Fish Physiology: Molecular Endocrinology of Fish

FISH PHYSIOLOGY VOLUME XZZZ Molecular Endocrinology of Fish CONTRIBUTORS BENOIT AUPERIN K.-M. C H A N SHU JIN CHAN T ...

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FISH PHYSIOLOGY VOLUME XZZZ Molecular Endocrinology of Fish

CONTRIBUTORS BENOIT AUPERIN K.-M. C H A N SHU JIN CHAN T H O M A S T. C H E N CLARA M. C H E N G S T E P H E N J . DUGUAY HARRY P. E L S H O L T Z J. N. FRYER C H O Y L. H E W S H U I C H I HIRAOKA H I R O S H I KAWAUCHI KAORU KUBOKAWA YVES LE D R E A N K. L E D E R I S D A V I D W. L E S C H E I D S O N A L I MAJUMDAR ADAM MARSH J O H N E. McRORY T H O M A S P. M O M M S E N Y 0SH ITAKA NAGAH AM A

Y. OKAWARA MASAO O N 0 FARZAD P A K D E L D A V I D B. PARKER PATRICK P R U N E T D. R I C H T E R CHR. SCHONROCK MIKE SHAMBLOTT NANCY M. S H E R W O O D D O N A L D F. S T E I N E R KUNIMASA S U Z U K I M I N O R U TANAKA Y.-L. T A N G AKIHISA URANO YVES VALOTAIRE GRAHAM F. W A G N E R FEI X I O N G MASAKANE YAMASHITA B.-Y. YANG MICHIYASU YOSHIKUNI

FISH PHYSIOLOGY Edited by N A N C Y M. S H E R W O O D DEPARTMENT OF BIOLOGY UNIVERSITY OF VICTORIA VICTORIA, BRITISH COLUMBIA, CANADA

C H O Y L. H E W DEPARTMENT OF BIOCHEMISTRY RESEARCH INSTITUTE, HOSPITAL FOR SICK CHILDREN, TORONTO, AND DEPARTMENTS OF CLINICAL BIOCHEMISTRY AND BIOCHEMISTRY UNIVERSITY OF TORONTO TORONTO, ONTARIO, CANADA

Series Editors ANTHONY P. FARRELL DEPARTMENT OF BIOLOGICAL SCIENCES SIMON FRASER UNIVERSITY BURNABY, BRITISH COLUMBIA, CANADA

DAVID J. RANDALL DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, BRITISH COLUMBIA, CANADA

VOLUME XIII Molecular Endocrinology of Fish

ACADEMIC PRESS San Diego New York Boston

London

Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Cataloging-in-Publication Data (Revised for vol. 13) Hoar, William Stewart, date. Fish physiology. (v. 13: Fish physiology series) Beginning with v. 8 editors vary. Includes bibliographies and indexes. Contents: v. 1. Excretion, ionic regulation, and metabolism.--[etc.]--v. 12. The cardiovascular system (2 v.)--v. 13. Molecular endocrinology of fish. 1. Fishes--Physiology--Collected works. I. Randall, David J., date. 11. Conte, Frank P., date. 111. Title. IV. Series. 597'.01 76-84233 QL 639. I .H6 ISBN 0-12-350405-8 (v. 5) ISBN 0-12-350437-6

PRINTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 Q W 9 8 7 6

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CONTENTS CONTRIBUTORS

xi

PREFACE

xv xvii

OF OTHERVOLUMES CONTENTS

I. Brain Hormones 1.

Molecular Evolution of Growth Hormone-Releasing Hormone and Gonadotropin-Releasing Hormone Nancy M . Sherwood, David B . Parker, John E . McRory, and David W. Lescheid I. Introduction

11. GHRH-PACAP

111. Gonadotropin-Releasing Hormone IV. Intertwining of Function in the GnRH and GHRH Families References

2.

3 4 29 50 51

Corticotropin-Releasing Factors Acting on the Fish Pituitary: Experimental and Molecular Analysis K . Lederis,]. N . Fryer, Y. Okawara, Chr. Schonrock, and D . Richter I. Introduction

11. ACTH-Releasing Peptides and Their Receptors

111. CRF, Its Protein Precursors, cDNAs, and Genes IV. Evolutionary Considerations for CRF-UI References V

68 69 78 90 94

CONTENTS

vi

Expression of the Vasotocin and Isotocin Gene Family in Fish Akihisa Urano, Kaoru Kubokawa, and Shuichi Hiraoka

3.

I . Introduction Genes, cDNAs, and Precursors Divergence of V T and IT Gene Expression VT and IT Gene Expression in Osmotic Adaptation Conclusion References

11. 111. IV. V.

102 108 117 122 127 128

11. Pituitary Hormones 4. Control of Teleost Gonadotropin Gene Expression Fei Xiong, Kunimasa Suzuki, and C h o y L. H e w I. Introduction 11. Duality of Teleost Gonadotropins 111. Genomic Organization of Teleost Gonadotropins IV. Control of Gonadotropin Gene Expression V. Conclusion References

5.

The Somatolactin Gene Masao Ono and Hiroshi Kawauchi

I. Somatolactin 11. Somatolactin Gene 111. Regulation of Somatolactin Gene Expression IV. Conclusion References

6.

135 136 140 142 153 1S4

159 164

168 173 174

Structure and Evolution of Fish Growth Hormone and Insulinlike Growth Factor Genes T h o m a s T . C h e n , A d a m Marsh, Mike Shamblott, K . - M . C h a n , Y.-L. Tang, Clara M . Cheng, and B.-Y. Yang I. Introduction

11. Conserved Domains of Fish Growth Hormones

179 181

CONTENTS

111. IV. V. VI. VII. VIII. IX.

Conserved Domains of Fish Prolactins and Somatolactins Genomic Organization of Fish GH, PRL, and SL Genes Ancestral Gene of the Fish Growth Hormone Gene Family A Functional Model of Fish Growth Hormone Gene Family Fish IGF I and IGF I1 mRNAs Age- and Tissue-Specific Levels of Five IGF mRNAs Concluding Remarks References

vii 185 189 191 194 197 200 202 203

111. Other Hormones Structure and Expression of Insulinlike Growth Factor Genes in Fish Shu ] i n Chan and Donald F . Steiner

7.

I. Introduction 11. IGF Activity in Fish 111. Cloning of Fish IGF cDNAs and Genes

IV. Expression and Regulation of IGF V. Summary and Perspective References

213 214 215 220 22 1 222

8. Molecular Aspects of Pancreatic Peptides Stephen J. Duguay and Thomas P . Mommsen I. Introduction 11. Insulin 111. Glucagon and Glucagonlike Peptide

IV. Somatostatin V. Pancreatic Polypeptide and Related Peptides References

9.

226 226 231 250 258 262

The Molecular Biology of the Corpuscles of Stannius and Regulation of Stanniocalcin Gene Expression Graham F . Wagner I. Introduction

11. A Brief History of Discovery

111. Molecular Cloning of Eel and Salmon Stanniocalcin IV. Structural Comparisons of Eel and Salmon Stanniocalcin

273 275 276 278

CONTENTS

viii V. Studies on Tissue-Specific Expression of the Stanniocalcin Gene VI. Localization of Stanniocalcin mRNA in CS Cells by in Situ Hybridization VII. Calcium Regulation of Stanniocalcin Cell Activity VIII. Conclusions References

IV. 10.

289 30 1 302

Comparative Aspects of Pituitary Development and Pit-l Function Sonali Majumdar and Harry P . Elsholtz I. Introduction

111. Differentiation of Adenohypophysial Cell Types IV. Transcription Factor Pit-1 V. Comparison of Pit-1 in Mammals and Teleost Fish: Studies on the PRL Target Gene VI. Conclusion References

309 310 311 313 320 324 325

Structure and Regulation of Genes for Estrogen Receptors Yves Le Drkan, Farzad Pakdel, and Yves Valotaire I. Introduction

11. The Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor

111. The Rainbow Trout Estogen Receptor Gene 1V. Conclusion References

12.

285

Hormone Regulation

11. Comparative Organization of the Pituitary Gland

11.

283

331 337 349 357 357

Prolactin Receptors Patrick Prunet and Benoit Auperin

I. Introduction 11. Prolactin Receptors in Mammalian Tissues 111. Prolactin Receptors in Fish References

367 369 372 385

CONTENTS

ix

Regulation of Oocyte Maturation in Fish Yoshitaka Nagahama, Michiyasu Yoshikuni, Masakane Yamashita, and Minoru Tanaka

13.

I. Introduction 11. 111. IV. V.

Phenomenology Structure of Follicles Gonadotropin: Primary Mediator of Oocyte Maturation Maturation-Inducing Hormone (MIH): Secondary Mediator of Oocyte Maturation VI. Maturation-Promoting Factor (MPF):Tertiary Mediator of Oocyte Maturation VII. Conclusions References

393 394 395 398 400 4 19 428 430

AUTHORINDEX

44 1

SYSTEMATIC INDEX

473

SUBJECTINDEX

479

This Page Intentionally Left Blank

CONTRIBUTORS Numbers in purentheses indicute the puges on which the authors' contributions begin.

Benoit Auperin (367), Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cbdex, France K.-M. Chan' ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202

Shu Jin Chan (213), Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Thomas T. Chen ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Clara M. Cheng ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Stephen J. Duguay (225), Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Harry P. Elsholtz (309), Department of Clinical Biochemistry and Banting G Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada M5G 1L5

'

Present address: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong.

xi

xii

CONTRIBUTORS

J. N. Fryer (67),Department ofAnatomy and Neurobiology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 Choy L. Hew (135), Department of Biochemistry, Research Institute, Hospital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada MSG 1 L5 Shuichi Hiraoka (1O l ) , Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060, Japan, and Laboratory of Molecular Biology, Ocean Research l n stitute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan Hiroshi Kawauchi (159), Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, lwate 022-01, Japan Kaoru Kubokawa (1 O l ) , Laboratory of Molecular Biology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan Yves Le Drean (331), Laboratoire de Biologie Molkculaire, U R A , CNRS 256, Universitk de Rennes I , 35042 Rennes Cddex, France

K. Lederis (67),Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 1N4 David W. Lescheid (3),Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2 Sonali Majumdar (309), Department of Clinical Biochemistry and Banting G Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada MSG 1L5 Adam Marsh (179), Center of Marine Biotechnology, University of Maryland Biotechnology lnstitute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 John E. McRory (3), Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2 Thomas P. Mommsen (225),Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6

CONTRIBUTORS

xiii

Yoshitaka Nagahama (393), Laboratory of Reproductive Biology, De-

partment of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444, Japan Y. Okawara (67),Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 Masao Ono (159), Department of Molecular Biology, School of Medi-

cine, Kitasato University, Sagamihara, Kanagawa 228, Japan Farzad Pakdel(331), Laboratoire de Biologie Molkculaire, URA, CNRS 256, Universitk de Rennes I , 35042 Rennes Ckdex, France David B. Parker2 (3),Department of Biology, University of Victoria,

Victoria, British Columbia, Canada V8W 2Y2 Patrick Prunet (367), Laboratoire de Ph ysiologie des Poissons, INRA,

Campus de Beaulieu, 35042 Rennes Ckdex, France D. Richter (67), lnstitut f u r Zellbiochemie und Klinische Neurobiologie, Universitats-Krankenhaus Eppendorf, Universitat Hamburg, W-20246 Hamburg, Federal Republic of Germany Chr. Schonrock (67), Institut f u r Zellbiochemie und Klinische Neuro-

biologie, Universitats-Krankenhaus Eppendorf, Universitat Hamburg, W-20246 Hamburg, Federal Republic of Germany Mike Shamblott (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21 202 Nancy M . Sherwood (3),Department of Biology, University of Victo-

ria, Victoria, British Columbia, Canada V8W 2Y2 Donald F. Steiner (213), Howard Hughes Medical Institute and De-

partment of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Kunimasa Suzuki (135),Department of Biochemistry, Research Insti-

tute, Hospital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L5

’

Present address: The Clayton Foundation, Laboratory for Peptide Biology, The Salk Institute, La Jolla, California 92037.

xiv

CONTRIBUTORS

Minoru Tanaka (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444,Japan Y.-L. Tang3 (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Akihisa Urano (1Ol), Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060, Japan Yves Valotaire (331),Laboratoire de Biologie Moleculaire, U R A , CNRS 256, Universitk de Rennes I , 35042 Rennes Ckdex, France Graham F. Wagner (273),Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1 Fei Xiong (135), Department of Biochemistry, Research Institute, Hos-

pital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L5 Masakane Yamashita (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okaxaki 444,Japan

B.-Y. Yang (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Michiyasu Yoshikuni (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444, Japan

Present address: American Red Cross, 15601Crabs Branch Way, Rockville, Maryland 20855.

PREFACE

In this volume our aim is to highlight some of the exciting research that has emerged on molecular biology of fish hormones, their receptors, and regulation. Like studies in biomedical sciences, comparative vertebrate studies have found that molecular biological techniques are a powerful and indispensable tool for advancing our knowledge of gene structure, evolution, and regulation of fish hormones. Comparative studies of the structure of these hormones, at both the protein and the DNA level, provide important clues about the structurefunction relationship of the hormones, as well as their evolutionary history and mechanisms of action. Similarly, elucidation of regulatory DNA sequences is a prerequisite for studying tissue and celltype specificity, temporal expression ofthese hormones, and regulation by various factors. Largest in number and most diverse of the vertebrates, fish have an immense variety of life cycles, developmental stages, body structures, and physiological mechanisms. Clearly, fish offer a natural laboratory for elucidating the role of hormones in adaptation to a variety of environments. As shown in this book, knowledge of the structural basis of fish hormones has made possible major advances in the understanding of fish neuropeptides (Chapters 1-3); pituitary hormones, including the novel somatolactin (Chapters 4-6); and hormones related to growth, metabolism, and ion regulation (Chapters 7-9). Pioneering work on regulation by hormones and of hormones is presented in Chapters 4 and 10-13. Important advances are expected in this area in the next five to ten years. Finally, recent data on the estrogen and prolactin receptors are presented (Chapters 11 and 12).Here we see the intricate balance that exists between hormones and receptors, and the physiological implications of their relationship. xv

xvi

PREFACE

Finally, we thank Dave Randall and Tony Farrell for the invitation to prepare this volume and for their kindly guidance. We also acknowledge with gratitude the help, suggestions, and patience of Dr. Charles Crumly and Heidi Inman of Academic Press. NANCY M . SHERWOOD CHOY L. HEW

CONTENTS OF OTHER VOLUMES Volume I The Body Compartments and the Distribution of Electrolytes W. N . Holmes and Edward M . Donaldson The Kidney Cleveland P . Hickman, Jr., and Benjamin F . Trump Salt Secretion Frank P . Conte The Effects of Salinity on the Eggs and Larvae of Teleosts F . G. T . Holliday Formation of Excretory Products Roy P . Forster and Leon Goldstein Intermediary Metabolism in Fishes P . W. Hochachka Nutrition, Digestion, and Energy Utilization Arthur M . Phillips, Jr. AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECT

INDEX

Volume I1

The Pituitary Gland: Anatomy and Histophysiology J . N . Ball and Bridget 1. Baker The Neurohypophysis A . M . Perks Prolactin (Fish Prolactin or Paralactin) and Growth Hormone J . N . Ball Thyroid Function and Its Control in Fishes Aubrey Gorbman xvii

xviii

CONTENTS OF OTHER VOLUMES

The Endocrine Pancreas August Epple The Adrenocortical Steroids, Adrenocorticotropin and the Corpuscles of Stannius I. ChesterJones, D. K . 0. Chan, I. W. Henderson, a n d ] . N . Ball The Ultimobranchial Glands and Calcium Regulation D. Harold C o p p Urophysis and Caudal Neurosecretory System Howard A. Bern AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume I11

Reproduction William S. Hoar Hormones and Reproductive Behavior in Fishes N . R . Liley Sex Differentiation

Toki-o Yamamoto Development: Eggs and Larvae 1.H . S. Blaxter Fish Cell and Tissue Culture Ken Wolfand M . C. Quimby Chromatophores and Pigments Ryozo Fujii Bioluminescence J . A. C . Nicol Poisons and Venoms Findlay E . Russell AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume IV Anatomy and Physiology of the Central Nervous System Jerald J. Berstein


T h e Pineal Organ James Clarke Fenwick Autonomic Nervous System Graeme Campbell T h e Circulatory System D . J . Randall Acid-Base Balance C . Albers Properties of Fish Hemoglobins Austen Riggs

Gas Exchange in Fish D . J . Randall T h e Regulation of Breathing G . Shelton Air Breathing in Fishes Kjell Johansen The Swim Bladder as a Hydrostatic Organ Johan B . Steen Hydrostatic Pressure Malcolm S. Gordon Immunology of Fish John E. Cushing AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume V

Vision: Visual Pigments F . W. Munz Vision: Electrophysiology of the Retina T . Tomita Vision: The Experimental Analysis of Visual Behavior David lngle Chemoreception Toshiaki J . Hara

xix

xx


Temperature Receptors R . W. Murray Sound Production and Detection William N . Tavolga The Labyrinth 0. Lowenstein The Lateral Line Organ Mechanoreceptors Ake Flock The Mauthner Cell I . Diamond Electric Organs M . V . L. Bennett Electroreception M . V. L. Bennett AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume VI The Effect of Environmental Factors on the Physiology of Fish F. E . J . Fry Biochemical Adaptation to the Environment P . W. Hochachka and G . N . Somero Freezing Resistance in Fishes Arthur L. DeVries Learning and Memory Henry Gleitman and Paul Rozin The Ethological Analysis of Fish Behavior Gerard P . Baerends Biological Rhythms Horst 0. Schwassmann Orientation and Fish Migration Arthur D . Hasler Special Techniques D . J . Randall and W. S. Hoar AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX


Volume VII Form, Function, and Locomotory Habits in Fish C. C. Lindsey Swimming Capacity F . W . H . Beamish Hydrodynamics: Nonscombroid Fish Paul W . Webb Locomotion by Scombrid Fishes: Hydromechanics, Morphology, and Behavior John J . Magnuson Body Temperature Relations of Tunas, Especially Skipjack E . Don Stevens and William H . Neil1 Locomotor Muscle Quentin Bone The Respiratory and Circulatory Systems during Exercise David R . Jones and David J. Randall Metabolism in Fish during Exercise William R . Driedzic and P . W. Hochachka

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume VIII Nutrition C. B . Cowey and J . R . Sargent Feeding Strategy Kim D . Hyatt The Brain and Feeding Behavior Richard E . Peter Digestion Ragner Fange and David Grove Metabolism and Energy Conversion during Early Development Charles Terner Physiological Energetics J . R . Brett and T . D . D. Groves

xxi

xxii


Cytogenetics J . R. Gold Population Genetics Fred W. Allendorf and Fred M . Utter Hormonal Enhancement of Growth Edward M . Donaldson, U Y H . M . Fagerlund, David A. Higgs, and J . R. McBride Environment Factors and Growth 3. R. Brett Growth Rates and Models W. E . Ricker AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume IXA Reproduction in Cyclostome Fishes and Its Regulation Aubrey Gorbman Reproduction in Cartilaginous Fishes (Chondrichthyes) J . M . Dodd The Brain and Neurohormones in Teleost Reproduction Richard E . Peter The Cellular Origin of Pituitary Gonadotropins in Teleosts P. G. W. J . v a n Oordt and J. Peute Teleost Gonadotropins: Isolation, Biochemistry, and Function David R. l d l e r and T . B u n Ng The Functional Morphology of Teleost Gonads Yoshitaka Nagahnma The Gonadal Steroids A. Fostier, B.Jalabert, R. Billard, B. Breton, and Y . Zohar

Yolk Formation and Differentiation in Teleost Fishes T . B u n Ng and David R . Idler An Introduction to Gonadotropin Receptor Studies in Fish G l e n V a n D e r Kraak AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX


xxiii

Volume IXB

Hormones, Pheromones, and Reproductive Behavior in Fish N . R . Liley and N. E . Stacey Environmental Influences on Gonadal Activity in Fish T. J . L a m Hormonal Control of Oocyte Final Maturation and Ovulation in Fishes Fredrick W. Goetz Sex Control and Sex Reversal in Fish under Natural Conditions S. T . H . Chan and W. S . B. Yeung Hormonal Sex Control and Its Application to Fish Culture George A. Hunter and Edward M . Donaldson Fish Gamete Preservation and Spermatozoan Physiology Joachim Stoss Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish Edward M . Donaldson and George A . Hunter Chromosome Set Manipulation and Sex Control in Fish Gary H. Thorgaard AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume XA

General Anatomy of the Gills George Hughes Gill Internal Morphology Pierre Laurent Innervation and Pharmacology of the Gills Stefan Nilsson Model Analysis of Gas Transfer in Fish Gills Johannes Piiper and Peter Scheid Oxygen and Carbon Dioxide Transfer across Fish Gills David Randall and Charles Daxboeck Acid-Base Regulation in Fishes Norbert Heisler

xxiv


Physicochemical Parameters for Use in Fish Respiratory Physiology Robert 6 . Boutilier, Thomas A. Heming, and George K . Iwama AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume XB Water and Nonelectrolyte Permeation Jacques Isaia Branchial Ion Movements in Teleosts: The Role of Respiratory and Chloride Cells P. Payan, J. P. Girard, and N . Mayer-Gostan Ion Transport and Gill ATPases Guy de Renzis and Michel Bornancin Transepithelial Potentials in Fish Gills W. T . W. Potts The Chloride Cell: The Active Transport of Chloride and the Paracellular Pathways J . A. Zadunaisky Hormonal Control of Water Movement across the Gills J. C. Rankin and Liana Bolis Metabolism of the Fish Gill Thomas P. Momnzsen The Roles of Gill Permeability and Transport Mechanisms in Euryhalinity David H . Evans The Pseudobranch: Morphology and Function Pierre Laurent and Suzanne Dunel-Erh Perfusion Methods for the Study of Gill Physiology S . F . Perry, P. S.Davie, C. Daxboeck, A . G . Ellis, and D. G . Smith AUTHOR

INDEX-SYSTEMATIC

INDEX-SUBJECT

INDEX

Volume XIA Pattern and Variety in Development J . H . S . Blaxter Respiratory Gas Exchange, Aerobic Metabolism, and Effects of Hypoxia during Early Life Peter J . Rombough


xxv

Osmotic and Ionic Regulation in Teleost Eggs and Larvae D. F . Alderdice Sublethal Effects of Pollutants on Fish Eggs and Larvae H. von Westernhagen Vitellogenesis and Oocyte Assembly Thomas P. Mommsen and Patrick J . Walsh Yolk Absorption in Embryonic and Larval Fishes Thomas A . Heming and Randal K . Buddington Mechanisms of Hatching in Fish Kenjiro Yamagami AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX Volume XIB The Maternal-Embryonic Relationship in Viviparous Fishes John P. Worums, Bryon D. Grove, and Julian Lombardi First Metamorphosis John H . Youson Factors Controlling Meristic Variation C . C . Lindsey The Physiology of Smolting Salmonids W . S . Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes David L. G . Noakes and Jean-Guy J . Godin AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX Volume XIIA

The Heart Anthony P. Farrell and David R. Jones The Arterial System P. G . Bushnell, David R. Jones, and Anthony P. Farrell The Venous System Geoffrey H . Satchel1 The Secondary Vascular System J . F. Steffensen and J . P. Lomholt

xxvi


Cardiac Energy Metabolism William R. Driedzic Excitation-Contraction Coupling in the Teleost Heart Glen F . Tibbits, Christopher D . Moyes, and Leif Hove-Madsen AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume XIIB

Fish Blood Cells Ragnar Funge Chemical Properties of the Blood D . G. McDonald and C. L. Milligan Blood and Extracellular Fluid Volume Regulation: Role of the Renin-Angiotensin System, Kallikrein-Kinin System, and Atrial Natriuretic Peptides Kenneth R. Olson Catecholamines D . J. Randall and S. F. Perry Cardiovascular Control by Purines, 5-Hydroxytryptamine, and Neuropeptides Stefan Nilsson and Susanne Holmgren Nervous Control of the Heart and Cardiorespiratory Interactions E . W. Taylor Afferent Inputs Associated with Cardioventilatory Control in Fish Mark L. Burleson, Neal J . Smatresk, and William K . Milsom AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

BRAIN HORMONES


1 MOLECULAR EVOLUTION OF GROWTH HORMONE-RELEASING HORMONE AND GONADOTROPIN-RELEASING HORMONE Nancy M . Sherwood, David B . Parker,]ohn E . McRory, and David W. Lescheid Department of Biology, University of Victoria Victoria, British Columbia, Canada

I. Introduction 11. GHRH-PACAP

A. Identification B. Phylogenetic Studies C. Structural Analysis D. Functional Roles of GHRH and PACAP E. Evolution of GHRH and PACAP 111. Gonadotropin-Releasing Hormone A. Identification B. Phylogenetic Studies C. Structural Analysis D. Questions Regarding Localization and Function of GnRH E. Evolution of GnRH and GHRH Families IV. Intertwining of Function in the GnRH and GHRH Families References

I. INTRODUCTION This chapter is concerneG with two of the most funamental processes in life: growth and reproduction. Single cells grow and divide whether they are isolated or part of a multicellular organism, but the emergence of the nervous system in multicellular animals provided a new and overriding control on growth and reproduction. The mechanism by which the nervous system coordinated these slow processes of growth and reproduction was by the secretion of neuropeptides, and a number of such neuropeptides have been identified in inverte3 FISH PHYSIOLOGY, VOL. XI11

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction Ln any form reserved.

