knockout mice, a greater than 50% reduction in ECaC-1/TRPV5 expression was found compared to control mice [229]. Additionally, renal ECaC-1/TRPV5 expression in 1a-hydroxylase KO mice is normalized after treatment with 1,25(OH)2D3 [229]. Similar findings were seen when examining calbindin D28K expression which colocalizes to the same distal tubule cells as ECaC-1/TRPV5 [179,229]. However, ECaC-1/TRPV5 is not regulated through vitamin D

effects on calbindin D28K. Calbindin D28K KO mice and cyclosporine A induced down-regulation of calbindin D28K has no effect on ECaC-1/TRPV5 expression [235]. Others have suggested that calcium also regulates ECaC-1 expression. Quantitative PRC techniques showed reduced expression of ECaC-1 in VDR KO mice compared to control mice. When fed high-calcium diets, VDR KO mice had normalization of ECaC-1 concentrations [252].

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482 TABLE 26.6


Distribution of Plasma Membrane Calcium Pump in Transporting Epithelia as Assessed by Immunohistochemistry

Tissue

Source

Cell type

Location in cell

Reference

Kidney

Rat, human

Distal convoluted tubule, principal cell

Basolateral

[18,157e160]

Intestine

Rat, chick

Absorptive cell

Basolateral

[239,240]

Trophoblast

Rat, human

Syncytiotrophoblast

Basal

[241]

Choroid plexus

Cat Human

Choroid plexus Secretory cell

Apical

[242]

Shell gland

Chick

Principal cell

Apical

[243]

Bone

Human

Osteoblast

Not vectorially oriented

[244]

Bone

Chick

Osteoclast

Not vectorially oriented

[245]

The 25-Hydroxyvitamin D3- and 1,25Dihydroxyvitamin D3-24-Hydroxylase The 24-hydroxylase enzyme is widely distributed in a number of renal and nonrenal tissues [70e78,87, 88,253]. Pioneering work by DeLuca’s laboratory showed that the renal 24-hydroxylase was regulated by calcium and phosphorus such that elevated or normal calcium levels induced the 24-hydroxylase whereas low calcium levels inhibited it [72e74,254]. Similarly, elevated serum phosphorus concentrations increased the synthesis of the 24-hydroxylase enzyme whereas low-phosphorus diets decreased the activity of the enzyme [255]. We have used antibodies against the 24-hydroxylase cytochrome P-450 to examine the distribution of the enzyme in the human kidney, and found exceptionally high concentrations of the cytochrome P-450 in distal tubular cells [161]. Lower amounts were found in the proximal tubule. Using enzymatic methods, several investigators have found 24-hydroxylase activity in kidney cells of the proximal tubule of the rat nephron. Iida et al. were unable to find 24-hydroxylase activity in microdissected distal tubule segment [256]. The reason for this apparent discrepancy between human and rat tissues is uncertain. Certainly, it would make biologic sense for the 24hydroxylase to be present in the distal tubule where other elements of the vitamin-D-responsive system are present. Other enzymes responsible for the transformation of 25-hydroxyvitamin D are also present in the kidney; they mediate the reactions shown in Table 26.3.

CONCLUSION The kidney plays a vital role in the conservation of calcium and phosphorus. Besides being the site of synthesis of 1,25(OH)2D3, the kidney responds to the hormone by increasing the efficiency of calcium and

phosphorus reabsorption. Elements of the calcium transport systems including calbindin D28K, calbindin D9K, the epithelial calcium channel, and the plasma membrane calcium pump all localize to the distal portion of the nephron and are regulated directly or indirectly by 1,25(OH)2D3.