4

NANCY M. SHERWOOD ET AL.

brates from coelenterates to protochordates. In vertebrates the orderly sequence of maturational changes that lead to growth and reproduction is also influenced by neuropeptides that act as releasing factors, neuromodulators, and even local hormones in nonneural tissue. Hence, vertebrates and invertebrates share the use of neuropeptides whether the action is direct or indirect for altering growth and reproduction. Fish are a pivotal group in which to consider neuropeptides related to growth and reproduction. Two distinct families of peptides in fish have been associated with the neural control of reproduction or growth, gonadotropin-releasing hormone (GnRH) and growth hormonereleasing hormone (GHRH). These hormones have specific actions in releasing gonadotropins or growth hormone from the pituitary, although some crossover of function has been reported. The primary structures of fish GnRHs and GHRHs have been only recently identified, but they have clear structural similarities to those of other vertebrates so that shared ancestral hormones can be postulated. Fish are also intermediates in the deuterostome line of evolution that is thought to have led to mammals. The relationship of fish GnRH and GHRH to peptides in invertebrates is not yet clear, but it is assumed that both have links to invertebrate peptides. In any event, fish provide a varied group for consideration of the origin, function, and evolution of the neuropeptides of GnRH, GHRH, and a newly discovered peptide, pituitary adenylate cyclase activating polypeptide (PACAP), which is related to GHRH in fish and found in the same precursor. Mammals have two separate genes, one of which encodes only the classic GHRH peptide, whereas the other gene encodes a precursor with both PACAP and a PACAP-related peptide (PRP), the latter having sequence similarity to GHRH. In contrast, fish (salmon, catfish, and sturgeon) have a precursor that contains a GHRH-like peptide in addition to the PACAP hormone, but to date a second gene that encodes only GHRH has not been found. The question is whether the niammalian GHRH gene arose from a gene duplication after the bony fish separated from the tetrapod line.

11. GHRH-PACAP A. Identification 1. GHRH IN TETRAPODS In 1959 Seymour Reichlin provided one of the first indications that growth hormone is under the control of the brain. He showed that

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

5

rats with hypothalamic lesions grew less well than control animals (Reichlin, 1960a,b). By 1964 it was known that rat hypothalamic extracts contained a substance that specifically caused the release of growth hormone from rat pituitary cells in vitro (Deuben and Meites, 1964). Several different growth hormone-releasing substances were isolated and partially purified (Dhariwal et al., 1965; Frohman et al., 1971; Schally et al., 1971; Stachura et al., 1972), but none proved to be authentic GHRH. The isolation and characterization of a growth hormone-releasing factor proved elusive until 1982. At that time two independent groups, working with separate pancreatic islet cell tumors, isolated and characterized GH-releasing peptides. Vale and co-workers found a 40-aminoacid peptide with a free carboxy terminus within their tumor extract (Rivier et al., 1982) (Table I). Guillemin and colleagues also found a CHRH,-4,OH form in the same pancreatic tumor (Esch et al., 1982). In addition, the Guillemin group isolated a 44-amino-acid, amidated GHRH peptide, as well as 40-amino-acid and 37-amino-acid peptides with free carboxy terminals in a different tumor (Guillemin et al., 1982). By 1984 the hypothalamic form of GHRH was shown to b e identical to the pancreatic hormone (Ling et al., 1 9 8 4 ~ )To . date, seven mammalian GHRH peptides and one nonmammalian GHRH-like peptide have been isolated and sequenced (Fig. 1A). Further information on GHRH has been obtained from molecular studies, but is mainly limited to mammals. The sequence of GHRH cDNA is known for human pancreatic tumor (Gubler et al., 1983; Mayo et al., 1983),rat hypothalamus (Mayo et al., 1985b), rat placenta (Gonzhles-Crespo and Boronat, 1991), mouse hypothalamus (M. A. Frohman et al., 1989), and mouse placenta (Suhr et al., 1989). Only the human (Mayo et al., 1985a)and rat (Mayo et al., 198513) genes are known (Table I). For birds, reptiles, and amphibians, indirect evidence suggests that a GHRH-like molecule also exists and has similar functions compared with mammalian GHRH peptides. For example, synthetic hGHRH,-,,NH2 stimulated the release of GH either in vivo or in vitro from the pituitary of the chicken (Perez et al., 1987), dwarf chicken (Harvey et al., 1984), turtle (Denver and Licht, 1991), and frog (Malagon et al., 1991). 2. GHRH IN FISH Although the structure of GHRH for a nonmammalian species was not published until 1992, there was substantial indirect evidence that fish contained a GHRH-like substance with similar function, immuno-

Table I Identification of PRP/GHRH/PACAP Sequcences Source of sequence

Q,

Peptide

Species

Tissue

Form

PRP

Human

Testes

1-29, 1-48

Peptide

cDNA

X X

1-29, 1-48 Sheep Rat

Brain Brain

Human

Tumor"

GHRH

Tumor" Tumora Brain Tumor" Tumor" Rat

Brain Brain

1-29, 1-48 1-29, 1-48 1-44 NH2 1-40 OH 1-37 OH 1-40 OH 1-40 OH 1-44 NH2 1-44 NH2 1-44 NH2 1-44 NH2 1-43 OH 1-43 OH 1-43 OH

Gene

X X

References Ohkubo et ul. (1992) Hosoya et al. (1992) Ohkubo et u1. (1992) Ohkubo et al. (1992) Ogi et al. (1990)

X

Guillemin et al. (1982)

X X X

Rivier et al. (1982) Esch et ul. (1982) Ling et al. ( 1 9 8 4 ~ ) Gubler et al. (1983) Mayo et al. (1983) Mayo et nl. (1985a) Spiess et al. (1983) Mayo et al. (198513) Mayo et al. (1985b)

X X X

X X X

Mouse

PACAP

cow Pig Goat Sheep Carp Salmon Human

Sheep

Rat Salmon Frog ~

Pancreatic tumor.

Placenta

1-43 OH

X

Brain Placenta Brain Brain Brain Brain Brain Brain

1-42 OH 1-42 OH 1-44 NHZ 1-44 NH, 1-44 NHZ 1-44 NH, 1-45 OH 1-45 OH

X X

Testes Brain Brain Brain Brain Brain Brain Brain Brain Brain

1-38 1-38 1-38 1-38 1-38 1-27 1-38 1-38 1-38 1-38

Gonzales-Crespo and Boronat (1991) M. A. Frohman et d . (1989) Suhr et u1. (1989) Esch e t a / . (1983) Bohlen et a / . (1983) Brazeau et al. (1984) Brazeau et d . (1984) Vaughan et u/. (1992) Parker et a / . (1993)

X X X X X X X

X X X X

Kimura et a / . (1990) Kimura et u/. (1990) Ohkubo et al. (1992) Hosoya et al. (1992) Miyata et d . (1989) Miyata et a / . (1990) Kimura et d . (1990) Ogi et ul. (1990) Parker et a / . (1993) Chartrel et (I/. (1991)

8

NANCY hl. SHERWOOD ET A L .

A SALMON

25 30 35 40 45 I I I I I EADGMFNKAYRKALGQLSARKYLHSLMAKRVGGGSTMEDDTEPLS-OH

CARP

H

STURGEON H

5

10

15

20

I

I

I

I

MI

N

-OH

S

-OH

EEEEN ENS

-OH

T V

I

V

V S

CATFISH

H

MOUSE

HV A 1 TTN

RAT

H

A 1 TSS

RI

SHEEP

Y

A 1 TNS

I

L QDI NRQQ ERNQEQGAKVR --NHz

GOAT

Y

A 1 TNS

V

L QDI NRQQ ERNQEQGAKVR --N&

cow

Y

A 1 TNS

V

L QDI NRQQ ERNQEQGAKVR --N&

PIG

Y

A 1 TNS

V

L QDI SRQQ ERNQEQGARVR --NH2

HUMAN

Y

A 1 TNS

V

L QDI SRQQ ESNQERGARAR --N&

LLDR L D I V

B SALMON

S

L S

Y

IQDI NKQ- ERIQEQ--RAR

Y

L

5

10

15

20

25

30

I

I

I

I

I

I

D

G

I

F

T

-OH

E I NRQQ ERNQEQ--RSRFN-OH

D

S

Y

S

35 I

R

Y

FROG

K

IK

HUMAN

K V K

CATFISH

H

T V

R

R

~

F

Fig. 1. Comparison of vertebrate GHRH and PACAP peptides. (A) Salmon GHRH amino acids are compared with those oftwo other fish and seven mammals. (B) Comparison of amino acids for the known forms of PACAP. Residues are coded by a single letter and only residues that differ from the salmon sequence are shown. For maximal alignment, a dash is inserted to shift the sequence.

reactivity, and chromatographic behavior compared with mammalian GHRH peptides. For example, in 1966 Olivereau and Ball showed that severing the connections between the hypothalamus and pituitary resulted in poor growth rates and a significant decrease in the number of growth hormone cells in the molly Poecilia formosa. This suggested

1.


GHRH

AND

GnRH

9

that the hypothalamus in this species may exert a dominant stimulatory influence over growth hormone secretion (Donaldson et aZ., 1979). In 1984 Peter and associates demonstrated that intraperitoneal injections of hGHRH,_,,NH, stimulated GH release from sexually regressed goldfish, Carussius auratus (Peter et al., 1984), although hGHRH,-,,NH, did not release GH fi-om goldfish pituitaries in vitro (Marchant and Peter, 1989). Immunoreactive GHRH neurons were detected in a number of fish species and irGHRH-like molecules were partially characterized with HPLC (Table I). By 1990 a carp GHRH-like molecule was available (even though its sequence had not yet been published) and it was shown that the carp GHRH,-,,OH and carp GHRH1-2yNH2forms not only caused GH release from cultured rainbow trout (Oncorhyncus mykiss) pituitary cells (Luo et al., 1990; Luo and McKeown, l99la), but the 45-aminoacid form stimulated GH release from goldfish (C. auratus) pituitaries both in citro and in cico (Vaughan et al., 1992). However, a number of hormones, such as thyroid hormones, glucocorticoids (Donaldson et al., 1979; Nishioka et al., 1985; Luo and McKeown, 1991b), and insulinlike growth factor I (McCormick et aZ., 1992), can influence the release of GH in some teleosts under certain conditions, making it difficult to determine the true GH releaser in fish. cDNAs have been ioslated for a GHRH-like peptide in three fish: sockeye salmon (Oncorhynchus nerka) (Parker et al., 1993),Thai catfish (Clarias macrocephaZus) ( J . E. McRory personal communication), and white sturgeon (Ascipenser transmontanus) (D. W. Lescheid personal communication). The physiological studies of these molecules are in progress.

3. PACAP An unexpected discovery in mammals was the presence of an mRNA coding for a precursor with two peptides: one peptide had sequence similarity with GHRH and the other was pituitary adenylate cyclase activating polypeptide (PACAP). The mammalian GHHH-like peptide (named PACAP-related peptide, PRP) has not been shown to release GH, whereas PACAP released GH and three other pituitary hormones as well (Hart et aZ., 1992). PACAP was originally isolated based on its ability to increase cyclic AMP (CAMP)accumulation in cultured rat pituitary cells. This approach was unique because the other hypothalamic neurohormones had been isolated using assays for specific physiological functions, like the release of growth hormone or gonadotropins. In 1989 Miyata and co-workers isolated and characterized the 38-amino-acid form of PACAP from sheep hypothalami (Miyata et al., 1989)(Table I and Fig. 1B). A 27-amino-acid form, identical with the N-terminal region of

10

NANCY M. SHEHWOOD E?' AL.

PACAP1-38, was isolated from the same ovine hypothalamic extracts the following year (Miyata et al., 1990). Sheep (Kimura et al., 1990), rat (Ogi et al., 1990), and human (Kimura et al., 1990; Ohkubo et al., 1992) PACAP cDNAs have been characterized. By 1992 the human PACAP gene had been isolated (Hosoya et al., 1992), but the essential biological function of PACAP is still unknown. In other tetrapods, a PACAP1-38peptide has been isolated from the European green frog (Chartrel et al., 1991). In fish, PACAP has been isolated from three species. Our laboratory has cloned PACAP cDNAs for sockeye salmon (0.nerka) (Parker et al., 1993), Thai catfish (C. macrocephalus) ( J . E. McRory, personal communication), and white sturgeon (A. transmontanus) (I>. W. Lescheid, personal communication). We have recently isolated the GHKH/ PACAP gene from sockeye salmon (0.nerka) (D. B. Parker and N. M. Sherwood, personal communication). B. Phylogenetic Studies

1.

IMMUNOCYTOCHEMISTKY OF

GHRH

IN

TETRAPODS

Immunoreactive GHRH perikarya have been detected in the arcuate and ventromedial nuclei in humans, monkeys (Bloch et al., 1983, 1984;Bresson et al., 1984),and rats (Ishikawaetal., 1986).The paraventricular nucleus in guinea pigs also contains GHRH (Beauvillain et al., 1987).The GHRH nerve fibers that originate in these nuclei project to the median eminence and terminate on the portal vascular system. In addition, immunoreactive GHRH is present in the duodenum (Bruhn et n l . , 1985), testis (Berry and Pescovitz, 1988; Moretti et al., lYgOb), ovary (Moretti et al., l99Ob), and placenta (Baird et al., 1985; Meigan et al., 1988), suggesting alternative functions for the peptide. In the only amphibian species (Rana temporaria) examined for immunoreactive GHRH neurons, cells were detected in the magnocellular portion of the preoptic nuclei and gave rise to nerve fibers running in both the external and internal layers of the median eminence (Marivoet et ul., 1988).

2. IMMUNOCYTOCHEMISTRY OF GHRH IN FISH In fish, immunocytochemistry was used to detect a GHRH-like niolecule in the brain of the cod (Gadus morhua) (Pan et al., 1985a,b), sea bass (Dicentrarchus Zabrax) (Marivoet et al., 1988),rainbow trout (0.mykiss) (Luo and McKeown, 1989; Olivereau et al., 1990), and eight other species of teleost fish, including eels (Anguilla anguilla, A. rostrata), goldfish (C. auratus), carp (Cyprinus curpio), chinook

1.


GHKH

AND

GnRH

11

salmon (0.tshawytscha),trout ( S a l m o f a r i o ) ,mullet (Mugil rumada), and sculpin (Myoxocephalus octodecimspinosus) (Olivereau et al., 1990). In cod there was cross-reactivity in neurons of the preoptic area and lateral tuberal nucleus and in fibers of the pars nervosa of the pituitary with an antiserum made against the GHRH,-,,OH molecule, but only cells in the rostral pars distalis stained with a GHRH,-,,NH, antiserum (Pan et al., 1985a). In most teleost studies, however, human GHRH1-,,NH, antiserum stains immunoreactive perikarya in the preoptic nuclei and to a lesser extent in the lateral tuberal nucleus. In the eel, carp, goldfish, and salmon, the irGHRH fibers did not enter the rostral pars distalis and only a few fibers were seen in close association with the somatotrophs (Olivereau e t al., 1990).Instead, the immunoreactive fibers from these species and a variable number of fibers from mullet and sculpin terminated in the intermediate or neurointermediate lobe of the pituitary.

3. IMMUNOCYTOCHEMISTRY OF PACAP Immunocytochemical methods were used originally to show the presence of immunoreactive PACAP in the hypothalamus and septum of sheep (Koves et al., 1990). A dense network of PACAP fibers was seen in both external and internal zones of the median eminence, pituitary stalk, and in close contact with the hypophysial capillaries. PACAP immunoreactivity was not limited to the hypothalamus, but was seen also in the posterior pituitary. Within spider monkey and human brains, a similar distribution of PACAP-immunoreactive elements existed in the supraoptic and paraventricular nuclei (Vigh e t ul., 1991). In rats, PACAP perikarya in the hypothalamus were located in the supraoptic, paraventricular, anterior commissural, periventricular, and perifornical nuclei (Koves et al., 1991). Extrahypothalamic regions that have immunoreactivity to PACAP include the central thalamic nuclei, amygdaloid complex, bed nucleus of stria terminalis, septum, hippocampus, cingulate cortex, and entorhinal cortex (Koves et al., 1991). PACAP-immunoreactive fibers outside of the brain were detected in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep, and squirrel monkeys (Uddman et al., 1991). Also, immunoreactive PACAP was found in rats in the following tissues, which are listed from highest to lowest concentration: testis, posterior pituitary, adrenal gland, duodenum, stomach, jejunum, ileum, anterior pituitary, colon, ovary, epididymis, and lung. Other organs also had immunoreactive PACAP, but the concentration was less than 1ng/g wet tissue (Arimura et al., 1991). PACAP localization in fish has not been reported.

12

NAKCY M. SIIEKWOOD ET AL.

4. CHKOMATOGKAPHY OF GHRH

IN

FISH

Liquid chromatography has been utilized to isolate and characterize irGHRH-like molecules from a few fish species. Pan et uZ. (1985b) used exclusion chromatography of extracted codfish G. nzorhuu brains to isolate three fractions that reacted with a hGHRH antiserum. Codfish brains were also analyzed with high performance liquid chromatography (HPLC) by Ackland et al. (1989) to partially purify an irGHRH molecule that had a similar HPLC retention time in comparison to hGHRH,_,,NH,. Chum salmon (Oncorhynchus ketu) and coho salmon (0.kisutch) were shown to have an irGHRH-like molecule that could be detected with a hGHRH,-,,NH, antiserum (Parker and Sherwood, 1990). It was not until Vaughan et ul. (1992) extracted 16,000 carp (C. curpio) hypothalami and used HPLC methods that a nonmammalian GHRH-like peptide was identified and sequenced for the first time. PACAP from fish has not been studied using chromatography.

C . Structural Analysis 1. P w r I m

SEQUENCES

GHRH peptides have been characterized in human, rat, mouse, cow, sheep, pig, goat, and carp (C. carpio) (Fig. 1A). Most of the peptides are 44 amino acids long, with tyrosine as the initial aniinoterminal residue and an aniidated carboxy terminus. The exceptions to this rule are rat, mouse, and carp GHRH, which are 43, 42, and 45 amino acids long, respectively, with histidine at the N terminus and free acid at the C terminus. Of the mammalian GHRHs, pig GHRH is closest to human GHRH with only three amino acid differences, whereas mouse GHRH is the most distinct with 18 amino acid changes. The carp GHRH peptide is only 41% (18/44) identical to human GHRH,-,,NH,. PACAP peptides have been iodated only from ovine hypothalamus and frog brain. In the ovine hypothalamus, the two forms of PACAP are identical in the first 27 amino acids, but one form is extended to 38 aniino acids. Both PACAP1-38and PACAP,-27 are encoded by the same exon in humans. It is not yet known whether PACAP,_2-;is a posttranslational derivative of PACAP,-38 or whether it is directly cleaved from a common precursor (Hosoya et al., 1992; Okazaki et uZ., 1992) (Fig. 2). Frog PACAP is 38 amino acids long and has only 1 amino acid substitution compared to ovine PACAP.