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[267] T. Yoshida, N. Yoshida, T. Monkawa, M. Hayashi, T. Saruta, Dietary phosphorus deprivation induces 25-hydroxyvitamin D (3) 1alpha-hydroxylase gene expression, Endocrinology 142 (5) (2001) 1720e1726. [268] M.Y. Zhang, X. Wang, J.T. Wang, et al., Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alphahydroxylase gene expression in the proximal renal tubule, Endocrinology 143 (2) (2002) 587e595. [269] H. Kawashima, S. Torikai, K. Kurokawa, Calcitonin selectively stimulates 25-hydroxyvitamin D3-1 alpha-hydroxylase in proximal straight tubule of rat kidney, Nature 291 (5813) (1981) 327e329. [270] R. Bland, E.A. Walker, S.V. Hughes, P.M. Stewart, M. Hewison, Constitutive expression of 25-hydroxyvitamin D3-1alphahydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium, Endocrinology 140 (5) (1999) 2027e2034. [271] B. Lund, O.H. Sorensen, J.E. Bishop, A.W. Norman, Stimulation of 1,25-dihydroxyvitamin D production by parathyroid hormone and hypocalcemia in man, J. Clin. Endocrinol. Metab. 50 (3) (1980) 480e484. [272] N. Yoshida, T. Yoshida, A. Nakamura, T. Monkawa, M. Hayashi, T. Saruta, Calcitonin induces 25-hydroxyvitamin D3 1alpha-hydroxylase mRNA expression via protein kinase C pathway in LLC-PK1 cells, J. Am. Soc. Nephrol. 10 (12) (1999) 2474e2479. [273] N.D. Adams, R.W. Gray, J. Lemann Jr., The effects of oral CaCO3 loading and dietary calcium deprivation on plasma 1,25-dihydroxyvitamin D concentrations in healthy adults, J. Clin. Endocrinol. Metab. 48 (6) (1979) 1008e1016. [274] B. Sauveur, M. Garabedian, C. Fellot, P. Mongin, S. Balsan, The effect of induced metabolic acidosis on vitamin D3 metabolism in rachitic chicks, Calcif. Tissue Res. 23 (2) (1977) 121e124. [275] H.P. Weber, R.W. Gray, J.H. Dominguez, J. Lemann Jr., The lack of effect of chronic metabolic acidosis on 25-OH-vitamin D metabolism and serum parathyroid hormone in humans, J. Clin. Endocrinol. Metab. 43 (5) (1976) 1047e1055. [276] L. Castillo, Y. Tanaka, H.F. DeLuca, M.L. Sunde, The stimulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase by estrogen, Arch. Biochem. Biophys. 179 (1) (1977) 211e217. [277] N.D. Adams, T.L. Garthwaite, R.W. Gray, T.C. Hagen, J. Lemann Jr., The interrelationships among prolactin, 1,25dihydroxyvitamin D, and parathyroid hormone in humans, J. Clin. Endocrinol. Metab. 49 (4) (1979) 628e630. [278] R. Kumar, W.R. Cohen, F.H. Epstein, Vitamin D and calcium hormones in pregnancy, New Engl. J. Med. 302 (20) (1980) 1143e1145. [279] E. Spanos, K.W. Colston, I.M. Evans, L.S. Galante, S.J. Macauley, I. Macintyre, Effect of prolactin on vitamin D metabolism, Mol. Cell Endocrinol. 5 (3-4) (1976) 163e167. [280] P.C. Eskildsen, B. Lund, O.H. Sorensen, J.E. Bishop, A.W. Norman, Acromegaly and vitamin D metabolism: effect of bromocriptine treatment, J. Clin. Endocrinol. Metab. 49 (3) (1979) 484e486. [281] J.M. Gertner, R.L. Horst, A.E. Broadus, H. Rasmussen, M. Genel, Parathyroid function and vitamin D metabolism during human growth hormone replacement, J. Clin. Endocrinol. Metab. 49 (2) (1979) 185e188. [282] C. Menaa, F. Vrtovsnik, G. Friedlander, M. Corvol, M. Garabedian, Insulin-like growth factor I, a unique calciumdependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells, J. Biol. Chem. 270 (43) (1995) 25461e25467. [283] L. Condamine, C. Menaa, F. Vrtovsnik, F. Vztovsnik, G. Friedlander, M. Garabedian, Local action of phosphate

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C H A P T E R

27 Vitamin D and the Parathyroids Justin Silver, Tally Naveh-Many Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem 91120, Israel

INTRODUCTION The action of 1,25(OH)2D3 or its analogs to decrease PTH secretion is part of the management of all patients with chronic kidney disease in order to prevent or suppress their secondary hyperparathyroidism. There is ongoing academic and commercial activity in the development of drugs that may have more selective actions on the parathyroid whilst leading to less hypercalemia. These attempts are of great clinical and pharmaceutical interest but still remain to be proven by rigorous scientific testing and therefore despite the extensive literature on the subject, the final word on the analogs awaits prospective outcome studies [1,2] and are not discussed in detail in this chapter. However, there is discussion of the subject in Chapters 70 and 81. In this chapter we shall discuss the PTH gene and the regulation of PTH gene expression by 1,25(OH)2D3.

and cleavage occurs during protein synthesis, very little intact preproPTH is found within the parathyroid cell. Mature PTH has a molecular mass of approximately 9600 daltons and is the only form that is secreted from the parathyroid cell. The amino acid sequence has been determined in several species, and there exists a high degree of identity among species, particularly in the amino-terminal region of the molecule. The parathyroids synthesize another protein that is also secreted [5]. This protein, secretory protein I, is identical to chromogranin A isolated from the adrenal medulla, and it is present in other endocrine cells and neoplasms as well. Its function is not known, but it is stored and secreted with PTH despite its differential transcriptional regulation relative to PTH. A 26-kDa N-terminal fragment of chromogranin A (CgA) secreted by bovine parathyroid glands, when added to dispersed, parathyroid cells in primary culture, inhibited the low-calcium-stimulated secretion of both PTH and CgA, suggesting an autocrine or paracrine regulation of secretion [6].

PARATHYROID HORMONE BIOSYNTHESIS

THE PARATHYROID HORMONE GENE

Parathyroid hormone, a protein of 84 amino acids, is synthesized as a larger precursor, preproparathyroid hormone [3,4]. PreproPTH has a 25-residue “pre” or signal sequence, and a 6-residue “pro” sequence. The signal sequence, along with the short pro sequence, functions to direct the protein into the secretory pathway. Like other signal sequences, the pre sequence binds to a signal recognition particle during protein synthesis. The signal recognition particle then delivers the nascent peptide chain to the rough endoplasmic reticulum, where it is threaded through a protein-lined aqueous pore. During this transit, the signal sequence is cleaved off by a signal peptidase, and the pre sequence is rapidly degraded. Because the process of transport


The PTH Gene The human parathyroid hormone (PTH) gene is localized on the short arm of chromosome 11 at 11p15 [7,8]. The human and bovine genes have two functional TATA transcription start sites, and the rat only one. The two homologous TATA sequences flanking the human PTH gene direct the synthesis of two human PTH gene transcripts both in normal parathyroid glands and in parathyroid adenomas [9]. The PTH genes in all species that have been cloned, have two introns or intervening sequences and three exons [10]. Strikingly, even though fish do not have discrete parathyroid glands, they do synthesize PTH using two distinct genes that

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27. VITAMIN D AND THE PARATHYROIDS

share the same exoneintron pattern found in tetrapod PTH genes [11,12]. The locations of the introns are identical in each case [13]. Intron A splits the 50 untranslated sequence of the mRNA five nucleotides before the initiator methionine codon. Intron B splits the fourth codon of the region that codes for the pro sequence of preProPTH. The three exons that result, thus, are roughly divided into three functional domains. Exon I contains the 50 untranslated region. Exon II codes for the pre sequence, or signal peptide and exon III codes for PTH as well as the 30 untranslated region. It is interesting that the human gene is considerably longer in both intron A and the 30 untranslated region of the cDNA compared to the bovine, rat, and mouse. The genes for PTH and PTHrP (PTH-related protein) are located in similar positions on sibling chromosomes 11 and 12. It is therefore likely that they arose from a common precursor by chromosomal duplication.