Salmon GHRH/PACAP Precursor 22

1

173 R

R K R K K R

KR

RRKKKGKR

GRR

1 1 1 1 I 111 l l l l l

Sign1

Human GHRH Precursor 108

20

1

RR Signal

RK

RK

R RRRGR

I I I I IIII

Fig. 2. Comparison of the salmon and human proteins for GHRH, PRP, and PACAP precursors. The number ofamino acids for each precursor is at the upper right. Possible cleavage sites are indicated by single lines for one amino acid or black bands for two amino acids. The single-letter code is used for amino acids: R, arginine, K, lysine, and G for glycine, the amino acid that donates the final amide group. Possible mature peptides are shown below each precursor. For the peptides shown, the only ones that have been isolated from normal tissue are PACAP,* and PACAP,; from sheep; GHRH,, from rat; GHRH, from human, cow, pig, goat, and sheep; and GHRH45from carp.

14

NANCY M. SHERWOOD ET AL.

2. DNA SEQUENCES

The cDNAs for GHRH and PRP/PACAP have been isolated from a number of mammals (Table I). Although the mature forms of the PRP peptide have not been isolated, the peptide structure can be deduced from the cDNA sequence. In the cDNA, PRP is just upstream of' the region that encodes PACAPl-38 in the human, rat, and sheep (Figs. 2 and 3). Human PRP is only 48% similar to human GHRH (Ohkubo et al., 1992). Like the mammalian PRP/PACAP cDNAs, the fish GHRH-like/ PACAP cDNAs for sockeye salmon (0.nerka), Thai catfish (C. macrocephalus),and white sturgeon (A. transmontanus)contain four distinct consecutive regions: a signal peptide, a cryptic peptide, a GHRH-like region, and a PACAP region (Fig. 3). These precursors are similar to the glucagon (Heinrich et al., 1984) and the VIP (Bodner et al., 1985; Itoh et al., 1983) precursors that also contain consecutive coding regions for two different mature neuropeptides. Of all the fish GHRH-like regions sequenced to date, sturgeon (A. transmontanus) GHRH is closest (45%) to human GHRH1-,,NH2; sockeye salmon (0.nerka) and carp (C. carpio) GHRH are a close second (41%); and Thai catfish ( C . macrocephalus) GHKH has the least (32%) sequence identity. A comparison between human PRP and fish GHRH-like peptides shows that the similarity ranges from 62% for sturgeon to 55% for carp. 3. GENESTRUCTURE AND COPYNUMBER Several genes have been isolated and sequenced (Table I ) , including a human and rat GHRH gene, a human PRPIPACAP gene, and a salmon (0.nerka) GHRH-like/PACAP gene. In humans, GHRH is a single gene, 10 kilobases long, and separated into five exons. Splicing of the human GHRH transcript can occur to yield either a 107- or 108amino-acid preproGHRH (Gubler et al., 1983; Mayo et al., 1985a).The rat GHRH transcript encodes a 104-amino-acid preproGHRH (Mayo et al., 1985b). PRP/PACAP in humans is encoded on a single gene, contains five exons (Fig. 4), and is transcribed into a preprohormone of 176 amino acids. The salmon GHRH-like/PACAP gene also has five exons and has at least two copies or, alternatively, allelic polymorphism (Parker et al., 1993). The salmon gene can be transcribed into an mRNA that encodes a 173-amino-acid preprohormone. The human and rat GHRH genes have conventional TATA and CCAAT boxes, required for the accurate initiation of transcription in most eukaryotic promoters, whereas the human PRP/PACAP and the salmon GHRH-like PACAP genes do not contain a TATA or CCAAT

1.


GHRH

AND

GnRH

15

HUMAN PRP/PACAP

OVlNE PRP/PACAP

RAT PRP/PACAP

SALMON GHRH-ILe/PACAP

STURGEON GHRH-like/PACAP

CATFISH GHRH-k?/PACAP

Fig. 3. Comparison of mammalian PHP/PACAP precursors and fish GIIIIJ\VII(lia\.saii ef u l . , 1UY.3\. 'l'hc t r m i . ; l i i t i o i i start >ite h r i i i c t h i o n i i i c . i s i i i i t l c . r l i r i c * t l . :\stc-risk.; indicate, t r a i i . ; c . r i p t i o i i start h i t v s clc.tc.riiiinc.tl l o r t h c c d i i c k e i i 1c;F-l C C I I C * .

~

~

7.

STRUCTURE AND FUNCTION OF INSULINLIKE GROWTH FACTOR

219

a 642-nucleotide open reading frame encoding a 214-aa precursor was obtained. The deduced sequence of mature IGF in this clone was 82% identical to human IGF-11. Additional sequence homology was found when the trout E peptide was compared to human proIGF-I1 E peptide (Fig. 4). However, the sequences were not as well conserved as was the case for human and fish proIGF-I E peptides. The gene organization for rainbow trout IGF-I1 has not yet been reported. Nonetheless, these results indicate that teleosts contain homologues to both IGF-I and -11. In more primitive fish, an IGF cDNA has been cloned from the Atlantic hagfish, which is a representative agnathan or jawless vertebrate (Nagarnatsu et al., 1991).Although the signal peptide was incomplete, predicted sequences for a 71-aa mature IGF and 30-aa E peptide were obtained. Sequence comparisons revealed that hagfish IGF was 60% identical to either human IGF-I or -11, but no significant homology was found when the E peptides were compared. The lack of a specific sequence bias in hagfish IGF toward either IGF-I or -11 raised the possibility that it is descended from a putative common ancestral IGF gene that subsequently duplicated to form the IGF-I and -11 genes. At present, IGF genes in species more primitive than hagfish have not been identified and it has been proposed that IGF may have emerged early in vertebrate evolution from a hybrid insulin/IGF gene, similar to the gene found in amphioxus (Chan et al., 1990).

Signal peptide Human Trout

MGIPMGKSMLVLLTFLAFASCCIA MERQRKHEYklSVCHTCRRTENTRMKVKM-SS-NR--VIA--LTLTY-V

Human Trout

AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRV*SRRS*'R

0 domain

Human Trout

C domain

EVASA----------A-----E---------T--SN----QNA domain D domain GIVEECCFRSCDLALLETYCATPAKSE -------------N---Q---K-----

E peptide

Human Trout

RDVSxxx**~xXTPPTVLPDNFPRYPVGKFFQYDTWx~QST~RL~RGLP ----ATSLQIIPMV--1KQ-VPRKHVTV-YSK-EA-Q-KAA------V-

Human Trout

ALLRARRGEVLAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMASNRL -I----KFRRQ-VKIKAQEQ-MF-----T--SKL-PVLPPTDNYV-HN Fig. 4. Comparison of human and trout preproIGF-I1 sequences.

220

SHU TIN CHAN AND DONALD F. STEINEK

IV. EXPRESSION AND REGULATION OF IGF In mammals the expression of IGF-I and -11 genes is regulated both temporally and in a tissue-specific manner. IGF-I1 is expressed in multiple tissues predominately during embryogenesis. In contrast, IGF-I is also expressed in many tissues and during embryogenesis but is synthesized predominately in the postnatal liver under the control of growth hormone. The expression of IGF-I in teleosts appears to follow a similar pattern. In coho salmon, IGF-I mRNA has been detected by the RTPCR technique in embryos and in multiple tissues from juvenile and adult fish, including adipose tissue, brain, heart, kidney, liver, muscle, ovaries, testes, and spleen (Duguay et al., 1992). Ribonuclease protection assays showed that the IGF-I mRNA level is highest in the liver (Duan e t al., 1993). As stated previously, multiple IGF-I transcripts were found in all tissues owing to alternative RNA splicing. However, the structure of the secreted hormone is unaffected because the coding sequence for mature IGF-I is identical in all the transcripts. There is also good evidence that growth hormone regulates the expression of IGF-I in teleosts. Cao et al. (1989) showed that the injection of bovine growth hormone into coho salmon induced a sixfold increase in liver IGF-I mRNA level. In contrast, Duan et al. (1993) and Duguay et al. (1994) showed that the relative amount of IGF-I mRNA in heart, fat, brain, kidney, ovaries, and spleen was not affected b y growth hormone. They also found that two other pituitary hormones, prolactin and somatolactin, had no effect on the hepatic IGF-I mRNA level. In salmonid fishes, growth hormone appears to facilitate the adaptive osmoregulation from seawater to fresh water and this action is independent of its effects on somatic growth (Bolton et al., 1987). Sakamoto and Hirano (1993) reported that the osmoregulatory action of growth hormone may be mediated by an increased IGF-I mRNA level in gills and body kidney in rainbow trout. However, it should be noted that the effect was relatively small in that the stimulated IGF-I mRNA levels in kidney and gills were still 20 times less than that found in liver. I n contrast to IGF-I, a teleost IGF-I1 mRNA has only recently been cloned from rainbow trout and little has been reported on its expression. Shamblott and Chen (1992) indicated that liver RNA isolated from rapidly growing juvenile rainbow trout contained higher levels of IGF-I1 than IGF-I mRNA based on Northern blot analysis.

7.

S T R U C T U R E A N D F U N C T I O N OF I N S U L I N L I K E G R O W T H F A C T O R

221

This is similar to the situation found in adult hiinian liver, which expresses high levels of IGF-I1 mRNA, whereas adult rats and mice contain very low levels of hepatic IGF-11. The expression of IGF-I1 in other teleost tissues has not been reported. In the hagfish, Nagamatsu et al. (1991) investigated the tissue expression of I G F mRNA using RNA blot analysis and the more sensitive RT-PCR technique. IGF mRNA was detected in hagfish liver, but not in brain, heart, muscle, or islet tissue. Since growth hormone has not been identified in hagfish, it is not known whether I G F expression is regulated by this hormone. Although the restricted tissue expression of IGF was surprising, it should be noted that only RNAs isolated from adult hagfish tissues were assayed. It is possible that the IGF mRNA may be expressed in different hagfish tissues in the younger larval stage. To further investigate the structure and regulation of IGF in agnathans, we have recently cloned an I G F cDNA from the sea lamprey Petromyzon murinus and assayed for tissue expression by RT-PCR. Preliminary analysis indicates that lamprey I G F mRNA is expressed predominately in the liver but it can also be found in other tissues, notably brain and pancreas (S. J. Chan and J. Youson, unpublished results, 1994).

V. SUMMARY AND PERSPECTIVE The IGFs have long been suspected of playing important roles in promoting tissue growth and development. The recent results obtained with “knock-out’’ mice models have now provided definitive evidence that this is, indeed, the case in mammals. Transgenic mice with null mutations in IGF-I or -11 expressed deficiencies in normal growth, whereas double mutants as well as mice with a mutation in the type I IGF receptor were found to be nonviable (DeChiara et al., 1990; Liu et al., 1993).The identification of I G F genes in teleosts and agnathans, reviewed here, extend these studies and filrther suggests that I G F may play an essential developmental role in all vertebrate species. To define this role, however, it is important to further characterize the physiology and biological actions of IGF in fish. In particular, the relative expression of IGF-I and -11 teleosts during embryogenesis and their actions in specific tissues need to be clarified. It would also be interesting to determine whether primitive vertebrates, such as hagfish and lamprey, contain only a single IGF gene.

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ACKNOWLEDGMENTS We thank Florence Rozenfeld for expert assistance in preparing the manuscript. Research from our laboratory was supported by the Howard Hughes Medical Institute and USPHS Grants DK13914 and DK20595.

REFERENCES Allendorf, F. W., and Thorgaard, G. H. (1984).Tetraploidy and the evolution of salmonid fishes. In “The Evolutionary Genetics of Fishes” (B. J. Turner, ed.), pp. 1-53. Plenum, New York. Bautista, C. M., Mohan, S., and Baylink, D. J. (1990).Insulin-like growth factors I and I1 are present in the skeletal tissues of ten vertebrates. Metabolism 39, 96-100. Bolton, J. P., Young, G., Nishioka, R. S., Hirano, T., and Bern, H. A. (1987). Plasma growth hormone levels in normal and stunted yearling coho salmon, Oncorhynchus kisutch. /. Erp. Zool. 242, 379-382. Bowsher, R. R., Lee, W.-H., Apathy, J. M., O’Brien, P. J., Ferguson, A. L., and Henry, D. P. (1991). Measurement of insulin-like growth factor-I1 in physiological fluids and tissues. Endocrinology (Baltimore)128, 805-814. Cao, Q.-P., Duguay, S. J., Plisetskaya, E. M., Steiner, D. F., and Chan, S . J. (1989). Nucleotide sequence and growth hormone-regulated expression of salmon insulinlike growth factor I mRNA. M o l . Endocrind. 3, 2005-2010. Chan, S. J., Cao, Q.-P., and Steiner, D. F. (1990).Evolution ofthe insulin superfamily: Cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus. Proc. Natl. Acad. Sci. U.S.A. 87, 9319-9323. Chan, S. J., Nagamatsu, S., Cao, Q,-P., and Steiner, D. F. (1992). Structure and evolution of insulin and insulin-like growth factors in chordates. Prog. Brain Aes. 15-24. Chen, T. T., Marsh, A,, Shamblott, M., Chan, K. M., Tang, Y. L., Cheng, C. M., and Yang, B. Y. (1994).Structure and evolution of fish growth hormone and insulinlike growth factor genes. In “Fish Physiology” (N. Sherwood and C. Hew, eds.), Vol. 13, Chapter 6 . Academic Press, San Diego. Cohick, W. S., and Clemmons, D. R. (1993).The insulin-like growth factors. Annu. Rec. Physiol. 55, 131-153. Daughaday, W. H. (1972). Somatomedin: Proposed designation for sulphation factor. Nature (London)235, 107. Daughaday, W. H., and Rotwein, P. (1989). Insulin-like growth factors I and 11. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Reu. 10,68-91. Daughaday, W. H., Kapadia, M., Yanow, C. E., Faraick, K., and Mariz, I. K. (1985). Insulin-like growth factors I and I1 of nonmammalian sera. Gen. Comp. Endocrinol. 59, 316-325. DeChiara, T. M., Efstratiadis, A,, and Robertson, E. J. (1990). A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor I1 gene disrupted by targeting. Nature (London) 345, 78-80. Drakenberg, K., Sara, V. R., Lindahl, K. I., and Kewish, B. (1989). The study of insulinlike growth factors in tilapia, Oreochromis mossambicus. Gen. Comp. Endocrind. 4, 173-180.

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Duan, C., and Hirano, T. (1990).Stimulation of=S-sulfate uptake by mammalian insulinlike growth factor I and I1 in cultured cartilages of the Japanese eel, Anguilla japonica. J. E x p . Zool. 256,347-350. Duan, C., Duguay, S. J., and Plisetskaya, E. M. (1993). Insulin-like growth factor I (IGFI ) mRNA expression in coho salmon, Oncorhynchus kisutch: Tissue distribution and effects of growth hormoneiprolactin family proteins. In “Fish Physiology and Biochemistry,” pp. 371-379. Kugler, Amsterdam. Duguay, S. J., Park, L. K., Samadpour, M., and Dickhoff, W. W. (1992). Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon. Mol. Endocrinol. 6, 1202-1210. Duguay, S. J . , Swanson, P., and Dickhoff, W. W. (1994). Differential expression and hormonal regulation of alternatively spliced IGF-I mRNA transcripts in salmon. J. Mol. Endocrinol. 12, 25-37. Funkenstein, B., Silbergeld, A., Cavari, B., and Laron, Z. (1989). Growth hormone increases plasma levels of insulin-like growth factor I (IGF-I) in a teleost, the gilthhead seabream (Sparus aurata).J. Endocrinol. 120, R19-21. Furlanetto, R. W., Underwood, L., Van Wyk, J. J., and D’Ercole, A. J. (1977). Estimation of somatomedin C levels in normals and patients with pituitary disease by radioimmunoassay. J. Clin. Invest. 60, 648-657. Gray, E. S., and Kelley, K. M. (1991).Growth regulation in the gobiid teleost, Gillichthys mirabilis: Roles of growth hormone, hepatic growth hormone receptors and insulinlike growth factor-I. J. Endocrinol. 131, 57-66. Iwami, M., Kawakami,T., Ishizaki, H.,Takahashi, S. Y.,Adachi, R., Susuki, Y., Nagasawa, H., and Suzuki, A. (1989). Cloning of a gene encoding bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori with prothoraciocotropic activity. Dev. Growth Differ. 31, 31-37. Jansen, M., vanSchaik, F. M. A., Ricker, A. T., Bullock, B., Woods, D. E., Gabbay, K. H., Nussbaum, A. L., Sussenbach, J. S., and Van den Brande, J. L. (1983).Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature (London) 306,609-611. Kajimoto, J., and Rotwein, P. (1989). Structure and expression of a chicken insulin-like growth factor I precursor. Mol. Endocrinol. 3, 1907-1913. Kajimoto, Y., and Rotwein, P. (1990).Evolution of insulin-like factor I (IGF-I): Structure and expression of an IGF-I precursor from Xenopus laevis. Mol. Endocrinol. 4, 217-226. Kajimoto, Y., and Rotwein, P. (1991). Structure of the chicken insulin-like growth factor I gene reveals conserved promoter elements. J. Biol. Chem. 266, 9724-9731. Kavsan, V. M., Koval, A. P., Grebenjuk, V. A., Chan, S. J., Steiner, D. F., Roberts, C. T., Jr., and LeRoith, D. (1993).Structure of the chum salmon insulin-like growth factor I gene. DNA Cell Biol. 12, 729-737. Lagueux, M., Lwoff, L., Meister, M., Gotzent., F., and Hoffnian, J. A. (1990). cDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulins. Eur. J. Biochern. 187, 249-254. Liu, 1.-P., Baker, J., Perkins, A. S., Robertson, E. J., and Efstratiadis, A. (1993). Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igflr). Cell (Cambridge, Mass.) 75, 59-72. McCormick, S. D., Kelley, K. M., Young, G., Nishioka, R. S., and Bern, H. A. (1992a). Stimulation of coho salmon growth by insulin-like growth factor I. Gen. Comp. Endocrinol. 86. 398-406.

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XlcCormick, S. D., Tasi, P. I., Kelley, K ., Yonng, G., Nishioka, K. S., and Bern, H. A. (1992b). Hormonal control of sulfate incorporation in hranchial cartilage of coho salmon: Role of IGF-I. J. E x p . Zool. 262, 166-171. Aloriyama, S., Swanson, P., Nishi, M., Takahashi, A,, Kawauchi, H . , Dickhoff, W. W., and Plisetskaya, E. M. (1993). Development of coho sahnon insulin-like growth factor-I radioimmunoassay. A m . Zool. 33, 11A. Nagarnatsu, S., Chan, S. J., Falkmer, S., and Steiner, D. F. (1991). Evolution of the insulin gene superfamily: Sequence of a preproinsulin-like growth factor cDNA from the Atlantic hagfish. J. B i d . Chem. 266,2397-2402. Rechler, M. M., and Nissley, S. P. (1985). The nature and regulation of the receptors for insulin-like growth factors. Annu. Rec. Physiol. 47, 425-442. Sakamoto, T., and Hirano, T. (1993). Expression of insulin-like growth factor 1 gene in osmoregulatory organs during seawater adaptation of the salmonid fish: Possible mode of osmoregulatory action of growth hormone. Proc. Natl. Acud. Sci. U . S . A . 90, 1912-1916. Shamlilott, M. J., and Chen, T. T. (1992). Identification of a second insulin-like growth factor in a fish species. Proc. Natl. Acad. Sci. U.S.A. 89, 8913-8917. Shuldiner, A. R., Nirula, A., Scott, L. A., and Roth, 3. (1990). Evidence that Xenopus laeuis contains two different nonallelic insulin-like growth factor-I genes. Biochem. Biophysic. Res. Commun. 166,223-230. Siegfried, J. M., Kasprzyk, P. G., Treston, A. M., Mulshine, J. L., Quinn, K. A., and Cuttitta, F. (1992). A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone. Proc. Natl. Acad. Sci. U.S.A. 89, 8109-8111. Smit, A. B., Vreugdemjo, E., Ehberink, R. H. M., Gaeraerts. W. P. M.,Klootwijk, j., and Joosse, J. (1988). Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide. Nature (London)331, 535-538. Upton, Z., Chan, S. J., Steiner, D. F., Wallance, J. C., and Ballard, F. J . (1993). Evolution of insulin-like growth factor binding proteins. Growth Regul. 3, 27-30. Wallis, A. E., and Devlin, R. H. (1993). Duplicate insulin-like growth factor 1 genes in salmon display alternative splicing pathways. Mol. Endocrind. 7, 409-422. Wilson, D. M., and Hintz, R. L. (1982). Inter-species comparison of somatomedin structure using immunological probes. J . Endocrind. 95, 59-64. Zangger, I., Zapf, J., and Froesch, E. R. (1987). Insulin-like growth factor I and I1 in 14 animal species and man as determined by three radiohgand assays and two bioassays. Actu EndocrinoL 114, 107-112.

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES STEPHEN 1.DUGUAY The Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois

THOMAS P. MOMMSEN Depaitment of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canadz1

I. Introduction 11. Insulin A. Gene Structure B. Messenger RNA Transcripts and cDNA Sequences C. Peptide Sequences D. Biosynthesis E. Secretion F. Physiological Actions 111. Glucagon and Glucagonlike Peptide A. Gene Structure and cDNA Sequences €3. Gene Expression C . Glucagon Processing and Message Transduction D. Physiological Actions of Glucagon E. Glucagonlike Peptide Processing and Message Transduction F. Physiological Actions of Glucagonlike Peptides G. Conclusion IV. Somatostatin A. Gene Structure B. Messenger RNA Transcripts and cDNA Sequences C . Biosynthesis D. Secretion E. physiological Actions V. Pancreatic Polypeptide and Related Peptides A. The Pancreatic Polypeptide Family B. cDNA and Peptide Sequences 225 Copyright 0 1994 by Academic Presq, Inc. All right* of reproduction i n any fomm rerewed.

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C. Prohormone Processing D. Immunohistochemical Identification of Peptides E. Physiological Actions References

I. INTRODUCTION As the field of molecular biology has developed and matured in recent years, the repertoire of experimental approaches and techniques available to investigators studying piscine systems has been expanded enormously. Many laboratories engaged in fish research are now utilizing molecular methods, and others will certainly adopt these techniques in the future. The purpose of this chapter is to review the current knowledge of the molecular biology of insulin, glucagon, glucagonlike peptide, somatostatin, and pancreatic peptide in piscine systems. Gene and messenger RNA structures will be emphasized when available. Other molecular aspects that are particularly well characterized in piscine systems, such as biosynthesis, or exciting developments such as glucagon receptor studies or structure-function relationships of glucagons and glucagonlike peptides (GLPs) will also be discussed. It is hoped that this review will be a useful resource for those investigators wishing to initiate research and expand our knowledge on molecular aspects of pancreatic peptides in fish.