The PTH mRNA Complementary DNA encoding for human [14,15], bovine [16,17], rat [18], mouse [19], pig [18], chicken [20,21], dog [22], cat [23], horse [24], macaca [25], fugu fish [11], and zebrafish [12] PTH have all been cloned [13]. The PTH gene is a typical eukaryotic gene with consensus sequences for initiation of RNA synthesis, RNA splicing, and polyadenylation. The primary RNA transcript consists of RNA transcribed from both introns and exons, and then RNA sequences derived from the introns are spliced out. The product of this RNA processing, which represents the exons, is the mature PTH mRNA, which will then be translated into preproPTH. There is considerable identity among mammalian PTH genes, which is reflected in an 85% identity between human and bovine proteins and 75% identity between human and rat proteins. There is less identity in the 30 noncoding region. A more extensive review of the structure and sequences of the PTH gene has been published elsewhere [13] and in the book, Molecular Biology of the Parathyroid [26].

DEVELOPMENT OF THE PARATHYROID AND TISSUE-SPECIFIC EXPRESSION OF THE PTH GENE The thymus, thyroid, and parathyroid glands in vertebrates develop from the pharyngeal region, with contributions both from pharyngeal endoderm and from neural crest cells in the pharyngeal arches. Studies of gene knockout mice have shown that the hoxa3, pax 1, pax 9, and Eya1 transcription factors are needed to form parathyroid glands as well as many other pharyngeal pouch derivatives, such as the thymus. Glial cells

missing2 (Gcm-2), a mouse homolog of Drosophila Gcm, is a transcription factor whose expression is restricted to the parathyroid glands [27]. A human patient with a defective Gcm B gene, the human equivalent of Gcm-2, exhibited hypoparathyroidism and complete absence of PTH from the bloodstream [28]. The parathyroid gland of tetrapods and the gills of fish both express Gcm-2 and require this gene for their formation [29]. They also showed that the gill region expresses mRNA encoding the two PTH genes found in fish, as well as mRNA encoding the calcium-sensing receptor.

PROMOTER SEQUENCES Regions upstream of the transcribed structural gene often determine tissue specificity and contain many of the regulatory sequences for the gene. For PTH, analysis of this region has been hampered by the lack of a parathyroid cell line. It has been shown that the 5 kb of DNA upstream of the start site of the human PTH gene was able to direct parathyroid-gland-specific expression in transgenic mice [30]. Analysis of the human PTH promoter region identified a number of consensus sequences by computer analysis [31]. These included a sequence resembling the canonical cAMPresponsive element 50 -TGACGTCA-30 at position e81 with a single residue deviation. This element was fused to a reporter gene (CAT) and then transfected into different cell lines. Pharmacological agents that increase cAMP led to an increased expression of the CAT gene, suggesting a functional role for the cAMP responsive element (CRE). Specificity protein (Sp) and the nuclear factor-Y (NF-Y) complex are thought to be ubiquitously expressed transcription factors associated with basal expression of a host of gene products. Sp family members and NF-Y can cooperatively enhance transcription of a target gene. There is a highly conserved Sp1 DNA element present in mammalian PTH promoters [32]. Coexpression of Sp proteins and NF-Y complex leads to synergistic transactivation of the hPTH promoter, with alignment of the Sp1 DNA element essential for full activation [32]. The presence of a proximal NF-Y-binding site in the hPTH promoter highlights the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription [33]. Several groups have identified DNA sequences that might mediate the negative regulation of PTH gene transcription by 1,25-dihydroxyvitamin D (1,25(OH)2D3). Demay et al. [34] identified DNA squences in the human PTH gene that bind the 1,25(OH)2D3 receptor (VDR). Nuclear extracts containing the VDR were examined for binding to sequences in the 50 -flanking region of the

IV. TARGETS

REGULATION OF PTH GENE EXPRESSION

hPTH gene. A 25-bp oligonucleotide containing sequences from e125 to e101 from the start of exon 1 bound nuclear proteins that were recognized by monoclonal antibodies against the VDR. The sequences in this region contained a single copy of a motif (AGGTTCA) that is homologous to the motifs repeated in the up-regulatory VDR response element (VDRE) of the osteocalcin gene. When placed up-stream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediated transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. Therefore, this down-regulatory element differs from up-regulatory elements both in sequence composition and in the requirement for particular cellular factors other than the VDR for repressing PTH transcription [34]. Russell et al. [35] have shown that there are two negative VDREs in the rat PTH gene. One is situated at e793 to e779 and bound a VDR/ RXR heterodimer with high affinity and the other at e760 to e746 bound the heterodimer with a lower affinity. Transfection studies with VDRE-CAT constructs showed that they had an additive effect. Liu et al. [36] have identified such sequences in the chicken PTH gene and demonstrated their functionality after transfection into the opossum kidney (OK) cell line. They converted the negative activity imparted by the PTH VDRE to a positive transcriptional response through selective mutations introduced into the element. They showed that there was a p160 protein that specifically interacted with a heterodimer complex bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a VDR/RXR heterodimer to interact with accessory factor proteins. Further work is needed to demonstrate that any of these differing negative VDREs function in this fashion in parathyroid cells. The transrepression by 1,25(OH)2D3 has also been shown to be dependent upon another promoter element. Kato’s laboratory have identified an E-box (CANNTG)like motif as another class of nVDRE in the human 1a (OH)ase promoter [37,38]. In sharp contrast to the previously reported DR3-like motif in the hPTH gene promoter, a basic helix-loop-helix factor, designated VDR interacting repressor (VDIR), transactivates through direct binding to this E-box-type element (1anVDRE). However, the VDIR transactivation function is transrepressed through ligand-induced proteine protein interaction of VDIR with VDR/RXR. In the absence of 1,25(OH)2D3, VDIR appears to bind to 1anVDRE for transactivation through the histone acetylase (HAT) coactivator, p300/CBP. Binding of 1,25(OH)2D3 to VDR induces interaction with VDIR and dissociation

495

of the HAT coactivator, resulting in recruitment of histone deacetylase (HDAC) corepressor for ligandinduced transrepression [37]. They have also characterized the functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters [39]. They identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE e87 to e60 bp) and in the hPTHrP promoter (hPTHrPnVDRE e850 to e600 bp e463 to e104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. They concluded that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression [39]. These studies were specific to a mouse proximal tubule cell line and await the development of a parathyroid cell line to confirm them in a homologous cell system.

REGULATION OF PTH GENE EXPRESSION 1,25-Dihydroxyvitamin D PTH regulates serum concentrations of calcium and phosphate, which, in turn, regulate the synthesis and secretion of PTH. 1,25(OH)2D3 has independent effects on calcium and phosphate levels and also participates in a well-defined feedback loop between 1,25(OH)2D3 and PTH [40]. 1,25(OH)2D3 potently decreases transcription of the PTH gene. This action was first demonstrated in vitro in bovine parathyroid cells in primary culture, where 1,25(OH)2D3 led to a marked decrease in PTH mRNA levels [41,42] and a consequent decrease in PTH secretion [43e46]. The physiological relevance of these findings was established by in vivo studies in rats [47]. The localization of VDR mRNA to parathyroids was demonstrated by in situ hybridization studies of the thyroparathyroid and duodenum. VDR mRNA was localized to the parathyroids in the same concentration as in the duodenum, the classic target organ of 1,25(OH)2D [48]. Rats injected with amounts of 1,25 (OH)2D3 that did not increase serum calcium had marked decreases in PTH mRNA levels, reaching T 913G>C 1016G>A 1031C>T 1070G>A 1634C>T 1696G>A 1772A>G 1771G>A 2337C>G 2546C>A 2582G>C 2605C>T 2925C>T 2946C>T 2946C>G 2947G>A 3299C>T 331A>G 3430G>A 3359G>C 3430C>T 3680T>G 3917C>G 246G>T

Q65H G73R R107H P112L G125E P143L D164N E189G E189K T321R S323Y R335P L343F P382S R389C R389G R389H T409I Y413G R453H R429P R453C V478G R492P P479R