11. INSULIN A. Gene Structure The chum salmon (Oncorhynchus keta) insulin gene has been shown to consist of three exons separated by two introns with a total length of approximately 1560 base pairs (bp) (Fig. 1). Exon 1 codes for most of the 5' untranslated (5' UT) region. Exon 2 codes for the remaining 5' UT region as well as the signal peptide, B-chain, and first six amino acids of the C-peptide. Exon 3 encodes the remainder of the C-peptide as well as the A-chain and 3' UT region. Intron 1 occurs in the 5' UT region and is 393 b p long. Intron 2 interrupts the codon for the seventh amino acid of the C-peptide and is 287 bp in length (Koval et al., 1989a,b).This gene structure has been remarkably well conserved during evolution. In fact, with the exception of the special case of the rat I and mouse I insulin genes, all known vertebrate insulin genes consist of three exons separated by two introns. The

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MOLECULAR ASPECTS OF PANCREATIC PEPTIDES Insulin Gene Structure

\

\

\ \

Insulin mRNA

SP

B

I

C

A

Fig. 1. Structure of salmon insulin gene, insulin mRNA, and preproinsulin. Exons are indicated by numbered boxes and introns 1 and 2 are represented by thin lines connecting exons. The TATA box and putative Nir and Far boxes are shown in the 5’ end of the gene. Dashed lines indicate the exon splicing pattern used to generate the insulin mRNA. The start codon (AUG) and poly-A tail ofthe mRNA are labeled. Regions ofthe mRNA coding for the signal peptide (SP) and B-, C-, and A-chains are indicated. The processing sites for preproinsulin are labeled by an open arrow for the site of signal peptide cleavage and solid arrows for cleavage are dibasic residues to remove the Cpeptide.

positions of the introns have been highly conserved as well, interrupting the 5' UT region and the C-peptide in all cases (Chan et ul., 1992). Even the amphioxus (Brunchiostoma californiensis) insulinlike peptide gene structure is similar, with an intron interrupting the seventh codon of the C-peptide (Chan et al., 1990). Considerable allelic polymorphisms can be found in chum salmon gene sequences. Kashuba et al. (1986) identified 16 point mutations in the untranslated and translated regions of three cDNA clones. Also, the gene sequence reported by Koval e t al. (1989b) differs by more than 30% in the 3' UT region with the chum salmon cDNA sequence reported by Sorokin et al. (1982),suggesting the presence oftwo genes. It has been shown by genomic analysis with Southern blotting and polymerase chain reaction (PCR) that there are indeed two insulin genes in this species, probably as a result of chromosomal duplication (Kavsan et al., 1993).

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B. Messenger RNA Transcripts and cDNA Sequences Insulin cDNAs have been cloned from anglerfish (Lophius ainericanus), hagfish (Myxine glutinosa), chum salmon (Oncorhyncus kitsutch), and carp (Cyprinus carpio) (Hobart et al., 1980b; Chan et al., 1981; Sorokin et al., 1982; Hahn et al., 1983). Sequence analysis indicates that the insulin molecule has been well conserved during evolution. For instance, the B- and A-chains of hagfish insulin share 65% amino acid sequence identity with human counterparts. In contrast, C-peptides vary greatly in sequence and length, but are always flanked b y dibasic residues that serve as processing sites (see Fig. 1 and following). Fish preproinsulin signal peptides also differ considerably in amino acid sequence but retain features considered essential for function, such as a positively charged amino terminus and a hydrophobic core. Northern blot analysis of Brockmann body RNA from anglerfish, hagfish, and salmon indicates a single mRNA transcript of 840 nucleotides (nt), 1050 nt, and 760 nt, respectively, for insulin of each species (Hobart et al., 1980b; Chan et al., 1981; Sorokin et al., 1982).

C. Peptide Sequences Insulin peptides have been purified and sequenced from over 60 species of vertebrates. All have a two-chain structure consisting of a B-chain of approximately 30 residues linked to an A-chain of about 21 amino acids by two cystine bridges. An intramolecular A-chain disulfide linkage has also been found in all insulin molecules. A total of' 17 amino acid residues have been identified as invariant (Chan et al., 1992).A comparison of insulin sequences from representative teleosts, holocephalans, elasmobranchs, and agnathans can be found in Mommsen and Plisetskaya (1991).

D. Biosynthesis 1. REGULATIONOF SYNTHESIS a. Gene Transcription. The promoter region of the salmon insulin gene contains a standard TATA box transcription initiation site at -34 bp (Fig. 1) (Koval et al., 1989b). Although promoter analysis has not been conducted on fish insulin genes, mammalian insulin promoters have been studied extensively and binding sites for both

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positive and negative regulatory factors have been identified. Two important enhancer elements that have been identified are the Nir and Far boxes, also referred to as I E B l and IEB2, which confer celltype specific expression of the insulin gene. The Nir and Far box sequence CANNTG appears to define the binding site for members of the basic helix-loop-helix family of transcription factors (Moss et al., 1988; Karlsson et al., 1989; Aronheim et al., 1991; German e t al., 1991; Clark and Docherty, 1992). Inspection of the chum salmon insulin gene sequence reveals potential Nir and Far motifs at -124 bp and -143 bp of the promoter region (Fig. 1).

h. Protein Translation. In general, translation of proteins destined for the secretory pathway is initiated by ribosomes at an AUG codon of the mRNA and proceeds until the nascent signal peptide interacts with the signal recognition particle (SRP), leading to SRP-mediated translational arrest. The SRP-signal peptide complex then interacts with SRP receptors on the surface of the rough endoplasmic reticulum (RER),at which point translation resumes and the nascent polypeptide is translocated into the lumen ofthe RER. Translation initiation, elongation, and SRP-mediated arrest have been shown to be important regulatory points in the biosynthesis of proinsulin in mammalian systems and can be influenced by glucose and other nutrients (Itoh, 1990; Steiner, 1990). In experiments on hagfish ( M y x i n e glutinosa), neither glucose nor amino acids stimulated proinsulin biosynthesis (Emdin and Falkmer, 1977). 2. CONVERSION OF PREPROINSULIN TO INSULIN a. Signal Peptide Cleavage. Detailed studies using anglerfish (Lophius americanus) and sea raven (Hemitripterus americanus) Brockmann body mRNA in cell-free translation systems have shown that an 11.5-kDa protein can be immunoprecipitated by anglerfish insulin antisera. When cell-free translation is performed in the presence of pancreatic microsomes containing signal peptidase activity, the signal peptide is cleaved after 23 (anglerfish) or 25 (sea raven) residues to produce proinsulin (Shields and Blobel, 1977). Likewise, a 12- to 14-kIla protein that is immunoreactive with carp insulin antisera was produced in a cell-free translation system utilizing carp ( Cy p r i n u s carpio) Brockmann body mRNA (Rapoport et al., 1976). This preproinsulin molecule was converted to the 9-kDa proinsulin molecule during in uitro translation with pancreatic microsomes (Prehn e t al., 1980).

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b. Endoproteolytic Removal of C-Peptide. Proinsulin is the biosynthetic precursor of insulin and has the primary structure NH,-Bchain-C-peptide-A-chain-COOH. Inter- and intrachain disulfide bonds are formed after proinsulin enters the endoplasmic reticulum. Proinsulin is then transported through the Golgi compartment and packaged into secretory granules, where it is converted to insulin. Conversion to mature insulin is accomplished by endoproteolysis of dibasic residues at the B-chain/C-peptide and C-peptide/A-chain junctions (Fig. 1)(Steiner, 1990). This cleavage is mediated by the prohormone convertases PC2 and PC3 (also known as PC1). PC2 and PC3 are calcium-dependent serine proteases that cleave only at dibasic residues (Steiner et a/., 1992). Cleavage occurs on the carboxyl side of the second basic residue of the pair. The basic residues remaining on the carboxy terminus of the B-chain are then removed by a carboxypeptidase (Steiner, 1990). The available evidence indicates that the proinsulin processing pathway described here, which has been studied extensively in mammals, is also utilized in fish. Proinsulin molecules have been detected in islets of hagfish (Myxine glutinosa), carp (Cyprinus carpio), and anglerfish (Lophius americanus) (Yamaji et al., 1972; Steiner et al., 1973; Lukowsky et al., 1974), and conversion of proinsulin to insulin has been demonstrated in hagfish (Steiner et al., 1973).Also, PC2-like and carboxypeptidaselike processing enzymes capable of converting proinsulin to insulin have been isolated from anglerfish secretory granules (Mackin and Noe, 1987a; Mackin et d., 1991b). An interesting variation on this processing theme may be found in the ratfish, Hydrolagus colliei. Four insulin peptides have been isolated from this species that are probably generated by multiple cleavages of a single proinsulin and/or degradation by carboxypeptidase. The dibasic sequence normally present at the B-chain/C-peptide junction has been replaced by isoleucine-arginine. This cleavage site is apparently still recognized by a processing enzyme in the ratfish islet (Conlon et al., 1989). E. Secretion Secretion of insulin from the secretory granules of islets is stimulated by nutrients and may be modulated by hormones and neurotransmitters. For many fish species, amino acids are more effective than carbohydrates as secretagogues, and these agents often elicit a typical mammalian-type biphasic response. Although amino acids and glucose also stimulate insulin release from mammalian islets, there appear to

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be important differences in the mechanism of action of secretagogues between fish and mammals. For example, arginine stimulates insulin secretion from mammalian islets indirectly by stimulating glucagon release. In salmon, arginine-stimulated insulin release operates independently of any effect on glucagon (cf. Mommsen and Plisetskaya, 1991). F. Physiological Actions Insulin is the major anabolic hormone in fish. It stimulates the uptake of glucose and amino acids by skeletal muscle and liver and increases the rate of protein synthesis in these tissues. Insulin also acts to suppress hepatic gluconeogenesis and glycogenolysis. In addition to these actions on carbohydrate and protein metabolism, insulin exerts a positive effect on the flux of fatty acids into hepatic lipids. The physiological actions of insulin in fish have been reviewed in detail by Mommsen and Plisetskaya (1991) and Plisetskaya and Duguay (1993).

111. GLUCAGON AND GLUCAGONLIKE PEPTIDE In contrast to the situation just described for insulin and for somatostatin in the following, comparatively little is known about the molecular biology ofpiscine glucagons or glucagonlike peptides (GLP). Generally, for members of the glucagon family of hormones, attention has been focused on evolutionary aspects of peptide occurrence and peptide sequence, sites, and modes ofaction, as well as immunocytochemical localization of production sites, rather than on gene structure, processing, or posttranslational modification. Therefore, to present an overview of the glucagon complex in fishes, we give examples from mammals and other vertebrate groups. We also incorporate specific aspects of physiological and other functions of these peptides, especially where piscine systems offer new and exciting views on aspects of these somewhat neglected pancreatic and gut hormones. Glucagon is the best-known member of a constantly expanding superfamily of peptide hormones that includes secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), growth hormone-releasing hormone, peptide histidine methionine, helospectin, helodermin, pituitary adenylyl cyclase-activating peptide (PACAP), PACAP-related peptide and closely related peptides. Many of these have been described for several groups of vertebrates includ-

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ing fishes (see Chapter 1, this volume) (Conlon, 1988; Plisetskaya, 1990a; Jonsson, 1991; Parker et al., 1993a). In addition, a number of related peptides are co-encoded in the preproglucagon gene of all vertebrates. These include, starting from the 5' end of the gene, at times with overlapping sequences, glicentin-related polypeptide (GRPP), glicentin, glucagon (preproglucagon 33-61), oxyntomodulin (preproglucagon 33-70), and one or two glucagonlike peptides. Fulllength GLP-1 in mammals spans from residue 72 to residue 108 in the preproglucagon sequences, whereas GLP-2, if present, is from 126 to 159. Glucagon also displays some sequence homology with prealbumin (Jornvall et al., 1981).

A. Gene Structure and cDNA Sequences The human proglucagon gene, deduced from a genomic library, contains a total of six exons separated by five introns (White and Saunders, 1986), a pattern that is found in numerous other mammals (cf. White and Saunders, 1986) (Fig. 2A). Exons 2 to 5 encode the following four regions of the preproglucagon sequence: exon 2 encodes the signal peptide and part of the N-terminal region; exon 3 covers the remainder of the N-terminal peptide and glucagon, as well as the first four amino acids of an intervening peptide; the remaining six amino acids of the intervening peptide and the full-length glucagonlike peptide 1 are encoded by exon 4; and exon 5 covers the intervening peptide between GLP-1 and GLP-2, as well as GLP-2. In mammals, but not necessarily in the fishes (see the following), glucagon and the two glucagonlike peptides show strong sequence conservation and are considered to have arisen from two independent duplications of an ancestral gene coding for glucagon (Lopez et al., 1984). The rat glucago11 gene contains three DNA control elements in the 5' flanking sequence of the glucagon gene: a promoter, which accounts for A-cellspecific expression, as well as two enhancerlike elements (Philippe and Rochat, 1991). The Brockmann bodies of the anglerfish (Lophius americanus) express two nonallelic preproglucagon genes, leading to the production ofthree proglucagon transcripts of630,650, and 670 bases, respectively (Lund et al., 1983), coding for two closely related glucagons plus some related peptides (see the following). The single mRNA with 650 bp was shown to code for the so-called glucagon I, whereas the other two sequences were detected using a probe coding for glucagon 11. It is not known whether the length difference in the two transcripts for gliicagon I1 is due to different genes encoding this glucagon precursor,

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differential splicing of the mRNA, or the degree of polyadenylation of the 3’ end of the mRNAs. Nevertheless, compared with the proglucagon mRNA transcripts of mammals, which contain around 1300 bases, the fish transcript is small (Fig. 2B). Part of the size difference can be explained through the absence of regions coding for glucagonlike peptide 2 (GLP-2) and the intervening peptide (between GLP-1 and GLP-2) in the fish transcripts, corresponding to exon 5 in mammals (cf. Fig. 2A). Because GLP-2 (34residues), plus the preceding intervening peptide (6 residues), accounts for only about 120 base pairs, the bulk of the size difference between anglerfish and mammal is due to an extended untranslated 3’ sequence in the mammals. The size of the untranslated region in mammals exceeds 400 bases, but amounts to only 186 bases from the stop codon immediately following GLP to the consensus polyadenylation motif AATTAAA at the 3’ end of anglerfish glucagon I. In the case of anglerfish glucagon 11, the same region is only 92 bases long (from stop codon to AATAAA sequence). The role of the comparatively larger untranslated region in the mammals is under debate because deletion of variable amounts of the 3‘ untranslated region (plus all 3’ flanking regions) will produce read-through mRNA transcripts with compromised 3’ ends (Lee and Drucker, 1990). Although these deletions have little effect on the relative amounts of rat proglucagon transcripts measured in BHK fibroblast or islet cell lines, the study by Lee and Drucker (1990) shows quite conclusively that as few as 50 bases of the 3’ flanking region are essential for accurate formation of the glucagon mRNA 3’ end and polyadenylation. In all teleostean and elasmobranch fishes analyzed to date, peptides corresponding to GLP-2 have not been found (Fig. 2B). This holds true for peptides isolated from Brockmann bodies or the intestinal tract of numerous species of fishes as well as for the preproglucagon cDNAs analyzed for the anglerfish. The anglerfish cDNAs from proglucagon gene I and gene I1 contain stop codons immediately following the sequence of GLP. Interestingly, the intron connecting exons 4 (GLP-1, see earlier) and 5 (GLP-2) in preproglucagon genes of mammals (human and rat) contains a stop codon (Bell, 1986). Therefore it can be imagined that faulty processing of this intron, that is, retaining this stop codon, would result in glucagon precursors lacking peptides corresponding to GLP-2. Unfortunately, to date an analysis of fish genomic DNA to support or refute this idea has not been published. A similar situation may exist in the birds, which also appear to lack GLP-2, but in contrast to the fishes, the chicken contains an exceptionally long intervening peptide (28 amino acid residues, including the

234 STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN h

8.

235


B 3 UT

KR

Signal peptide

RK

Glucagon

RR

KR

KR

IP

UT

GLP

Fig. 2. (A) Structure of human preproglucagon gene and proglucagon mRNA. Exons are indicated by numbers. Modified from Bell (1986) and White and Saunders (1986). Processing sites (K or R) are indicated by vertical bars. For reasons of clarity, the dibasic (RR) processing site within glucagon is indicated by the amino acids only. Glucagon is stippled. The N-terminal region of GLP-1, which is removed to produce the biologically active form, is indicated by striping. IP, intervening peptides. (2B) Structure ofanglerfish (Lophius arnericanus) proglucagon. Modified from Lund et al. (1983) and Andrews and Ronner (1985). Abbreviations as in Part A. UT, untranslated regions. Numbers on top of the translated box indicate residue number of the proglucagon.

two flanking, dibasic processing sites) joining glucagon and GLP-1 (Hasegawa et al., 1990). The presence of GLP-2 in all vertebrates, excepting fishes and birds, appears to be a bit of an enigma. Whenever present, the sequence of GLP-2 is highly conserved, which is usually taken as an indication of increased evolutionary pressure and important physiological function. Alas, to date no clear functions and/or targets for GLP-2 have been identified, with the exception of the apparent ability of GLP-2 to regulate DNA synthesis under specialized conditions (Lund et al., 1993) and to stimulate adenylyl cyclase in rat hypothalamus and pituitary (Hoosein and Gurd, 1984).Future research will undoubtedly charify whether these activities constitute generalized features of GLP-2 peptides in higher vertebrates (except birds). In spite of the strong sequence homology with GLP-1 (cf Table I11 for the human GLPs), GLP-2 neither binds to the GLP-1 receptor nor interferes with the binding of GLP-1 or glucagon to their respective membrane receptors. In spite of the many similarities in proglucagon cDNA between mammals and fishes, there are a number of differences in the processing and sequence of GLP-1 (mammals) or piscine GLP. Exon 4 of the mammalian proglucagon gene encodes a short intervening peptide of 10amino acids, followed by the full-length glucagonlike peptide 1 with 37 residues. However, during posttranslational processing at sites removed from the endocrine pancreas, inactive GLP-1 is cleaved at Arg' to produce a biologically highly potent truncated GLP-l(7-37) (tGLP). Therefore, the exons encode a 16-residue spacer (a 10-residue intervening peptide and a 6-residue N-terminal truncation) between functional glucagon and functional (truncated) glucagonlike peptide

236

STEPHEN J. DUGUAY AND THOMAS P . MOMMSEN

Table I Comparison of Base and Amino Acid Sequences of' Intervening Peptide between Human Glucagon and GLP-1 and Anglerfish Gliicagon and GLP" 0.ryntomodulin

1 Intervening peptide

N-terminal esten\ion of GLP-I

GLP

C A C GAT GAA TTT GAG ACA

CAC His'

Arg

G AAT AAC AcnAsn

ATT GCC I AAA CGT IleAld LysArg

An)rlerfish I

AGC Ser

GGT GTC; GI) Val

GCA GAA AAG CGT A l a C l u Lys A r g

N o t present

c 4c

Anylrrfi\h I1

AAT Asn

GGT TTA GlyLeu

T T T - - - AGA CGC Phe - - - A r g A r g

Uot p r r w n t

C.4T Hi\'

Humm

AG

I

H I \ AspGlnPheGInAiy

HIS'

B a w nratches human \.erru* anglerfish intervening peptide. ,Anglmfidi 1

**

.Atipl?rfi\h I 1

*

* *

* I *

1

* I

*

***

* **

" 1 Indicates the 3' e n d of exon 3. 1 signifies the C-terminal end of oxyntomodulin, the extended form of glucagon (glucagon 1-37). Modified from White and Saunders (1986) and Lund et al. (1983).

1. In the anglerfish proglucagon cDNAs, the intervening peptide is even shorter (gene I: 7 residues; gene 11: 6 residues), but apart from the two basic amino acids critical for processing (Lys-Arg),little homology is discernible between fish and mammalian intervening peptides. Since the time that the existence of glucagonlike peptide(s) was first demonstrated from the cDNAs of anglerfish and mammalian proglucagon genes, the sequences for numerous GLPs have been published for fishes, including cyclostomes, elasmobranchs, and teleosts. In all cases, a sequence corresponding to the 6-residue N-terminal extension of the mammalian GLP-1 was absent (cf. Table I). It is removal of this extension that appears to be critical in developing the insulinotropic activity of GLP-1 in all mammalian (and amphibian) systems; this proteolytic processing does not occur at the site of GLP-1 production. Again, if the intervening peptide found in the two anglerfish glucagons can serve as a general example for the situation in fishes, few similarities, if any, exist between the intervening peptide and the amino acids truncated from the N-terminal His' of the biologically active GLP-1. The notion that this situation has wider application finds full support in amino acid sequences described for numerous species of teleostean fishes. Homology of fish GLPs with mammalian GLP-1 is greatest if aligned with His' of the truncated mammalian peptide (Table 111).

8.