1 2 2 2 2 3 3 3 3 5 6 6 6 7 7 7 7 8 8 8 8 8 9 9 9

Nonsense mutations 2014G>A 2561G>A 3372G>A

W241X W328X W433X

4 6 8

Deletions 212delG 958delG 1609delC 1921delG 1984delC 3922delA

Frameshift after 55K Frameshift after 87Y Frameshift after 135K Frameshift after 209C Frameshift after 230V Frameshift after 498E

1 2 3 4 4 9

Insertions 3392-3398dup 3398-3406insCCCACCC 3398-3408insCCCACACCC

Frameshift after 443P Frameshift after 441H Frameshift after 441H

8 8 8

Frameshift after 66V

2

Frameshift Frameshift Frameshift Frameshift

Intron 2 Intron 3 Intron 6 Intron 7

Deletioneinsertion 897-901delGGGCG; 897-902insCTTCGG Splice-site mutations IVS2þ1 G to A (1083G>A) IVS3þ1 G to A (1796G>A) IVS6þ1 G to A (2715G>T) IVS7þ1 G to A (1083G>A)

after 129A after 196E after 379R after 405N

Sequence traces were aligned with the GenBank reference sequences of the CYP27B1 genomic DNA (AF027152). Mutations were numbered following the convention (http://www.hgvs.org/mutnomen/recs.html), which starts with the translation initiator methionine as amino acid þ1, and the A of the ATG codon as nucleotide þ1.

VIII. DISORDERS

1191

TREATMENT

TREATMENT Historically, patients with PDDR were treated with high doses of vitamin D (calciferol, 20 000 to 100 000 IU/day), in an attempt to overcome 1a-OHase deficiency, with a certain success [12,32]. Under such treatment, circulating levels of 25(OH)D increase sharply, with only minor changes in the levels of 1,25(OH)2D (Fig. 64.3). It’s likely that massive concentrations of 25(OH)D are able to bind to the VDR and induce the response of the target organs to normalize calcium homeostasis. However, because such therapy leads to progressive accumulation of vitamin D in fat and muscle tissues, adjustment in case of overdose is difficult and slow to come into effect. Furthermore, the therapeutic doses are close to the toxic doses and place the patient at risk for nephrocalcinosis and impaired renal function. There have been reports on the use of 25(OH)D3 as therapeutic agent in PDDR [33]. The doses used are smaller than those of vitamin D and induce a similar response. The action of 25(OH)D3 is likely to be similar to the one of vitamin D itself, by maintaining high serum concentrations of 25(OH)D3. The low availability and high cost of such a preparation have discouraged its widespread use as a long-term therapy for PDDR. The treatment of choice is replacement therapy with calcitriol. Before the compound became available from commercial sources, several investigators used the

monohydroxylated analog 1a-hydroxyvitamin D (1aOHD), which requires only liver hydroxylation at the 25 position (a step not affected by the PDDR mutation) to fully mimic 1,25(OH)2D [34]. The response is rapid with healing of rickets in 7e9 weeks, requiring a daily dosage of 2e5 mg. The maintenance dose is about half the initial dose. Withdrawal induces a reappearance of symptoms within 3 weeks. Thus, long-term compliance is a more important consideration than in the case of vitamin D treatment. On a weight basis, 1a(OH)D3 is about half as potent as 1,25(OH)2D, nullifying any possible economic advantage in favor of the monohydroxylated form. The reason for this difference in potency has not been investigated, but may be related to a difference in intestinal absorption or to a variable degree of 25-hydroxylation of 1a(OH)D3. Since 1,25(OH)2D3 (calcitriol) became commercially available in 1973, the treatment of choice for PDDR patients is replacement therapy with calcitriol. Treatment with calcitriol (either in liquid form or in capsules) is started at a dose of 1.0 mg per day, given in two doses of 0.5 mg. Subsequently, the calcitriol dose is modified according to the results of biochemical analyses. The aims of the treatment are to achieve normocalcemia, to maintain PTH levels within normal limits and to avoid hypercalciuria. In our experience, the median daily calcitriol dose is 0.50 mg per day (range: 0.2 mg to 1.0 mg) after 3 months of treatment, 0.25 mg after 1 year (range

A.D. 25

2.4

PTH (pmol/l)

Ca (mmol)

2.8

2.0 1.6

10

0 Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Nov

Dec

Jan

Feb

Mar

0 BMD (Z score)

3000 P’ase Alc (U/L)

15

5

1.2

2000

1000

-2 -4 -6 -8

Aug 1.0

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Ca (300 mg/d)

0.5

Rocaltrol Rx

0.0 Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Rocaltrol ( g/d)

0 Rocaltrol ( g/d)

20

1.0

Ca (300 mg/d)

0.5

Rocaltrol Rx

0.0 Aug

Sep

Oct

FIGURE 64.4 Biochemical response to treatment in a patient with PDDR treated with calcitriol.