237

B. Gene Expression In the rat, as in most other mammals analyzed to date, the glucagon gene is a single-copy gene that is expressed in a selective, cell-specific manner. The gene is expressed preferentially in the A-cells of the endocrine pancreas in the L-cells of the intestine, and in selected neurons of the brain. These tissues produce a single identical transcript. As demonstrated for the rat, expression differs substantially between tissues owing to processing at different dibasic amino acid processing sites. Intestinal L-cells, on the one hand, produce glicentin, oxyntomodulin (glucagon, 1-37), and GLP-1 and GLP-2, but not glucagon. The postulated, intestine-specific element for the glucagon gene has been located between -2000 and -1300 upstream from the start of transcription. In addition, the glucagon gene promoter has been located in an area together with numerous cis-acting domains. On the other hand, glucagon, full-length GLP-1, and a proglucagon fragment prevail as products of proglucagon processing in mammalian pancreatic tissue. Proglucagon and the major proglucagon fragment encompassing the two GLPs in mammals can be retained on lectin columns, suggestive of the fact that these peptides could occur in glycosylated forms (Patzelt and Weber, 1986). A number of fishes possess two proglucagon genes, which are nonallelic in the case of the anglerfish. Without invoking differential processing, at least five species (gar, anglerfish, daddy sculpin, eel, flounder) express two closely related glucagons and glucagonlike peptides (Tables I1 and 111).The similarities of glucagon sequences within the fishes and between elasmobranches and mammals have been used to develop an unrooted phylogenetic tree (Cutfield and Cutfield, 1993). As pointed out in the lower part of Table 11, the glucagon sequences of the primitive cartilaginous fishes reveal greater similarities to human glucagon than those of the other fishes. This fact has been interpreted to indicate a higher rate of molecular evolution of the gene in the teleosts than in other vertebrate groups. This accelerated rate of evolution is linked to the morphological development of the endocrine pancreas into the well-defined endocrine Brockmann bodies in these animals (Conlon and Thim, 1985). Tissue-specific expression in fishes differs from the mammalian picture in several ways. First, fish pancreas releases only one form of GLP, corresponding to the truncated GLP-1. Second, proglucagon gives rise to glucagon in gut tissue, at least in the dogfish (Scyliorhinus canicula). Little evidence has been found for the presence ofglicentinlike peptides in either tissue. Third, considering the gene structure

238


Table I1 Primary Structures of Fish Glucagons" Cvclostome 5

10

15

HSEGT

FTSDY

SKYLE

20 NKQAK

25 DFVRW

20 LMNA

HSEGT HSEGT

FTSDK FTSDY

SKYMD SKYLD

NRRAK NRRAK

DFVQW DFVQW

LMST LMNT

HTDGI HSEGT

FSSDY FSSDY

SKYLD SKYLD

NRRTK TRRAQ

DFVQW DFVQW

LLSTK LKNS

RNGAN

HSQGM HSQGM HSQGT HSQGT

FTNDK FTNDY FTNDY FTNDY

SKYLE SKYLE SKYMD SKYLD

EKRAK EKSAK TRRAQ TRRAQ

EFVEW EFliEW DFVQW DFVQW

LKNGK LKNGK LMST LMST

S

HSECT HSEGT HSEGT HSEGT HSQGT HSQGT HSEGT HSEGT HSEGT HSEGT

FSNDY FSNDY FSNDY FSNDK I T NDY FTNDY FSNDK FSNDY FSNDY FSNDY

SKYLE SKYLE SKYLE SKYQE SKYLE SKYQE SKY L E SKY L E SKY L E SKY L E

DRKAQ TRRAQ TRRAQ E RMAQ TRRAQ MKQAQ DRKAQ TRRAQ TRRAQ TRRAQ

E FVRW DFVQW DFVQW DFVQW DFVQW DLVQW DFVQW DFVQW DFVQW DFVQW

LMNN LKNS LM(NS) LMNS LMNS LMNSK LMNS LKNN LKNS LKNS

** *

* ***

* * I

HSQGT

FTS DY

SKY L D

1 Lamprey

Elasmohranchs Dogfish Ray

Holocephalans Ratfish Elephantfish Actinopterygians Paddlefish I Paddlefish I1 Bowfin Gar

T

S

Teleosts Anglerfish I

Anglerfish I1 Catfish Coho salmon Eel 1 Eel 11 Sculpin I Sculpin I1 Flounder Tuna I n \ ariant residues

a

h

*

a**

RNGSS

*

Xlammalian 1Iuman

S RRAQ

DFVQW

LMNT(KRNRNN1A)

' The eight-residue C-terminal extension of human glucagon has been included to indicate the full sequence of oxyntomodulin. Species: common dogfish, Scyliorhinus canicula (Conlon et al., 1987d); ray, Torpedo m a m o r a t a (Conlon and Thim, 1985); ratfish, Hydrolagus colliei (Conlon et al., 1989);elephantfish, Callorhynchus rnilii (Berks et al., 1989); paddlefish, Polyodon spathula (Nguyen et al., 1994); bowfin, Amia caloa (Conlon et al., 1993); gar, Lepisosteus spatula (Pollock et al., 1988); anglerfish, Lophius umericanus (Lund et al., 1983; Andrews et al., 1986; Nichols et al., 1988); channel catfish, lctalurus punctatus (Andrews and Honner, 1985); coho salmon, Oncorhynchus kitsutch (Plisetskaya et al., 1986); European ee1,Anguilla rostrata (Conlon et al., 1988b); flounder, Platichthys Jesus (Conlon et al., 1987b); daddy sculpin, Cottus scorpius (IIConlon et al., 1987c; I-Cutfield and Cutfield, 1993); tuna, Thunnus obesus (Navarro et al., 1991). a, acidic residue (glutamate or aspartate); b, basic residue (arginine or lysine).

8.

239


Table I11 Primary Structure of Fish Glucagonlike Peptides" Cycloctomes 1 5 HADGT

10 FTNDM

15 TSYLD

20 AKAAR

25 DFVSW

Elasmnhranchs Dogfish Ratfish

HAEGT HADGI

YTSDV YTSDV

DSLSD ASLTD

YFKAK YLKSK

RFVDS RFVES

LKSY LSNYN

RKQND

Actinnpterygians Bowfin* Gar Paddlefish

YADAP HADGT HADGT

YISDV YTSDV YTSDA

YSYLQ SSYLQ SSFLQ

DQVAK DQAAK EQAAR

K--- W KFCTW DFISW

LKSGQ LKQGQ LKKGQ

DRRE DRRE

Teleosts Anglerfish 1 Anglerfish I1 Catfish Coho salmon Eels Soulpin

HADGT HADGT HADGT HADGT HAEGT HADGT

FTSDV YTSDV YTSDV YTSDV YTSDV FTSDV

SSYLK SSYLQ SSYLQ STYLQ SSYLQ SSYLN

DQAIK DQAAK DQAAK DQAAK DQAAK DQAIK

DFVDR DFVSW DFITW DFVSW EFVSW DFVAK

LKAGQ LKAGR LKSGQ LKSGR LKTCR LKSKV

V(RRE) GRRE P A

**a**

f***

HAEGT HADGS

FTSDV FSDEM

Lamprey

Invariant residues (all fish,except bowfin) Mammals truncated human-I hunran-2

1,

SSYLE NTILD

GQAAK NLAAR

30

LARSD

*

EFIAW DFINW

KS

I,

LVKGRG LIQTK

I

a American eel, Anguilla rostrata; European eel, Anguilla anguilla. For other species names and references, refer to Table 111. f, aromatic amino acid ( Y or F); dash indicates deletions; a, acidic residue (glutamate or aspartate); b, basic residue (arginine or lysine).

and the low resemblance of the intervening peptide of fishes to the C-terminal extension of mammalian glucagon (i.e., the portion making up oxyntomodulin), the absence of an oxyntomodulin-type peptide in fishes does not come as a surprise. Possible exceptions to this hypothesis are a glucagonlike structure isolated from the pancreas of the holocepalan ratfish and an extended, unprocessed glucagon (possibly a storage form of glucagon) in an eel (cf. Table 11). The 36-residue peptide, which is thought to be a storage form of a 29-residue glucagon rather than a hormone in its own right, bears limited homology to oxyntomodulin in its 8-residue C-terminal extension. Obviously, an analysis of the gene structure of the proglucagon gene in this ancient group of fishes might give interesting comparative insights into the evolution of exon assembly and intron splicing because, in human and rat, part of the short C-terminal extension of oxyntomodulin is encoded by exon 3, whereas the remaining 6 amino acids are contributed by exon 4.

240


The mechanism of glucagon gene transcription is multifaceted and seems to differ substantially depending on the experimental system used. In a rat pancreatic cell line, for instance, activation of the gene appears to involve a protein kinase C pathway (Philippe et al., 1987), whereas in isolated rat islets, rat intestinal cells, and a mouse neuroendocrine cell line, a CAMP-dependent pathway is thought to prevail (Gajic and Drucker, 1993). As usual, the CAMP-dependent pathway involves a CAMPresponsive element (CRE) as well as the appropriate CRE binding protein. A new family of activating transcription factors has been identified that may bind specifically to CRE sequences. The appropriate sequence (5’-TGACGTCA-3‘) has been found to be a common theme in CAMP-activated genes (Meyer and Habener, 1993), including the glucagon gene (Drucker et al., 1991). It is the selectively phosphorylated form of this binding protein (or family members) that mediates increased rates of gene transcription, although CRE activity is further regulated by nucleotides flanking the core CRE octamer (hliller et al., 1993). In pancreatic A-cells, membrane depolarization can lead to induction of glucagon gene transcription. In this case the process is thought to depend on calcium influx and calciumicalmodulin-dependent protein kinase (Philippe et al., 1987; Schwaninger et ul., 1993). Although similar analyses on genomic D N A are sorely lacking, these results open up promising lines of inquiry for fish researchers interested in regulatory and evolutionary aspects of hormone action.

C. Glucagon Processing and Message Transduction In mammals glucagon is a highly conserved peptide of 29 amino acids with identical sequences in most species (Epple and Brinn, 1987). During the last few years, an impressive body of literature has accumulated dealing with structure-function analysis of mammalian glucagons. Unfortunately, the same cannot be said about their piscine counterparts, although the natural variability of peptide sequences found in a group of vertebrates as heterogeneous as the fishes is a powerful tool to analyze structure-function relationships. Such an analysis will also help identify conserved areas of the peptide as well as areas incurring larger species-dependent variability. In those cases where glucagon acts through a cell-surface receptor, as in hepatocytes, two processes combine to convey glucagon’s message to the interior of the cell. Hormone binding to the receptor is followed b y receptormediated changes in intracellular messengers. Although it is now estab-

8.


24 1

lished that in many mammalian cell types, glucagon regulates intracellular targets through different, likely interacting, mechanisms, including CAMP, inositolpolyphosphates, and intracellular calcium (Bygrave and Benedetti, 1993), most attention has been devoted to the analysis of receptor binding in conjunction with activation of adenylyl cyclase. By this route, selected areas of the glucagon molecule have been assigned different roles in receptor binding and adenylyl cyclase activation. Positions 1 through 5 are most critical to receptor recognition and binding, whereas Aspg is crucial for effective message transduction. One publication points out that it is the interaction between Asp' and His' that produces optimal receptor binding together with adenylyl cyclase activity of the peptide, whereas the positive charge of His' is essential for the activation of the adenylyl cyclase (Unson et al., 1993). Ultimately, most of glucagon's 29 residues are important to receptor binding, and not all biological functions of the hormone are localized to the N-terminal region of the peptide. C-terminally altered glucagons possess altered receptor-binding properties compared with the native hormone. Similarly, oxyntomodulin, the C-terminally extended glucagon (1-37) with specific actions directed toward the oxyntic cells (Jarrousse et al.,1985), will bind to the hepatic glucagon receptor, albeit with considerably reduced affinity. It should be kept in mind, however, that such structure-function studies have almost exclusively focused on receptor binding and activation of adenylyl cyclase, whereas other routes of message transduction and cross-talk between message transduction systems have been necessarily ignored given the experimental approach. The potential importance of non-CAMP message transduction systems to glucagon actions cannot be overstated. Working with three different species of teleostean fishes (American eel, Anguilla rostrata; rainbow trout, Oncorhynchus mykiss; brown bullhead, Zctalurus nebulosus),we have shown that a relatively poor correlation exists between cAMP increases and the concentrations of glucagon, or GLP. The rate of glucose output through endogenous glycogenolysis in isolated liver cells is activated significantly at low nanomolar (0.5 to 2) hormone concentrations, whereas significant increases in intracellular CAMP cannot be picked up until the hormone concentrations reach the mid-nanomolar range (20-100 nM) (Mommsen and Moon, 1990). This implies that a CAMPindependent route of message transduction may be involved at low (physiological) hormone concentrations. However, it is also possible that the route of analysis for determining cAMP masks hormonedependent changes in cAMP levels. Researchers normally determine total cAMP in the presence of a phosphodiesterase inhibitor to foil

242

STEPHEN J. DUGUAY A N D T H O M A S P. M O M M S E N

degradation of the cAMP formed and, thus, rely on detecting relatively large increases in total CAMPto study hormone effects. What is convenient for the researcher, however, may be irrelevant to the cell. It is likely that the fractional amount of free cAMP or the fraction bound to the regulatory subunit of protein kinase A constitutes the regulatory principle and not the total amount ofthis compound stashed on nonspecific or specific binding sites. For all vertebrates, many intracellular proteins are known that bind CAMP, ranging from CAMP-dependent regulatory elements on the nuclear DNA and their associated binding proteins, through phosphodiesterases to CAMP-dependent protein kinase A. These and similar proteins are likely to make up the bulk of total CAMP, with a considerably smaller fraction of the compound existing in the unbound form or sequestered by the regulatory subunits of protein kinase A. For instance, upon hormonal stimulation of adrenal cells isolated from the rat, the fraction ofcAMP bound to protein kinase A incurred the largest percentage increase of all cAMP pools assayed 1979).In conclusion, a role ofcAMP as intracellular messen(Sala et d., ger at low concentrations of glucagon cannot be excluded until further experimentation has been conducted on different intracellular cAMP fractions. As shown in Table 11, fish glucagons are relatively variable in sequence, but key amino acids are invariant in all species. Among these are, as expected, His' and Aspg, and the bulk of the N-terminal region. Ten of the first 13 amino acids are identical, giving credence to the idea of the overall importance of the N-terminal region of the peptide. By the same token, 60% of the last 10 amino acids are invariant (including an exchange of Asp2' for G1uZ1),pointing to the crucial role of the C-terminal region to glucagon's biological activity. Some interesting differences exist in the specific responses to glucagon and its synthetic analog. His' has been identified as essential to the biological activity of the peptide, both to receptor binding and to activation of adenylyl cyclase. Another critical role has been assigned to Aspg owing to chain length more than its charged environment. Deletion of His' and replacement of Asp9 with Glug yields a potent glucagon analog that retains some of the native peptide's receptor binding, measured by the ability of radioiodinated glucagon to displace analog binding, but is unable to activate adenylyl cyclase (Unson et al., 1991). As shown in Table 11, both His' and Aspg flank a rather conserved region even in the comparatively heterogeneous teleostean glucagons (cf. Table 11). In such potency and binding experiments, activation of adenylyl cyclase is normally measured with isolated liver membranes. When we tested the function of mammalian des-His'-

8.


243

Glu9 in fish liver cells, a different picture emerged. Whereas mammalian and teleostean glucagons are equipotent in their ability to activate gluconeogenesis and glycogenolysis in hepatocytes isolated from numerous species of teleostean fishes, the alleged antagonist behaved as a weak agonist. Its dose-response curve was right-shifted compared with unmodified glucagons by about two orders of magnitude (T. P. Mommsen, unpublished results). However, the two assay systems may not be directly comparable because the observed activation of glycogenolysis involves both receptor binding and activation of intracellular message transduction systems. Although glucagon action in fish systems is thought to involve mainly the activation of adenylyl cyclase, the activation of alternative pathways cannot be excluded. Mammalian glucagon possesses a dibasic processing site at Arg17Arg". In the rat liver it has been shown that glucagon processing by a specific endopeptidase (Blache et al., 1993) can generate a fragment (glucagon 19-29) with its own distinctive biological activity, that is, inhibition of the liver Ca2+ pump (Mallat et al., 1987). In addition, processing of oxyntomodulin at this dibasic site will also generate an active peptide with biological function similar to that of the full-length oxyntomodulin (Jarrouse et al., 1993). Although Arg" is conserved in all glucagons analyzed, with the exception of the lamprey, position 17 is variable. Some of the fish glucagons sequenced to date possess a dibasic processing region (KR, cf. Table 11),requiring a similar enzymatic process to produce the piscine equivalent of the mammalian mini" glucagons, whereas the remaining species require endopeptidases capable of recognizing a single Arg residue flanked by a number of different resides. However, when we tested the biological activity of different fish-derived miniglucagons, we failed to detect any activity in fish hepatocyte systems; the activity was measured by analyzing flux through glycogenolysis or gluconeogenesis (T. P. Mommsen and G. A. Cooper, unpublished). We did not attempt to determine the activity of the hepatocyte Ca2+pump in response to hormone exposure. Judging by the relative amounts of processed products (in this case glucagon) available in Brockmann bodies (exceeding about 7 nmol/g) and gut (less than 1 nmol/g) (Andrews and Ronner, 1985), one can assume that pancreatic cells contribute the bulk of glucagon present in the circulatory system. The pancreas also contains some 85 nmol of GLP/g. Although the ratio for the endocrine pancreas indicates that GLP outnumbers glucagon by over 10-fold, a much reduced ratio (about 3) is determined in plasma in the hepatic vein (Plisetskaya and Sullivan, 1989).This discrepancy may be due to (a) differential release ofthe two peptides, (b)different turnover ofthe peptides, (c) underesti"

244


mates for glucagon owing to determination of only one of the two nonallelic glucagons, or (d) postpancreatic processing of oxyntomodulin (where it exists) into glucagon. At any rate, the discrepancy is suprising, considering the similar role of the two peptides in fish metabolism and the origin of the two peptides from the same proglucagon sequence. In addition to the endocrine pancreas (A-cells), cells or cell groups with glucagonlike immunoreactivity have been detected in fish stomach (elasmobranch) and CNS (cyclostomes, elasmobranchs) (Conlon, 1988; Jonsson, 1991). The relative contributions of these different potential sources of glucagon in fish plasma are not known, and the actual release of the active hormone from the cells cannot be assumed. The regulation of glucagon secretion from the endocrine pancreas of fishes bears resemblance to that described for mammals: basic amino acids and KCI, all applied in supraphysiological concentrations, are potent secretagogues, whereas glucose is an inhibitor of glucagon release (Ince and So, 1984; Ronner and Scarpa, 1987; Plisetskaya et al., 1989).Further, epinephrine exerts a relatively strong glucagonotropic action in fish Brockmann bodies, albeit at pharmacological concentrations of the catecholamine (Mazeaud, 1964).

D. Physiological Actions of Glucagon The best-known and principal function of glucagon in fishes and mammals is a strong glycogenolytic action on liver resulting in hyperglycemia. It largely opposes the glucose-oriented actions of insulin. In addition, the hormone has numerous other functions, such as activation of lipolysis and other indirectly linked functions in fishes. With regard to the physiological roles of glucagon in fishes, attention has largely been focused on the hepatic action. However, if mammalian work can serve as a rough guide for future research, the brain and the endocrine pancreas, displaying some degree of glucagon binding capacity and expressing glucagon receptors, are worthy of attention. In the absence of data on receptor binding and glucagondependent gene expression in nonhepatic tissues of fishes, we will briefly summarize aspects of glucagon’s action on parenchymal hepatocytes. Glucagon injection results in a pronounced hyperglycemia in most species of fishes, a process brought about by activation of hepatic glycogenolysis and gluconeogenesis. Season, reproductive state, and temperature influence the targets and effectiveness of the hormone in piscine systems. At supraphysiological concentrations of glucagon, activation of glycogenolysis involved activation of adenylyl cyclase

8.


245

(Ottolenghi et al., 1990) and protein kinase A-mediated phosphorylation of glycogen phosphorylase; the latter process increases the proportion of the enzyme in the active @-form(Brighenti et al., 1991; Foster and Moon, 1990; Janssens and Lowrey, 1987). At the same time, glycogen synthase is inactivated by similar phosphorylation (T. Moon, G. Foster, and M. Vijayan, unpublished results). Flux through gluconeogenesis is enhanced through phosphorylation-dependent inhibition of pyruvate kinase and, in long-term experiments, through increases in the activity of phosphoenolpyruvate carboxykinase (PEPCK). Incidentally, the short-term induction observed for mammalian PEPCK is found wanting in fish liver. Exposure of fish systems to high concentrations of glucagon (largely bovine) increases lipolysis mediated via enhanced triglyceride lipase activity, the rate of amino acid uptake by the liver, and, finally, ureagenesis (reviewed in Mommsen and Moon, 1990). In addition to the hyperglycemia mentioned, in wiwo effects of the hormone are an accumulation of plasma unesterified fatty acids and glycerol as well as increases in ammonia excretion by treated fish. In fishes the hormone does not seem to change the rate of mitochondrial respiration as it does reproducibly in mammalian test systems. E. Glucagonlike Peptide Processing and Message Transduction Fish Brockmann bodies synthesize only one-short-GLP from a comparatively shorter proglucagon. If two GLPs or two glucagons are found, these are products of two nonallelic genes. Transcription of the proglucagon genes in the anglerfish will lead to the production of GLPs with 34 residues, terminating in G-R-G-R-R-E in the case of gene I1 and G-Q-V-R-R-E for gene I. Both peptides contain additional dibasic processing sites. As shown for gene I1 (Andrews et al., 1986), but not ruled out for gene I (Nichols et al., 1988), such processing indeed takes place in wiwo, leading to the production of two C terminally truncated GLPs. One of these has 31 residues and terminates in an amidated arginine; the amidation is likely produced via a stable 32-mer intermediate, derived from proteolytic processing of the original GLP at the additional dibasic processing site. As described next for fishes and as shown for GLP-1 in mammalian systems, the amino acid sequence at the C terminus and amidation do not compromise the biological activity of these peptides. Piscine glucagonlike peptides are not nearly as conserved in their amino acid sequences as glucagon (Table 111),with 10 invariant residues out of 31. With the exception of the unusual bowfin peptide,

246


which bears some resemblance to GIP and has a notably reduced biological potency (Conlon et al., 1993), the N-terminal region of the peptide is highly conserved. Of the first 9 amino acids, 8 positions are occupied by invariant residues and the C-terminal region is highly variable, with only 2 invariant positions for the entire remainder of the 31-residue peptide. The importance of the N terminus is supported by our observation for fish systems (T. P. Mommsen and A. Jardim, unpublished) and by those of others for mammals (Suzuki et al., 1989) that His' is essential to the biological action of GLP, be it glycogenolytic action in fishes or insulinotropic action in mammals. Further, the obvious variability in the C-terminal region of GLP in fishes can be taken as an indication that selection pressure is less strong to maintain the charge and lipophilic characteristics of the C terminus. That is exactly what was found experimentally when C-terminally altered mammalian GLPs were analyzed for their insulinotropic activity: large tracts of the C-terminal region were dispensible (Suzuki et al., 1989). There is some indication that CAMP-dependent pathways are involved in GLP message transduction in some species of fishes (Zctalurus sp., Anguilla), albeit only at supraphysiological concentrations of peptide. At physiological concentrations of GLP in these species and in other species (Oncorhynchus mykiss, Sebastes sp.), CAMPindependent routes are likely operative at all times (Mommsen and Moon, 1990). Contrary to numerous earlier reports, fish liver cells have been found to respond to selected hormones with a redistribution of intracellular calcium stores (Zhang et al., 1992a,b) and with liberation of' inositolpolyphosphates (T. W. Moon, personal communication). Thus, the stage is finally set for an in-depth analysis of intracellular message transduction systems in fish systems. A similar heterogeneous picture is emerging for mammals. In a pancreatic cell line, truncated GLP-1 was found to increase CAMP levels, whereas receptor affinity sensitivity to guanine nucleotide was taken to indicate the involvement of G-proteins in transduction (Goke et al., 1989). In marked contrast to glucagon binding to these cells, GLP exposure failed to alter intracellular Ca2+ levels and membrane potential remained unaffected by GLP treatment. However, in the presence of glucose, GLP may lead to membrane depolarization of subpopulations of pancreatic islet cells, likely through closing of ATPgated K+-channels (Holz et al., 1993), whereas in other experimental systems adenylyl cyclase as well as protein kinase C have been implicated as intracellular message transduction systems (Wheeler et al., 1993; Yada et al., 1993).

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F. Physiological Actions of Glucagonlike Peptides With regard to the function of GLPs in fishes and mammals, diametrically opposing roles have been described in which there is a predominant endocrine role in mammals as opposed to a clear metabolic role in fishes. In all mammals (Schmidt et al., 1985; Mojsov et al., 1987), amphibians, and reptiles (T. P. Mommsen and E. M. Plisetskaya, unpublished), the primary site of action of truncated GLP is in the pancreas. Nevertheless, judging from the expression of tissue receptors, some physiological role for tGLP in brain, stomach, kidney (removal site), and lung can be predicted. In pancreatic cells, the hormone increases insulin synthesis and secretion, while also suppressing glucagon gene transcription and glucagon secretion. In the course of the last year, GLP-selective receptors have been expression cloned and sequenced for different tissues and in different mammals (Thorens, 1992; Dillon et al., 1993) (human brain: S. Mojsov, personal communication). These receptors show sufficient resemblance to the receptors for parathyroid hormone, calcitonin, secretin, and glucagon ( Jelinek et al., 1993) to form a new subgroup of the family of G-protein coupled receptors with seven transmembrane domains. From different angles it has been confirmed that the liver is not a target for GLP action: first, liver shows no GLP binding; second, the tissue fails to express GLP receptors; third, intracellular message transduction systems are not recruited after GLP exposure; fourth, liver does not degrade GLP or remove it from the circulation; and finally, no biological action of GLP could be identified (Ruiz-Grande et al., 1990; Murayama et al., 1990; Blackmore et al., 1991; Thorens, 1992). On a metabolic level, the mammalian truncated GLP functions to accentuate insulin’s action. GLP does so by increasing insulin availability and concentration and by removing glucagon, one of insulin’s major antagonists. This situation illustrates the surprising principle that two peptide hormones with directly countering actions are derived from genes that evolved by duplication of a single gene. In contrast, fish liver has been identified unequivocally as the main target of GLP action (Mommsen and Moon, 1990; Brighenti et al., 1991), with little or no action of the peptide on endocrine pancreatic cells (Mommsen and Plisetskaya, 1993). Nonhepatic tissues (e.g., brain) cannot be entirely excluded as potential target tissues. In addition to being the site of strong metabolic rather than endocrine action, fish liver is also the primary site of GLP removal from the circulation. GLPs activate hepatic gluconeogenesis, glycogenolysis and lipolysis.

248

STEPHEN J. DUGUAY AND THOMAS P. MOXIMSEN

Thus the peptides have identical or similar targets to glucagon, but as implied earlier, they may differ in their intracellular message transduction pathways. It should be mentioned at this point that fish GLPs and mammalian truncated GLPs are entirely interchangeable in their specific actions: fish GLPs act as powerful insulinotropins in isolated mammalian islets (Plisetskaya and Duguay, 1993),and truncated GLPs are equipotent to fish GLPs in their glycogenolytic action in fish hepatocytes. Although both glucagon and GLPs act on fish liver in a similar fashion, they do not involve the same hepatic receptors. Radiolabeled glucagon that is bound to highly specific glucagon receptors on fish hepatocytes will only be minimally displaced by 1000-fold higher concentrations ofGLP (Navarro and Moon, 1994). However, the physiological actions of the two peptides can be discerned at identically low peptide concentrations (Mommsen and Moon, 1990; T. P. Mommsen and G. A. Cooper, unpublished). Exposure to GLP (or glucagon) changes metabolic output of liver cells immediately. Within 30 sec, increases in glucose output by the cells can be detected, and within a few minutes of the first exposure to hormone, the cells respond less and less readily to the hormone. After about 45 min the cells produce glucose at a slower rate than untreated control cells (Fig. 3 ) and are unresponsive even to much higher concentrations of the agonist (Mommsen and Plisetskaya, 1993). If a rat system can serve as a model, the obvious rapid decrease in fish hepatocyte responsiveness to GLP (Fig. 3 ) or glucagon (not shown) is due to desensitization of postreceptor mechanisms (Houslay et al., 1992).Down-regulation (decreased availability) of receptors can likely be ruled out as an explanation because internalization of glucagon is a relatively slow process in fish hepatocytes (Navarro and Moon, 1994). Nevertheless, fish liver has been shown to remove 75% of hepatic vein glucagon and more than 50% of its GLP in a single pass (Plisetskaya and Sullivan, 1989).

G. Conclusion Apart from the untranslated regions, preproglucagon gene structure appears to be similar in fishes and mammals, but small, physiologically important differences exist in the primary transcript and in processing sites. The glucagon sequence is highly conserved throughout all vertebrates, and in fishes the peptide assumes the same pivotal position opposite of insulin in carbohydrate and lipid metabolism. The hormone may do so b y slightly different intracellular routes and using

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249

MOLECULAK ASPECTS OF PANCREATIC PEPTIDES

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20

40

60

80

100

120

Time (minutes) Fig. 3. Time course of glucose production in response to truncated mammalian GLP in isolated rockfish (Sebastes caurinus) hepatocytes demonstrating the rapid desensitization of the cells to the hormone. Cells respond similarly to fish GLPs, mammalian glucagon, and fish glucagons. Cell behavior is tested as glucose release from endogenous glycogen. Glucose release is presented as an arbitrary rate per time interval (10 or 15 min), that is, a slope approaching zero indicates a constant rate of glucose production. Solid circles: control treated with vehicle; open circles: cells exposed to 5 nmol/liter of GLP1-37at 10 rnin. Data recalculated from Momrnsen and Plisetskaya (1993).

different intracellular targets, but ultimately by targeting the same tissues. The same cannot be said for the other important processing product of the proglucagon gene in fish pancreas-the GLPs. One gene product (GLP-2) is missing from the fish altogether. Fish GLP is structurally similar to the truncated GLP-1 of mammals, but different routes and sites of processing lead to the mature gene product. The fish gene is devoid of a region corresponding to the N-terminal sixamino-acid extension found in the mammalian proglucagon. Also, the mammalian pancreas secretes the full-length GLP-1, which is largely inactive. Fish proglucagon processing leads to storage and secretion of a biologically fully active short GLP from the pancreas. In the final analysis, GLP is an endocrine hormone and a powerful antagonist to

250


glucagon via its action on insulin synthesis and release. In fishes the same gene product (piscine and mammalian GLPs are freely interchangeable in their actions) functions as a true “glucagonlike” hormone. IV. SOMATOSTATIN

A. Gene Structure Although somatostatin genes have not been cloned from any fish species, there are several lines of evidence indicating that teleosts contain two somatostatin genes. Two cDNAs coding for different preprosomatostatins have been cloned and sequenced from anglerfish (Lophius americanus) (Goodman et al., 1980a; Hobart et al., 1980a; Goodman et al., 1982) and channel catfish (Zctalurus punctatus) (Taylor et al., 1981; Magazin et al., 1982; Minth et al., 1982). Southern blot analysis of Zctalurus genomic DNA using probes specific for each ofthe cloned cDNAs suggests that the corresponding genes are located on different fragments ofthe genome (Minth et al., 1982).Furthermore, multiple somatostatin peptides with different amino acid sequences have been isolated from coho salmon (Oncorhynchus kitsutch) (Plisetskaya et al., 1986), as well as daddy sculpin and flounder (Cottus scorpius and Platichthys flesus) (Conlon et al., 1987a).

I3. Messenger RNA Transcripts and cDNA Sequences Two anglerfish (Lophius americanus) somatostatin cDNAs have been sequenced and designated SST gene I and SST gene I1 cDNAs (Goodman et al., l980a, 1982; Hobart et al., 1980a). The SST gene 1 cDNA encodes a 121-amino-acid precursor containing a 25-amino-acid signal peptide, an 82-amino-acid propeptide region, and SST-14 at the carboxy terminus (Fig. 4). The sequence of anglerfish gene I SST-14 (aSST-14 I ) is identical to mammalian SST-14 and is preceded by the dibasic processing signal of arginine-lysine. There is also a single arginine residue upstream from the aSST-14 sequence that could be utilized to generate aSST-28 (see Section IV,C,2). The anglerfish gene I preprosomatostatin (aPPSS-I) sequence reported by Hobart et al. (1980a) differs at one nucleotide from the corrected sequence reported by Goodman et al. (1982). This nucleotide change is a glycine to glutamic acid substitution at residue 58 of the propeptide and the

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Producing Cell

aSST-14

aPPSS

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121 amino acid precursor -25

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- 1

20

60

02

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R

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4

96

I

t t

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Producing Cell

aSST-28

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125 amino acid precursor

-24

I

- 1

I

4

30

73

07

KR

R

RK

101

t aSST-28

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(h Fig. 4. Processing of preprosomatostatin precursors in SST-14 and SST-28 producing cells of the anglerfish (Lophius americanus) Brockmann body. Negative numbers refer to amino acid residues in the signal peptide. Positive numbers refer to residues of the prohormone and mature hormone. Open arrows indicate the location of signal peptide cleavage by signal peptidase. Large solid arrows indicate the major processing sites utilized to generate mature somatostatins. Small solid arrows indicate minor cleavage sites. The residues indicative of gene I somatostatins (Phe', Thr'") and the substitutions that are the hallmark of gene I1 somatostatins (Ty?', Gly24)are indicated in the respective mature aSSTs. The lysine hydroxylation site on aPPSS-I1 is indicated by OH.

discrepancy may be attributed to allelic variation or sequencing errors. Peptide sequencing data indicates that residue 58 is glutamic acid (Andrews and Dixon, 1987). Anglerfish gene I1 cDNA codes for a 125-amino-acid preprosomatostatin (aPPSS-11) containing a 24-residue signal peptide, an 87-amino-

252


acid propeptide, and aSST-14 at the carboxy terminus. Like aPPSS-I, this cDNA codes for mono- and dibasic residues that could be utilized to generate both aSST-14 and aSST-28 (see Section IV,C,2). However, anglerfish gene I1 SST-14 (aSST-14 11) contains two amino acid substitutions relative to aSST-14 I. Phenylalanine-7 has been replaced by tyrosine and threonine-10 has been replaced by glycine, which generates (Tyr7Gly")SST-14 (Hobart et al., 1980a). aPPSS-I and aPPSS-I1 share approximately 45% amino acid sequence identity. Northern blot analysis of Brockmann body RNA with probes specific for aPPSS-I and aPPSS-I1 indicate that gene I and gene I1 mRNAs comigrate with markers of approximately 700 nucleotides. However, gene I and gene I1 mRNAs have been localized to different cell populations in the Brockmann body. Gene I mRNAs were found in large clusters of cells that were distributed throughout the islet whereas gene I1 mRNAs were present in smaller clusters of cells. Gene I and gene I1 mRNAs do not appear to be colocalized to the same regions of the Brockmann body (Sevarino et al., 1989). Two cDNAs coding for distinct somatostatin peptides have been isolated from the channel catfish (Zctalurus punctatus). One cDNA codes for a 114- amino-acid precursor containing a signal peptide, a propeptide of approximately 75 amino acids, and SST-14, which is identical in sequence to aSST-14 I and the mammalian SST-14 peptides. This cDNA also codes for a pair of basic residues immediately preceding the SST-14 sequence. The catfish SST-14 cDNA lacks a monobasic processing site upstream of the SST-14 sequence and it is therefore not possible to produce catfish SST-22 from this product (Taylor et al., 1981; Minth et al., 1982). The second catfish somatostatin cDNA encodes a 105-amino-acid precursor containing a typical signal peptide, a pro region of approximately 57 amino acids, and the somatostatin sequence analogous to the SST-22 peptide previously isolated by Oyama et al. (1980). The SST-22 sequence is preceded by a single arginine residue. Catfish SST-22 and SST-14 differ in amino acid sequence at 7 out of 14 residues. The catfish SST-22 precursor cannot be processed to SST-14 owing to the replacement of the dibasic processing signal argininelysine with lysine-proline (Magazin et al., 1982). Northern blots of catfish Brockmann body RNA with a gene I (SST14) probe reveal a strong hybridization signal at 1000 nucleotides and two minor bands at 1375 and 810 nucleotides (Taylor et al., 1981).A single band of 880 nucleotides is detected using a probe of catfish gene I1 (SST-22) (Magazin et al., 1982).

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C . Biosynthesis 1. COTRANSLATIONAL PROCESSING

a . Signal Peptide Cleavage. The functionality of anglerfish preprosomatostatin signal peptides has been verified using in vitro translation systems. When anglerfish Brockmann body RNA is used in cell-free translation reactions, peptides of 14 to 18 kDa molecular mass are generated that can be immunoprecipitated with antisera to SST-14. When translation is performed in the presence of pancreatic microsoma1 membranes containing signal peptidase activity, the molecular weight of SST-14 immunoprecipitable proteins decreases by about 2 kDa (Goodman et al., 1980b; Shields, 1980). Signal peptide cleavage sites have been determined by sequence analysis of metabolically labeled aPPSS-I peptides (Noe et al., 1986a) and fast atom bombardment mass spectrometry (FABMS) of isolated aPPSS-I and aPPSS-I1 peptides (Andrews and Dixon, 1987; Andrews et al., 1987).Results indicate that the signal peptide cleavage ofaPPSS1occurs at the C ~ S ” - S ~bond. ? ~ The aPPSS-I1 signal peptide is cleaved at the Se?4-Gln25 bond (Fig. 4). 12. Lysine Hydroxylation. Anglerfish prosomatostatin I1 (aPSS-11) has been found to contain hydroxylysine at residue 23 of the SST-28 sequence (Fig. 4). The significance of this modification in terms of aSST-28 function is unknown. Hydroxylation of lysine residues is thought to be a cotranslational modification. The hydroxylation signal, X-Lys-Gly, is unique to anglerfish SST-28 because of a glycine substitution for the canonical threonine residue that is found in the analogous position of gene 1 and mammalian SST-14 (Andrews et al., 1984a; Spiess and Noe, 1985).Approximately 40% of the SST-28 in anglerfish Brockmann body contains hydroxylysine (Morel et al., 1984).

2. POSTTRANSLATIONAL PROCESSING u. Anglerfish Prosomatostatins. As described earlier, both aPSS-I and aPSS-I1 contain dibasic residues that could be cleaved to generate aSST-14 I and aSST-14 11, respectively. Both precursors also contain a monobasic cleavage site located 15 residues on the amino-terminal side beyond the dibasic processing site that could be utilized to produce aSST-28 I and aSST-28 11. aPSS-I and aPSS-I1 share 66% amino acid sequence identity between the carboxy-terminal 29 residues, which contain aSST-14, aSST-28, and putative processing signals. Despite this high degree of conservation, it has been shown that aPSSI and aPSS-I1 are processed to generate different products (Fig. 4).

254


High-pressure liquid chromatography (HPLC)and sequencing analysis of metabolically labeled peptides isolated from anglerfish Brockmann body indicate that aPSS-I is cleaved at Arga1Lys8' to generate aSST-14 I. aSST-28 I was not detected (Noe, 1981; Noe et al., 1986a). FABMS analysis of aPSS-I-derived peptides confirmed this observation; aSST-14 I, but not aSST-28 I, was present in Brockmann body extracts (Andrews and Dixon, 1987). Other peptides detected in these experiments were aPSS-I (1-27), (1-67), and (69-80). aPSS-I (1-27) is generated by cleavage at the monobasic residue Arg28.aPSS-I (1-67) is produced after cleavage at ArgW,the monobasic cleavage site that must be utilized to liberate aSST-28. Because aSST-28 I is apparently not produced, it is likely that cleavage at the dibasic site necessary to release aSST-14 I precedes cleavage at Arg68.Cleavage at both ofthese sites generates aPSS-I (69-80). Several studies indicate that, relative to aPSS-I, aPSS-11 is processed in a reciprocal manner. aSST-28 I1 has been isolated from Brockmann body extracts, but aSST-14-11 was not detected during the purification procedure (Morel et al., 1984). HPLC analysis of labeled Brockmann body peptides revealed that gene I1 SST is larger than aSST-14 (Noe and Spiess, 1983). FABMS experiments showed that aPSS-I1 is cleaved at Arg73to generate aSST-28 11. A minor cleavage site is which produces aPPSS-I1 (1-36). aSST-14 I1 was not detected (Andrews et al., 1987). In addition to differential processing, it is also apparent that aPPSSI and aPPSS-I1 are expressed in different cells of the Brockmann body. McDonald et al. (1987) showed by immunohistochemistry that aSST14 and aSST-28 are present in distinct areas of the islet and are not produced by beta, alpha, or pancreatic polypeptide cells. aSST-14 cells were found in large clusters distributed evenly throughout the islet. aSST-28 immunopositive cells were found individually or in small clusters and were often associated with glucagon-producing cells. The physiological significance ofthis association is not clear, and the mechanism responsible for cell-specific expression of SST gene I and gene I1 are unknown. These results have been corroborated by in situ hybridization with probes for anglerfish SST gene I and gene I1 mRNAs (Sevarino et al., 1989). The fact that aPPSS-I and aPPSS-I1 are posttranslationally processed at different sites, although both mono- and dibasic processing sites are present in each precursor, and that they are expressed in different cells raises the possibility that processing specificity may be dictated by (a) secondary structure surrounding the mono- and dibasic cleavage signals or (b)cellular factors, that is, the presence or absence

8.


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of appropriate processing enzymes. Sevarino et al. (1989) were able to distinguish between these factors by expressing cDNAs coding for aPSS-I and aPSS-I1 in mammalian cell lines. It had been previously determined that mouse corticotroph AtT20 cells generate both SST14 and SST-28 from transfected rat SST cDNA (rats have only one SST gene that can be processed to SST-14 or SST-28). The rat RIN 5F insulinoma cell line generates only SST-14 from transfected rat SST cDNA. When expressed in AtT2O cells, aPSS-I and aPSS-I1 were both processed to aSST-14 and aSST-28. In RIN 5F cells, both anglerfish precursors were processed to aSST-14, but aSST-28 was not detected. These results indicate that there are no inherent properties in the anglerfish prosoniatostatins molecules per se that preclude them from being processed to both aSST-14 and aSST-28. It is therefore likely that cellular factors are responsible for determining the patterns of aPSS processing. It is also interesting to note that the studies described here (Noe et al., 1986a; Andrews and Dixon, 1987; Andrews et al., 1987) provide evidence that both aPSS-I andaPSS-I1 are cleaved at mono- and dibasic sites to some extent (Fig. 4). This implies that regulation of processing might be achieved by (a) controlling expression of a repertoire of enzymes capable of distinguishing one monobasic site (or one dibasic site) from another by subtle differences in the context of the cleavage site or (b) closely regulating the level of expression of monobasic and dibasic processing enzymes.

b. Catfish Prosomatostatins. Both SST-14 and SST-22 have been isolated from channel catfish (Ictalurus punctatus) Brockmann bodies (Oyama et al., 1980; Andrews and Dixon, 1981). SST-14 and SST-22 share 50% amino acid sequence identity and cDNA analysis indicates that they are the products of different genes. The SST-14 prohormones contains a proline residue in place of the arginine residue necessary for generation of SST-22 and, therefore, cannot be processed to yield the larger peptide (Minth et al., 1982). In the SST-22 prohormone, the dibasic processing site utilized to produce SST-14 has been replaced by lysine-proline, eliminating conversion of this precursor to SST-14 (Magazin et al., 1982). SST-22 has been shown to be O-glycosylated on the threonine residue at position 5 (Andrews et al., 1984b). c . Other Fish Species. Four somatostatin peptides have been isolated from coho salmon (Oncorhynchus kitsutch). The predominant islet somatostatin is SST-25, which contains the T y P , GlyZ1sequence characteristic of anglerfish SST-28 11. In contrast to the situtation in anglerfish, salmon islets appear to be capable of producing SST-14 I1

256

STEPHEN J . DUGUAY AND THOMAS P. MOhlMSEN

from SST-25 I1 or its precursor. There is also evidence for very low levels of SST-28, an amino-terminal extension of SST-25. The fourth somatostatin present in salmon Brockmann body is SST-14 I, which is identical to anglerfish SST-14 I (Plisetskaya et al., 1986).Immunohistochemical studies revealed that SST-14 is located in the central part of the islet, in close association with beta cells, and SST-25 I1 is localized to the periphery of the islet and juxtaposed with alpha cells (Nozaki et al., 1988). Somatostatin peptides have been isolated from islet tissue of daddy sculpin (Cottus scorpius) and flounder (Platichthys jiesus). Both species contain SST-14 I peptides that are identical to anglerfish SST-14I (Conlon et al., 1987a; Cutfield et al., 1987). In addition, sculpin and flounder also produce SST-28 peptides that have 92% sequence identity to each other and 86% identity with anglerfish SST-28 11. These peptides contain the conserved dibasic residues necessary to produce an SST-14 I1 peptide, but they do not appear to be utilized as SST-14 11 was not detected. Both sculpin and flounder SST-28 I1 peptides contain Ty?' and Gly24residues, as does aSST-28 I1 (Conlon et al., 1987a). Two somatostatins have been isolated from the bowfin (Amia cuZva) and they appear to be derived from the same precursor. Bowfin SST14 is identical to aSST-14 1and bowfin SST-26 is an amino-terminally extended form of SST-14. The antisera used to identify somatostatin peptides during purification does not recognize gene Il-type SSTs (Tyr7Gly"). Therefore, the existence of additional SSTs in bowfin cannot be ruled out (Wang et al., 1993). Using the antisera specific to gene I-type SSTs, Conlon and colleagues have also identified SST-14 in the Pacific ratfish (Hydrolagus colliei),a holecephalan, and the elasmobranch (Torpedo marmoratu). Torpedo SST-14 is identical to aSST-14 I, whereas the ratfish SST-14 contains a serine substitution for asparagine at position 5 (Conlon et al., 1985; Conlon, 1990). Somatostatins have also been isolated from the oldest class ofvertebrates, the agnathans. The islet of the lamprey Petromyzon marinus contains three SSTs that appear to be derived from a single precursor. The largest and least abundant form is SST-37. SST-34 is generated from SST-37 or a common precursor by cleavage at a single arginine residue. SST-14 is produced by additional cleavage at a dibasic site to produce a gene 1-type molecule that differs from aSST-14 I by substitution of serine for threonine at position 12. SST-14 and SST34 are apparently produced by the same cells of the islet (Andrews et al., 1988).