VIII. DISORDERS

1192

64. PSEUDO-VITAMIN D DEFICIENCY

supplemented to ensure a daily supply of around 1 g of elemental calcium.

EVOLUTION OF PDDR UNDER TREATMENT FROM CHILDHOOD TO ADULTHOOD Short-term Effects of Treatment with Calcitriol

Radiographs of the right wrist (upper panel) and knee (lower panel) of a patient with PDDR, before treatment (left panel) and after only 3 weeks of treatment (right panel); healing of rickets is well under way.

FIGURE 64.5

0.1 mg to 1.0 mg), and 0.5 mg after 2 years (range 0.25 mg to 1.0 mg) (personal data). An important component of treatment is to ensure adequate calcium intake during the bone-healing phase. Dietary sources are

Replacement therapy with calcitriol results in rapid and complete correction of the abnormal phenotype, eliminating hypocalcemia, secondary hyperparathyroidism, and radiographic evidence of rickets within 3 months (Figs 64.4 and 64.5) [14]. Lumbar spine areal BMD also normalizes within 3 months (Fig. 64.6) [11], as described in children and adults who are treated for vitamin D deficiency [10]. The rapidity of the increase in BMD suggests that calcitriol treatment of PDDR patients initially leads to the mineralization of pre-existing unmineralized osteoid rather than the production of new bone matrix. Histological evidence of healing has been documented in PDDR patients [14] as well as in the animal model of PDDR [35]. The height deficit persists somewhat longer than the low areal BMD, but after 2 years of calcitriol treatment, height is also normalized (catch-up growth) and remains so until adulthood (Fig. 64.6) [11]. Severe enamel hypoplasia is only partially corrected if treatment, as is usually the case, is started around 12e15 months of age when permanent tooth enamel has already started to develop (Fig. 64.7).

From Childhood to Adulthood From childhood to adulthood, calcitriol doses are increased as needed to maintain PTH levels within

Evolution of height (in 12 patients) and areal BMD of the lumbar spine (in five patients) during the first 2 years of treatment with calcitriol in young children with PDDR, from [11] with permission.

FIGURE 64.6

VIII. DISORDERS

EVOLUTION OF PDDR UNDER TREATMENT FROM CHILDHOOD TO ADULTHOOD

FIGURE 64.7 Permanent incisors of a 9-year-old patient with PDDR in whom calcitriol treatment was initiated at age 14 months. The part of the enamel that was formed before treatment remains hypoplastic. Subsequent to treatment, normal enamel was produced.

1193

treatment history. With regard to serum levels of vitamin D metabolites under calcitriol treatment, 25(OH)D levels and 1,25(OH)2D are normal in most patients. As previously discussed, dental structural and/or developmental abnormalities are documented in adult patients treated late with calcitriol. Hypercalciuria is not infrequent during treatment with calcitriol, and changes in urinary calcium excretion are used to adjust the daily calcitriol dose. High levels of calcium excretion may lead to calcium deposition in tissues especially in cornea and kidneys. In our treatment protocol, renal ultrasound and slit-lamp examinations of the cornea are performed every 2 years to assess for the presence of nephrocalcinosis and corneal calcium deposits. This screening for potential adverse events of hypercalcemia revealed that one patient (4% of the study population of 25) had mild corneal calcium deposits and four patients (16%) had mild nephrocalcinosis on renal ultrasound.