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257

The most abundant somatostatin in Atlantic hagfish (Myxine glutinosa) islet tissue is SST-34. Monobasic and dibasic cleavage sites for the generation of SST-28 and SST-14 are present in this precursor. However, it appears that only the dibasic site is utilized to generate SST-14, which is identical to aSST-14 I (Conlon et al., 1988a).

3. PROHORMONE CONVERTASES The nature of the enzyme involved in converting prohormones to biologically active mature hormones has been the subject of extensive investigation for many years (Steiner et al., 1992), and the anglerfish (Lophius americanus) Brockmann body has been a valuable model system for studying these enzymes (Mackin et al., 1990). Two putative prohormone convertases (PCs) have been isolated from anglerfish Brockmann body secretory granules. One enzyme converts aPSS-I to aSST-14 I and can also process aPSS-I1 to the unnatural product aSST-14 11. The second enzyme converts aPSS-I1 to aSST-28 11. The aSST-14 I generating enzyme also converts anglerfish proinsulin to insulin by cleavage at dibasic residues flanking the C-peptide. The aSST-28 I1 generating enzyme does not recognize proinsulin as a substrate. This indicates that mono- and dibasic cleavages are performed by distinct PCs (Mackin and Noe, 1987b). The aSST-14 generating enzyme migrates as a doublet band of 67,65 kDa and at 57 kDa on SDS-polyacrylamide gels. Activity of this enzyme is calcium dependent, and the amino-terminal sequence is identical to that of the mammalian dibasic prohormone convertase PC2 in 11 of 17 residues (Mackin et aZ., 1991b). PC2 is also known to be calcium dependent and is activated b y removal of an aminoterminal pro region to produce the smaller, functional protease (Steiner et al., 1992). The aSST-28 I1 generating enzyme has been identified as a singlechain 39-kDa protein. This enzyme displays maximal activity toward monobasic sites at pH 4.2 and this activity is abolished by aspartyl protease inhibitors. Amino-terminal sequence analysis reveals significant homology to other known aspartyl proteases (Mackinet al., 1991a). An apparent homolog to the anglerfish monobasic aspartyl protease has been identified in the yeast Saccharomyces cerevisiae. When aPSS-I1 is expressed in yeast it is processed exclusively to aSST28 in both normal and kex2-deficient yeast strains (Bourbonnais et al., 1991). This is significant because the Kex2 enzyme is the yeast homolog of vertebrate serine proteases with specificity toward dibasic residues, that is, furin, PC1/3, and PC2. Bourbonnais et al. (1993) succeeded in isolating yeast mutants that were deficient in

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somatostatin-28 expression (sex mutants) and identified the defective gene as yeast aspartyl protease 3 (YAP3). They showed that yeast strains with a functional YAP3 gene could process aPSS-I to aSST-28 I and aPSS-I1 to aSST-28 11.

D. Secretion Regulation of secretion of somatostatin from fish islets has not been studied extensively. However, it appears that fish islets respond to secretagogues in a fashion similar to that of mammalian islets. Glucose evokes a biphasic release of SST-14 from perfused catfish (Ictalurus punctatus) Brockmann body whereas arginine has only a minor effect (Ronnerand Scarpa, 1982,1984). Mannose was also an effective secretagogue in this system; fructose and a-ketoisocaproate had minor effects; and alanine and leucine were ineffective (Ronner and Scarpa, 1987). Glucose also stimulates a biphasic release of SST-14 from perfused Brockmann body of the European silver eel (Anguilla anguilla) (Ince and So, 1984).

E. Physiological Actions Most of the physiological studies of Brockmann body somatostatins have focused on SST-25 in coho salmon (Oncorhynchus kitsutch). When injected into salmon, SST-25 decreases plasma insulin, glucagon, and GLP levels, depletes liver glycogen content, and causes hyperglycemia. Administration of SST-25 antiserum results in SST25 deficiency with associated increases in plasma insulin and liver glycogen content. The reader is referred to several reviews for further details (Plisetskaya, 1989; 1990a,b; Plisetskaya and Duguay, 1993).

V. PANCREATIC POLYPEPTIDE AND

RELATED PEPTIDES A. The Pancreatic Polypeptide Family The pancreatic polypeptide (PP) family is composed of three peptides that are 36 amino acids long, contain an amidated carboxy terminus, and share extensive sequence similarities. The common feature that defines this family, however, is the unique tertiary structure known as the PP-fold. Structural analysis using techniques such as model building, circular dichroism, secondary structure prediction

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algorithms, and X-ray crystallography indicate that members of the PP family contain an amino-terminal polyproline helix and a carboxyterminal a-helix joined by a type-I1 p-turn (Glover et al., 1985). In mammals, three members of the PP family have been characterized. PP is expressed in the pancreas. Peptide Y (PYY) is found in the intestine and the name reflects the fact that the first and last residues of the amino acid sequence are tyrosine (single-letter code = Y). Neuropeptide Y (NPY) is localized to the nervous system and its primary sequence also begins and ends with tyrosine (Larhammar et al., 1993). B. cDNA and Peptide Sequences PP family peptides and cDNA clones have been characterized from several piscine species. The sequences are shown in Table IV. All piscine sequences have been determined from peptides isolated from islet tissue and are therefore referred to as “PP” except the ray (Torpedo rnarrnorata) and goldfish (Carassius auratus) NPY sequences, which were deduced from cDNA clones obtained from libraries of nervous tissue. Also, the anglerfish peptide isolated from the Brockmann body has been named aPY by Andrews et al. (1985).On the basis of sequence comparisons of PP family proteins from fish, amphibians, birds, and mammals, Larhammar et al. (1993) have argued that Torpedo NPY may resemble the ancestral peptide of the PP family. Torpedo NPY is 94% identical to both porcine and goldfish NPY, indicating that the primary structure has been extremely well conserved during evolution. The pancreatic polypeptides isolated from several fish species are actually more similar in structure to NPY than PP (Table IV). C. Prohormone Processing NPY cDNA sequences have been determined for goldfish (Carussius auratus) and ray (Torpedo marmoratn). Both cDNAs encode a

28-amino-acid signal peptide followed by the 36-amino-acid sequence of mature NPY. The Torpedo cDNA codes for a carboxy-terminal extension propeptide that is 30 amino acids long. The Carussius propeptide is 28 residues in length. The mature peptide and the propeptide are separated by the sequence Gly-Lys-Arg. The Lys-Arg residues serve as the dibasic processing site, which may be cleaved by a member of the PC family of serine prohormone convertases. The carboxy-terminal glycine residue then donates the amide group to produce tyrosineamide (Blomqvist et aZ., 1992).

Table IV Primary Structure of PP Family Peptides" ~~

Porcine PP

-VY -

Porcine PYY Alligator NPY

-EA-

TP-Q SP- E

__ __

MAQ - SR-

- ML- -v-

AAE -AS -

(47%) (64%)

M-R- -R-

Porcine NPY Kay NPY

PAED ---E

Goldfish NPY

(92%) (94%)

LAKY

( 100%)

____

(94%)

-P- E -P- E

(86%) (86%) (83%)

Salmon PP

-P-E -P- E

Skate PP

AP- E

Eel PP

SP- E SP- -

Dogfish PP Gar PP Bowfin PP

Sculpin PP Anglerfi\h PY Invariant residues

SP1

*

*

*

*

*

*

(83%) (81%) (78%) (64%) (64%)

-

*

h

*

*

*

*

* *36

'I Amino acid alignment of PP family peptides. Residues are represented by single-letter code. Dashes indicate residues identical to ray sequence. Sequence identity relative to the ray sequence (in bold) is indicated in parentheses at right. Invariant residues are labeled with an asterisk. For porcine sequence references, see Larhammar et ul. (1993); ray, Torpedo rnarrnoruta (Blomqvist et ul., 1992); alligator, Alligcitor r,iississippiensis (Parker et ul., 1993b); goldfish, Curussius ciuratus (Blomqvist et ul., 1992); dogfish, Scqliorhinus cuniculu (Conlon et d . ,19911)); alligator gar, Lepisosteus spatula (Pollock et al., 1987); bowfin, Amiu culuu (Conlon et al., 1 991 ~)coho ; salmon, Oncorhynchus kitsutch (Kimmel et ul., 1986); skate, Ruju rhina (Conlon et ul., 1 9 9 1 ~ ); American eel, Anguilla rostruta (Conlon et ul., 1 9 9 1 ~ )daddy ; sculpin, Cottus scorpius (Conlon et al., 1986); anglerfish, Lophius urnericanus (Andrews and Dixon, 1986).

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In addition to mature PY, the putative proregion of this molecule has been isolated from anglerfish (Lophius americanus) Brockmann bodies and identified b y homology to the pro region of human proNPY (Andrews and Dixon, 1986). D. Immunohistochemical Identification of Peptides

PP family peptides have been isolated from the agnathan intestine (Conlon et al., 1991d), and there is immunohistochemical evidence for the existence of these peptides in islets of the lamprey. Using antisera to mammalian NPY and PP, and anglerfish PY, immunoreactivity was observed in the pancreas of Petromyzon marinus. All antisera stained the same cells and these cells were distinct from B- and Dcells (Cheung et al., 1991). However, Youson and Potter (1993) found cells that were immunopositive for NPY and aPY in the intestine but not the islet of two other lampreys (Geotria australis and Mordacia mordax). In the dogfish (Squalus acanthias) and the coho salmon (Omorhynchus kitsutch), PP immunoreactive cells are localized to the periphery of the islet. PP cells are associated with A-cells in the salmon (ElSalhy, 1984; Yi-Qiang et al., 1986). PP and glucagon are found in the same cells in small and intermediate islets of Sparus auratus but are not colocalized in the principal islet (Abad et al., 1988). In anglerfish (Lophius americanus),aPY is expressed in the islet and NPY immunoreactivity has been localized to islet nerves (Noe et al., 1986b). E. Physiological Actions

The physiological function of PP in fish is unknown. When tested in mammalian systems, piscine PPs exert NPY-like effects. These include increasing blood pressure and decreasing heart rate in rats, as well as stimulating appetite (Balasubramaniam et al., 1990). Injection of the dogfish (Scyliorhinus canicula) pancreatic NPY-like peptide into dogfish causes an increase in blood pressure (Conlon et al., 1991b).

ACKNOWLEDGMENTS We thank Erika Plisetskaya for stimulating discussion. S. J . D. is supported b y a Post-Doctoral Fellowship from the Howard Hughes Medical Institute. T. P. M. acknowledges the continued support through a research grant from the Natural Sciences and Engineering Research Council of Canada.

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Lopez, L. C., Li, W.-€I., Frazier, M. L., Luo, C.-C., and Saunders, G. F. (1984). Evolution of glucagon genes. M o l . Biol. Evol. 1, 335-344. Lukowsky, A., Prehn, S., and Rapoport, T. A. (1974). Biosynthesis of proinsulin in islets of Langerhans of the carp (Cyprinus carpio). Biochim Biophys. Acta 359,248-252. Lund, P. K., Goodman, R. H., Montminy, M. R., Dee, P. C., and Habener, J. F. (1983). Anglerfish islet pre-proglucagon 11. Nucleotide and corresponding amino acid sequence of the cDNA. J . Biol. Chem. 258,3280-3284. Lund, P. K., Hoyt, E., Simmons, J. G., and Ulshen, M. H. (1993). Regulation of intestinal glucagon gene expression during adaptive growth of small intestine. Digestion 54, 371-37:3. McDonald, J. K., Greiner, F., Bauer, G. E., Elde, R. P., and Noe, B. D. (1987). Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated. 1. Histoclaem. Cytochem. 35, 155- 162. Mackin, R. B., and Noe, B. D. (1987a). Characterization of an islet carboxypeptidase B involved in prohormone processing. Endocrinology (Baltimore) 120, 457-468. Mackin, R. B., and Noe, B. D. (1987b). Direct evidence for two distinct prosomatostatin converting enzymes. J . Biol. Chem. 262,6453-6456. Mackin, R. B., Noe, B. D., and Spiess, J. (1990). Purification of prosomatostatinconverting enzymes. Metabolism 39, Suppl. 2, 30-32. Mackin, R. B., Noe, B. D., and Spiess, J. (1991a). The anglerfish somatostatin-28generating propeptide converting enzyme is an aspartyl protease. Endocrinology (Baltimore) 129, 1951-1957. Mackin, R. B., Noe, B. D., and Spiess, J . (1991b). Identification of a somatostatin-14generating propeptide converting enzyme as a member of the kex2/furin/PC family. Endocrinology (Baltimore) 129, 2263-2265. Magazin, h4., Minth, C. D., Funckes, C. L., Deschenes, R., Tavianini, M. A., and Dixon, J . E. (1982). Sequence of a cDNA encoding pancreatic somatostatin-22. Proc. Natl. Acad. Sci. U.S.A.79, 5152-5156. Mallat, A,, Pavoine, C., Dufour, M., Lotersztajn, S., Bataille, D., and Pecker, F. (1987). A glucagon fragment is responsible for the inhibition of the liver Ca2+ pump by glucagon. Nature (London) 325,620-622. Mazeaud, F. (1964). Vitesse de production de I'hyperglucemie adrenalique en fonction de la temperature chez la carpe. C . R. SOC. Biol. 158, 36-40. Meyer, T. E., and Habener, J. F. (1993). Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription-activating deoxyribonucleic acid-binding proteins. Endocr. Reo. 14, 269-290. Miller, C. P., Lin, J . C., and Habener, J. F. (1993). Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins. Mol. Cell. Biol. 13, 7080-7090. Minth, C. D., Taylor, W. L., Magazin, M., Tavianini, M. A., Collier, K., Weith, H . L., and Dixon, J . E. (1982). The structure of cloned DNA complementary to catfish pancreatic somatostatin-14 messenger RNA. J . Biol. Chem. 257, 10372-10377. Mojsov, S., Weir, G. C., and Habener, J . F. (1987). Insulinotropin: Glucagon-like peptide I (3-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J . Clin. Invest. 79,616-619. Mommsen, T. P., and Moon, T. W. (1990). Metabolic response of teleost hepatocytes to glucagon-like peptide and glucagon. J . Endocrinol. 126, 109-1 15. Mommsen, T. P., and Plisetskaya, E. M. (1991). Insulin in fishes and agnathans: History, structure, and metabolic regulation. Rev. Aquat. Sci. 4,225-259.

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Mommsen, T. P., and Plisetskaya, E. M. (1993). Metabolic and endocrine functions of glucagon-like peptides-Evolutionary and biochemical perspectives. Fish Ph ysiol. Biochem. 11,429-438. Morel, A., Chang, J.-Y., and Cohen, P. (1984). The complete amino acid sequence of anglerfish somatostatin-28 11. F E B S L e t t . 175, 21-24. Moss, L. G., Moss, J. B., and Rutter, W. J. (1988). Systematic binding analysis of the insulin gene transcription control region: Insulin and immunoglobulin enhancers utilize similar transactivators. Mol. Cell. Biol. 8, 2620-2627. Murayama, Y., Kawai, K., Suzuki, S., Ohashi, S., and Yamashita, K. (1990).Glucagon-like peptide-1 (7-37) does not stimulate either hepatic glycogenolysis or ketogenesis. Endocrinol. Jpn. 37,293-297. Navarro, I., and Moon, T. W. (1994). Glucagon binding to hepatocytes from two teleost fishes: The American eel and the brown bullhead. J . Endocrinol. 140,217-227. Navarro, I., Gutierrez, J., Caixach, J., Rivera, J., and Planas, J. (1991). Isolation and primary structure of glucagon from the endocrine pancreas of Thunnus obesus. Gen. Comp. Endocrinol. 83,227-232. Nguyen, T. M., Mommsen, T. P., Mims, S. D., and Conlon, J. M. (1994).Characterization of insulins and proglucagon-derived peptides from a phylogenetically ancient fish, the paddlefish (Polyodon spathula). Biochem. J . (in press). Nichols, R., Lee, T. D., and Andrews, P. C. (1988). Pancreatic proglucagon processing: Isolation and structures of glucagon and glucagon-like peptide from gene I. Endocrinology (Baltimore)123, 2639-2645. Noe, B. D. (1981). Synthesis of one form of pancreatic islet somatostatin predominates. J . Biol. Chem. 256,9397-9400. Noe, B. D., and Spiess, J . (1983). Evidence for biosynthesis and differential posttranslational proteolytic processing of different (pre)prosomatostatins in pancreatic islets. J . B i d . Chem. 258, 1121-1128. h e , B. C., Andrews, P. C., Dixon, J. E., and Spiess, J. (1986a). Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue. J . Biol. Chem. 103, 1205-1211. Noe, B. D., McDonald, J. K., Greiner, F., Wood, J. G., and Andrews, P. C. (1986b). Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY. Peptides 7, 147-154. Nozaki, M., Miyata, K., Oota, Y., Gorbman, A., and Plisetskaya, E. M. (1988).Different cellular distributions of two somatostatins in brain and pancreas of salmonids, and their associations with insulin- and glucagon-secreting cells. Gen. Cornp. Endocrinol. 69, 267-289. Ottolenghi, C., Fahhri, E., Puviani, A. C., Gavioli, M. E., and Brighenti, L. (1990). Adenylate cyclase of catfish hepatocyte membrane: Basal properties and sensitivity to catecholamines and glucagon. M o l . Cell. Endocrinol. 60: 163-168. Oyama, H., Bradshaw, R. A,, Bates, 0.J., and Permutt, A. (1980).Amino acid sequence of catfish pancreatic somatostatin I. J . Biol. Chem. 255, 2251-2254. Parker, D. B., Coe, I. R., Dixon, C . H., and Sherwood, N. M. (1993a). Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily. Eur. J . Biochem. 215,439-448. Parker, D. B., McRory, J. E., Fischer, W. H., Park, M., and Sherwood, N. M. (1993h). Primary structure of neuropeptide Y from brains of the American alligator (Alligator mississippiensis). Regulat. P e p t . 45, 379-386. Patzelt, C., and Weber, B. (1986). Early 0-glycosidic glycosylation of proglucagon in pancreatic islets: An unusual type of prohormone modification. E M B O J . 5, 2103-2108.

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Philippe, J . , and Rochat, S. (1991). Strict distance requirement for transcriptional activation by two regulatory elements ofthe glucagon gene. DNA Cell Biol. 10, 119-124. Philippe, J., Drucker, D. J., and Habener, J. F. (1987). Glucagon gene transcription in an islet cell line is regulated via a protein kinase C-activated pathway.]. Biol. Chem. 262, 1823-1828. Plisetskaya, E. M. (1989). Pancreatic peptides. In “The Comparative Physiology of Regulatory Peptides” (S. Holmgren, ed.), pp. 174-202. Chapman & Hall, London and New York. Plisetskaya, E . M. (1990a). Clucagon and related peptides (an overview). In “Progress in Comparative Endocrinology,” pp. 67-72. Wiley-Liss, New York. Plisetskaya, E. M. (1990b).Endocrine pancreas of teleost fish: A model for interaction of islet hormones. J . E x p . Zool. S u p p l . 4, 53-57. Plisetskaya, E . M., and Duguay, S. J. (1993). Pancreatic hormones and metabolism in ectotherm vertebrates: Current views. I n “The Endocrinology of Growth, Development, and Metabolism in Vertebrates” ( M . P. Schreibman, C. G. Scanes, and P. K. T. Pang, eds.), pp. 265-287. Academic Press, New York. Plisetskaya, E. M., and Sullivan, C. V. (1989). Pancreatic and thyroid hormones in rainbow trout (Salmogairclneri): What concentration does the liver see? Gen. Comp. Endocrinol. 75,310-315. Plisetskaya, E. M., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., and Corbman, A. (1986).Isolation and structures of coho salmon (Oncorhynchus kisutch) glucagon and glucagon-like peptide. Regul. Pept. 14,57-67. Plisetskaya, E. M., Ottolenghi, C., Sheridan, M. A,, Mommsen, T. P., and Gorbman, A. (1989).Metabolic effects of salmon glucagon and glucagon-like peptide in coho and chinook salmon. Gen. Comp. Endocrinol. 73, 205-216. Pollock, H. C., Kimmel, J. R., Hamilton, J. W., Rouse, J. B., Ebner, K. E., Lance, V., and Rawitch, A. B. (1987). Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide. Gen. Comp. Enclocrinol. 67, 375-382. Pollock, H. C., Kimmel, J . R., Ebner, K. E., Hamilton, J. W., Rouse, J . B., Lance, V., and Rawitch, A. B. (1988).Isolation of alligator gar (Lepisosteus sputula) glucagon, oxyntomodulin, and glucagon-like peptide: Amino acid sequences of oxyntomodulin and glucagon-like peptide. Gen. Comp. Endocrinol. 69, 133-140. Prehn, S., Tsamaloukas, A,, and Rapoport, T. A. (1980). Demonstration of specific receptors of the rough endoplasmic membrane for the signal sequence of carp proinsulin. Eur. J . Biochem. 107, 185-195. Rapoport, T. A,, Hohne, W. E., Klatt, D., Prehn, S., and Hahn, V. (1976).Evidence for the synthesis of a precursor of carp proinsulin in a cell free translation system. F E B S Lett. 69, 32-36. Ronner, P., and Scarpa, A. (1982). Isolated perfused Brockmann body as a model for studying pancreatic endocrine secretion. Am. J . Physiol. 243, E352-E359. Ronner, P., and Scarpa, A. (1984). Difference in glucose dependency of insulin and somatostatin release. Am. J. Physiol. 246, E506-E509. Ronner, P., and Scarpa, A. (1987).Secretagogues for pancreatic hormone release in the channel catfish (Ictalurus punctatus). Gen. Comp. E d o c r i n o l . 65, 354-362. Rniz-Grande, C., Pintado, J., Alarcon, C., Castilla, C., Valverde, I., and Lopez-Novoa, J . M . (1990). Renal catabolism of human glucagon-like peptides 1 and 2. Can. J . Plzysiol. Pharmacol. 68, 1568-1573. Sala, G. B., Hayashi, K., Catt, K. J., and Dufau, M. L. (1979). Adrenocorticotropin action in isolated adrenal cells. The intermediate role of cyclic AMP in stimulation of corticosterone synthesis. J . Biol. Chem. 254, 3861-3865.