Pregnancies in Women with PDDR

FIGURE 64.8 Final height according to age of onset of calcitriol treatment in adult patients with PDDR, from [11] with permission.

normal limits. In our experience, in the period from 4 to 9 years of age, the median daily calcitriol dose increases from 0.25 mg to 0.50 mg. From 11 to 15 years of age, during the pubertal growth spurt, median calcitriol doses increase from 0.50 mg per day to 0.75 mg per day and remain so until adulthood [11].

Adult Patients with PDDR Adult height is significantly associated with the age at which calcitriol treatment is started (Fig. 64.8) [11]. The height of adult patients who receive calcitriol before the pubertal growth spurt is normal whereas patients who receive calcitriol only after the pubertal growth spurt are significantly shorter (Table 64.3). Lumbar spine areal BMD is normal in all adult patients whatever the

Female mice that are deficient in 1a-OHase (and that do not receive calcitriol) have uterine hypoplasia, absent corpora lutea, and thus are infertile [31]. This defect is at least partly due to hypocalcemia. Moreover, the 1aOHase gene is expressed in human endometrial stromal cells independent of the cycle phase but with a significant increase in early pregnant deciduas, suggesting a potential role of local production of 1,25(OH)2D in pregnancy establishment or maintenance [36]. The study of decidual tissues from two PDDR patients showed that it did not have the capacity to produce 1,25(OH)2D, indicating that decidua is a target for the PDDR mutation [37]. However, the physiologic importance of this defect is unclear. The importance of non-renal 1a-OHase and the role of local 1,25(OH)2D is discussed in Chapter 45. In our experience, pubertal development seemed to be normal in treated PDDR children. In our cohort, nine of 13 women with PDDR who were above 20 years of age have had 19 documented pregnancies [11]. It therefore appears that local production of 1,25(OH)2D in female reproductive organs is not critical for fertility, as systemic supplementation with calcitriol and normalization of serum calcium was sufficient to achieve fertility. During normal pregnancy, 1,25(OH)2D circulating levels steadily increase to about twice the control values. This adaptation to the specific needs of pregnancy can be mimicked in pregnant women with PDDR by increasing the daily calcitriol dose during the second half of pregnancy. During pregnancy, doses of calcitriol are adjusted according to the results of biochemical analyses (obtained every 4 weeks) to

VIII. DISORDERS

1194 TABLE 64.3

64. PSEUDO-VITAMIN D DEFICIENCY

Auxological, Biochemical, and Radiological Data of Adult Patients with PDDR Group 1

Group 2

Group 3

p

N

Value (range)

N

Value (range)

N

Value (range)

Number (female/male)

7

7 (1/6)

4

4 (4/0)

14

14 (9/5)

Age (years)

7

20.0 (17.0; 27.0)

4

20.5 (18.0; 26.0)

14

31.0 (24.0; 45.0)a,b

0.0009

Age of onset of calcitriol treatment (years)

7

1.0 (0.1; 1.6)

4

7.0 (4.0; 10.0)

14

23.0 (12.0; 31.0)

0.0001

Duration of calcitriol treatment (years)

7

19.1 (15.2e26.2)

4

14.8 (11.2e18.2)

14

11.0 (3.5e24.1)a

0.0025

Dose of calcitriol treatment (mg/day)

7

0.75 (0.75; 1.00)

3

1.25 (0.50; 1.50)

14

0.5 (0.50; 1.00)

Height (z-score)

7

0.3 (0.9; 1.0)

4

1.3 (1.6; 0.9)

14

2.2 (5.5; 0.9)

0.0008

Weight (z-score)

7

0.5 (1.4; 1.4)

4

1.0 (0.43; 2.18)

14

1.3 (3.0; 1.4)

0.011

Total calcium (mmol/l) (Norm: 2.25e2.63)

7

2.37 (2.29; 2.50)

4

2.33 (2.26; 2.55)

12

2.32 (2.10; 2.45)

0.39

Phosphate (mmol/l) (Norm: 1.23e1.62)

7

1.24 (0.87; 1.36)

4

1.07 (0.75; 1.22)

12

0.87 (0.74; 1.13)a

0.008

Alkaline phosphatase (UI/l) (

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