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Schmidt, W. E., Siegel, E. G., and Creutzfeldt, W. (1985). Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia 28, 704-707. Schwaninger, M., Lux, G., Blume, R., Oetjen, E., Hidaka, H., and Knepel, W. (1993). Membrane depolarization and calcium influx induce glucagon gene transcription in pancreatic islet cells through the cyclic AMP-responsive element. 1.B i d . Chem. 268,5168-5177. Sevarino, K. A., Stork, P., Ventimiglia,-R., Mandel, G., and Goodman, R. H. (1989). Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell (Cambridge, Mass.) 57, 11-19. Shields, D. (1980). In vitro biosynthesis of fish islet preprosomatostatin: Evidence of processing and segregation of a high molecular weight precursor. Proc. N a t l . Acad. Sci. U.S.A. 77,4074-4078. Shields, D., and Blobel, G. (1977). Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. Proc. N a t l . Acad. Sci. U.S.A. 74, 2059-2063. Sorokin, A. V., Petrenko, 0. I., Kavsan, V. M., Kozlov, Y. I., Dababov, V. G., and Zlochevskij, M. L. (1982).Nucleotide sequenceanalysis ofthe cloned salmon preproinsulin cDNA. Gene 20,367-376. Spiess, J . , and Noe, B. D. (1985). Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28. Proc. N a t l . Acad. Sci. U . S . A . 82, 277-281. Steiner, D. F. (1990).The biosynthesis of insulin. Handb. E x p . Pharmacol. 92,67-92. Steiner, D. F., Peterson, J . D., Tager, H., Edmin, S., Ostberg, Y., and Falkmer, S. (1973). Comparative aspects of proinsulin and insulin structure and biosynthesis. A m . Zool. 13,591-604. Steiner, D. F., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992).The new enzymology of precursor processing endoproteases. 1.Biol. Chem. 267,23435-23438. Suzuki, S., Kawai, K., Ohashi, S., Mukai, H., and Yamashita, K. (1989). Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas. Endocrinology (Baltimore) 125, 3109-31 14. Taylor, W. L., Collier, K. J., Deschenes, R. J., Weith, H. L., and Dixon, J . E. (1981). Sequence analysis of a cDNA coding for a pancreatic precursor to somatostatin. Proc. N a t l . Acad. Sci. U.S.A. 78, 6694-6698. Thorens, B. (1992). Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc. N a t l . Acad. Sci. U.S.A. 89, 8641-8645. Unson, C. G., Macdonald, D., Ray, K., Durrah, T. L., and Merrifield, A. B. (1991). Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding. J. Biol. Chem. 266,2763-2766. Unson, C. G., Macdonald, D., and Merrifield, R. B. (1993). The role of histidine-1 in glucagon action. Arch. Biochem. Biophys. 300, 747-750. Wang, Y., Youson, J . H., and Conlon, J . M. (1993). Prosomatostatin-I is processed to somatostatin-26 and somatostatin-14 in the pancreas of the bowfin, Amia calvu. Regul. P e p t . 47,33-39. Wheeler, M. B., Lu, M., Dillon, J. S., Leng, X.-H., and Boyd, A. E. (1993). The rat glucagon-like peptide-I (GLP-I)receptor can couple to adenylate cyclase, phospholipase C and the free cytosolic calcium level ([CA2']i). Digestion 54, 348-349. White, J. W., and Saunders, G. F. (1986). Structure of the human glucagon gene. Nucleic Acids Res. 14,4719-4730.

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9 THE M O L E C U L A R BIOLOGY OF THE C O R P U S C L E S OF STANNIUS A N D R E G U L A T I O N OF STANNIOCALCIN G E N E EXPRESSION GRAHAM F . WAGNER Department of Physiology, Faculty of Medicine University of Western Ontario London, Ontario, Canada

I. Introduction 11. A Brief History of Discovery 111. Molecular Cloning of Eel and Salmon Stanniocalcin IV. Structural Comparisons of Eel and Salmon Stanniocalcin V. Studies on Tissiie-Specific Expression of the Stanniocalcin G e n e VI. Localization of Stanniocalcin mRNA in CS Cells by i n Situ Hybridization VII. Calcium Regulation of Stanniocalcin Cell Activity A. Regulation of Stanniocalcin Secretion by Calcium B. Regulation of Stanniocalcin mRNA Levels by Calcium VIII. Conclusions References

I. INTRODUCTION For over 150 years, zoologists have been intrigued by the corpuscles ofstannius, endocrine glands that were first described by Stannius (1839) on the kidneys of teleostean and holostean fishes. The glands are unique to bony fishes as they have not been identified in other vertebrates and are derived embryologically from kidney tubule cells (Garrett, 1942; Kaneko et al., 1992). In salmon and trout, the corpuscles of Stannius are readily apparent as oval, cream-colored bodies situated midway along the ventral surface of each kidney. On average, a typical salmonid has 2-6 glands of varying size. In other fish, however, the glands can vary both in location (i.e., ureter) and number. Species such as the bowfin (Amin culua) can have more than 300 individual 273 FlSH P H Y S I O I L K Y , VOL XI11

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glands scattered throughout the posterior half of the kidney (Youson et al., 1976). But there is no evidence that variations in anatomical distribution have any bearing on their physiology. The corpuscles of Stannius (CS) play a major role in regulating calcium homeostasis through the synthesis and secretion ofstanniocalcin, a homodinieric, glycoprotein hormone with a novel primary structure. Stanniocalcin performs a function not unlike that of calcitonin in mammals, as one of its main roles is the prevention ofhypercalcemia. However, the two hormones acconiplish this through entirely different mechanisms. Whereas calcitonin inhibits osteoclastic bone resorption (Friedinan and Raisz, 1965; Milhaud et nl., 1965; Aliapoulios ct ul., 1966), stanniocalcin lowers the rate of gill calcium transport from the aquatic environnient (So and Fenwick, 1977, 1979). This underscores the differences between fish and mammals in the maintenance of calcium homeostasis. Unlike mammals, whicli rely on bone as a calcium reservoir, fish rely on the environnient as their principal source o f calcium and use the gills to draw from it according to metabolic needs. Despite the long passage oftinie since the CS discovery, the science o f their physiology has come of age only in the last 30 years beginning with Fontaine (1964), who first established a relationship between the C S and calcium homeostasis. The consequence of his discovery was renewed interest in these glands in laboratories all over the world. The CS have been extensively studied a s a result and are the subject of several reviews (Krishnamurthy, 1976; Wendelaar Bonga and Pang, 1986, 1991; Hirano, 1989; Wagner, 1993). In spite ofall that has been learned, however, the stanniocalcin field is still relatively unknown in comparison to other endocrine systems. The purpose of this chapter is to focus on recent developments concerning the molecular biology of the CS and stanniocalcin (STC), ’ specifically as they relate to tlie molecular cloning of STC, the loc,‘1I 1zation of STC mRNA in CS cells by i i i ,situ hybridization, and tlie regulation of STC gene expression by calcium. Because the niolecular biology of STC is a comparatively new field, there is not a large body of literature on the subject. Stanniocalcin has been cloned and sequenced from only two species of fish, salmon and eel. Therefore, readers should bear in mind that the perspective presented herein on comparative structure will be limited by the paucity of available data. Finally, few laboratories are presently engaged in studies on the regulation of STC gene expression, making it impossible to generalize with respect to salmonids, our chosen experiiiiental model, or bony fishes in gen-

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eral. As a consequence future reviews will be needed for periodic updates on this rapidly expanding and exciting field of research.

11. ,4 BRIEF HISTORY OF DISCOVERY Before Fontaine (1964) established a connection between the CS and calcium homeostasis, the glands were studied sporadically and by only a few laboratories around the world. Stannius (1839)believed that these structures were adrenal glands and, consequently, there were repeated efforts up until the inid-196Os to identify and characterize steroids and steroidogenic enzymes in CS tissue (Krishnamurthy, 1976). However, the ontogeny of CS cells was clearly different from that of fish interrenal tissue (Garrett, 1942), making it highly unlikely that they produced adrenal steroids at all. Ultrastructural studies then finally disclosed an extensive network of rough endoplasmic reticulum and Golgi and secretory granules in CS cells (Ogawa, 1967), all of which suggested that they synthesized polypeptides, not steroids. In a classical fashion, Fontaine (1964)demonstrated that surgically removing the CS in the European eel (Anguilln wnguilla) caused a form of hypercalceniia that could be alleviated simply by injecting CS extracts back into the animal. His findings established a connection between the CS and calcium homeostasis and inferred that the glands were the source of an antihypercalcenlic hormone, now known as stanniocalcin. Setting aside for the moment all queries as to the chemical nature of this active principle, efforts were focused on the cause ofthe hypercalceniia and the organs involved, again using the eel a s an experimental model. Originally it was believed that the hypercalcemia might be due to either decreased urinary calcium excretion or to increased mobilization of calcium froin bone. However, several laboratories independently concluded that removing the CS (stanniectomy) had no effect on the renal handling of calcium (Butler, 1969; Fenwick, 1974) or, for that matter, bone resorption (Fontaine et al., 1972). The environment was finally implicated as the source of calcium when it was observed that hypercalcemia did not develop if eels were transferred to low-calcium water following stanniectomy (Fenwick and So, 1974; Fontaine et al., 1972; Pang et nl., 1973). The gills were then pinpointed as the affected target organ when it was shown that the rate of gill calcium transport increased dramatically following stanniectomy, thereby revealing the true cause of the hypercalcemia. It was

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thus concluded that the CS were the source of an inhibitor of gill calcium transport ( Fenwick and So, 1974; So and Fenwick, 1977,1979). The active principle, STC, has since been purified from salmon and trout CS tissue and proven to be a potent inhibitor of gill calcium transport (Lafeber et al., 1988a,b; Wagner et al., 1986, 1988a, 1993). 111. MOLECULAR CLONING OF EEL AND

SALMON STANNIOCALCIN Literature searches turn up few studies of any kind dealing with nucleic acids in CS tissue prior to the molecular cloning of STC from the Australian eel, Anguillu australis (Butkus et al., 1987). There had been no attempts to even quantify KNA levels in CS cells, for example, or to identify the products of CS cells through in vitro translation of CS RNA. The few reports that existed at the time were mainly histological in nature. Tinctorial stains such as toluidine blue had been used to simply localize KNA in fixed tissue sections (Krishnamurthy, 1976). Furthermore, when Butkus and her colleagues began their quest for eel STC, the partial sequence of salmon STC had not yet appeared in press (Wagner et ul., 1986). As a consequence, they had little information at the outset regarding the size or structure of the hormone that could assist them in their cloning strategy. Accordingly, they devised an approach whereby the electrophoretic patterns of various eel tissue extracts were compared to that obtained with a CS extract. In this way they could identify proteins that were CS specific. Because of its sheer abundance in CS tissue, the STC band was selected for sequence analysis and proven to have a unique primary structure. On the basis of this sequence, an oligonucleotide probe (75-mer) was synthesized for screening an eel CS cDNA library and several positive clones were obtained. DNA sequence analysis of the largest clone yielded the complete primary structure ofeel STC, which consisted ofa 17-residue hydrophobic leader sequence, a 15-residue prosequence, and 231 amino acids comprising the mature protein core of the hormone. Northern blot analysis revealed that the eel STC message was 3.5 kilobases in length. Nothing further was accomplished in the field until the inolecular cloning of salmon STC (Wagner et al., 1992). Corpuscles of Stannius were collected from upstream migrating coho salmon (Oncorhynchus kisutch) as a source of RNA for library construction. Thereafter, the processes of oligonucleotide probe synthesis (50-mer) a n d library

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screening were made easier by knowledge ofthe N-terminal sequence of the coho salmon STC protein (Wagner et al., l988a). The entire salmon message was finally obtained in two cDNA clones that encompassed most of the 5' untranslated region, the complete protein coding region, and the entire 3' untranslated region. Northern blot analysis revealed that the salmon message was similar in size among representative salmonids ( 2 kb; Fig. l), but considerably smaller than in the Australian eel (3.5 kb).

-28s

-18s

Fig. 1. The rnRNA encoding STC is the same size ( 2 kb) among representative salmonids. Total CS RNA (30 w g per lane) from three representative salmonids-coho salmon (Oncorhynchus kisutch), arctic char (Salvelinus d p i n u s ) , and rainbow trout (0. mykiss)-was subjected to electrophoresis in 1%agarose/formaldehyde gels. The RNA was transferred to nitrocellulose and probed under conditions of high stringency with a "P-labeled cDNA corresponding to nucleotides 174-274 of coho salmon STC (see Fig. 2). Adapted from Wagner et u1. (1992).

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IV. STRUCTURAL COMPARISONS OF EEL AND SALMON STANNIOCALCIN The molecular cloning of eel STC was an important achievement in the field simply because it provided the first complete primary structure of the hormone. Knowledge of the sequence from various species prompted Genbank searches for possible homologues, but revealed instead that the STC sequence was unique among vertebrate proteins (Butkus et al., 1987; Wagner et nl., 1986, 1992) and put to rest years of speculation that STC was structurally related to parathyroid hormone (Lopez et al., 1984).With the cloning and complete character-

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180 190 200 210 220 CGT GCA CGC TTC TCA TCC AAC AGC CCC TCG GAT GTG GCT AGG TGT ARG ALA ARG pHE S E R S E R ASN SER PRO SER ASP VAL ALA ARG CYS +12 THR A L A SER 230 240 250 260 TTG AAT GGC GCT CTA GCC GTG GGA TGT GGT ACG TTT GCC TGC CTG LEU ASN GLY LEU ALA VAL GLY C Y S GLY THR PHE ALA CYS LEU +27 GLN SER ALA

270 280 290 300 310 GAG AAT TCT ACC TGT GAC ACT GAT GGC ATG CAT GAT ATC TGT CAA GLU A S N S E R THR C Y S ASP T?IR ASP GLY MET HIS ASP I L E CYS GLN +42 ASP GLU ARG

Fig. 2. T h e complete cDNA and deduced amino acid sequence of coho salmon STC niRNA. T h e mRNA encodes 256 residues, 223 ofwhich comprise mature stanniocalcin. T h e amino acid residues that differ or that are missing entirely (-) in the Australian eel are shown beneath the salmon sequence. Underlined amino acid residues include the initiator methionine, the N-terminal phenylalanine of mature salmon STC, the asparagine-linked glycosylation consensus sequence, potential dibasic and tribasic cleavage sites, and the polyadenylation signal. The cleavage site between pre and proSTC, which occurs between Ala.,6-Tyr-l, in the Australian eel, has not been determined in salmon. From Wagner et a / . (1992).

320

330

340

350

CTG TTC TTT CAC ACC GCA GCT ACC TTT AAC ACA CAG GGT AAG ACA LEU PHE PHE HIS THR ALA ALA THR PHE ASN THR GLN GLY LYS THR +57 SER LEU GLY LYS 360

370

380

390

400

TTT GTA AAG GAG AGT CTG AGG TGT ATT GCC AAC GGT GTC ACG TCT PHE VAL LYS GLU SER LEU ARG CYS ILE ALA ASN GLY VAL THR SER +72 LYs ILE 410

420

430

440

AAA GTC TTT CAG ACC ATC AGG CGC TGT GGA GTC TTC CAG AGA ATG +87

LYS VAL PHE GLN THR ILE ARG ARG CYS GLY VAL PHE GLN ARG MET LEU SER SER LYs 450

460

470

480

490

ATT TCT GAG GTC CAG GAG GAG TGT TAC AGT AGA CTG GAC ATC TGT ILE SER GLU VAL GLN GLU GLU CYS TYR SER ARG LEU ASP ILE CYS +lo2 LYS LEU 510

500

520

530

GGT GTG GCT CGC TCT AAC CCT GAG GCC ATT GGA GAG GTG GTG CAG GLY VAL ALA ARG SER ASN PRO GLU ALA ILE GLY GLU VAL VAL GLN +117 SER GLN MET ALA 540 550 560 570 580 GTC CCT GCA CAC TTC CCC AAC AGG TAC TAC AGC ACT CTG CTC CAG VAL PRO ALA HIS PHE PRO ASN ARG TYR TYR SER THR LEU LEU GLN +132 SER GLN 590

600

610

620

TCC CTG CTA GCC TGT GAT GAG GAG ACA GTG GCT GTG GTC AGG GCA SER LEU LEU A I A CYS ASP GLU GLU THR VAL ALA VAL VAL ARG ALA +147 THR ASP GLU GLN 630

640

650

670

660

GGG CTT GTT GCT AGG CTG GGG CCA GAC ATG GAA ACT CTC TTC CAG GLY LEU VAL ALA ARG LEU GLY PRO ASP MET GLU THR LEU PEE GLN +162 SER GLU GLU GLY VAL 680

690

700

710

TTG CTG CAG AAC AAA CAC TGC CCC CAG GGT TCT AAC CAG GGT CCT LEU LEU GLN ASN LYS HIS CYS PRO GLN GLY SER ASN GLN GLY PRO +177 THR ALA PRO SER ALA ALA GLY THR 720

730

750

740

760

AAC TCA GCC CCC GCT GGC TGG CGC TGG CCA ATG GGG TCG CCT CCT ASN SER ALA PRO ALA GLY TRP ARG TRP PRO MET GLY SER PRO PRO +192 GLY PRO VAL GLY GLY SER ARG CYS PRO TRP GLY 770

780

790

800

TCC TTC AAG ATC CAG CCC AGC ATG AGA GGA AGA GAC CCC ACC CAC SER PHE LYS ILE GLN PRO SER MET ARG GLY ARG ASP PRO THR HIS PRO CYS SER ARG SER SER PRO THR CYS ALA PRO GLY THR PRO PRO 810

820

830

840

850

CTA TTC GCT AGG AAA CGC TCT GTG GAG GCA TTG GAG AGA GTG ATG LEU PHE ALA ARG LYS ARG SER VAL GLU ALA LEU GLU ARG VAL MET THR SER LEU LEU ARG ASN ALA ARG PRO PRO ASN TYR H I S PRO GAG GLU +223 PRO ARG LEU ALA LEU MET ASP CYS PRO 864

a74

+207

+222

+231

884

894

904

914

TAGATTGGAG AAGAGGAGGC AGACATACAC ACCACTTATA CCTTAAGCAT ACATTCACAT

Fig. 2. Continued

280

GKAIIAXI F. \VAG;ZIER 974

GTACACACAC ACATACACAC ACCACAGCTA CCTTAAACAC AAAACACACT CATGATAGCT 1034

TTGCTCACAC ACACACTGAC TCACACGCAC ACTGACACAC ACACATTTTC ACACACATGC 1094

ACACACACAT AGCTTTACCC TCAAATGATT AAGGCTAAAT TATTAATGGA AGTTTGGGGC 1154

TGTTGTAATG TAGTATTTGA TTTGGGGAAG CATCTCTCTG TAAATGCTGT TGAAGGTATT 1214

TCTGTGTGGG TTGATACPTG ATGAAGGGGA GATGAAACCT GTTACCTAGA GCTTGAATGT 1274

GGAGGATTAT ATCTCCTCAG AATAGACTCG ACTAAACATG AGAGCTATTG AAAAGTCTGA 1334

ACATTTAATA TTAACAAGTG AAACA'MTCA AATGCCACCT AAGAAAACGA ACCATCACTG 1394

TAGTTCCATT GGATTTCAAC GTGGCCACTA CGGCCATAAC ATCCCCGTTT GGACCAGTCA 1454

TTAAAGACCG ATGGGTATAT TATTATAATA ATATTATTGA TATTTATTTT CTTACAGAAT 1514

G-ATTAAT

GATGTTGTGT TGTATCTAGT TGTAACTCGG "GAGTTTC

CCCCAGTGGG 1574

TGGGOITTGA CATCCTGATA TGACGTCACT GGCTGATGTA TTGCTCI'ATG

AGGATGTCAC 1634

CACCTCAGAG GGACAGTGTG ATGTCACCAT GCTGGTTGTG GGACTCACCG CGTCCCTCTC 1694

TGTCTl'CATC

TGTGTTAATG TAAGATCCTG TAGTGTGTAA AGACATTATA GAGTGATCCT 1754

TTGCTGTGTT CCTCAGATGT GGTTATGTGG TGTATGTTTT GAGATCCTGT GTGAGAATGT 1814

GTGCTAGTCA GGTACTATAC ACCTTGGGGC TTGGGGTCAT TCTCTCTGCT ATGAATACAT 1874

W G T G A C C T T C A T A A m T GTCTGAGGTG ATTTGTGTCG AGACTCACTG GTGATTAAAC 1934

GCTCACAGTT TCAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

Fig. 2. Continiicd

izatiori of salmon STC, it was finally possible to make some structural comparisons between species and pinpoint regions of the moleci1le that were more or less conserved. One such comparison is shown in Fig. 2, which illustrates the complete niicleotide and deduced protein sequence of coho salmon STC and amino acid substitutions a s they occur in the eel. Figure 3 , on the other harid, is a comparison of their nucleotide sequences, minus the 5' and 3' untranslated regions, which are completely divergent between the two species.

9.

28 1

R E G U L A T I O N OF S T A N N I O C A L C I N G E N E E X P R E S S I O N Coho Eel

87 122

ATGCTCGCAAAATTCGGCCTGTGCGCGGTCTTCCTCGTCCTGGGAACTGC 1 3 6 I I I I I I I I I I

I1 I 1

1 1 1 1 I 1 I 1

I I

I I

I I

I I

I l l 1 I l l 1

I I I I I I I I I I I I

.. .C&ACG&TMGCTGGTAACTGC 168

ATGCTGCGAATGAGTGGGCTAATC

Coho 1 3 7

CGCCACCTTCGACACCGACCCGGAGGAAGCTTCTCCTCGCCGTGCACGCT 1 8 6

Eel

169

TGCCTACGAGCAGGATGAGAGCGAGCCCTTATCTCCAAGGACA&G&&& 2 18

Coho 1 8 7

TCTCATCCAACAGCCCCTCGGATGTGGCTAGGTGTTTGAATGGCGCTCTA 2 3 6

Eel

219

TCTCCGCCAGCAGCCCATCTGATGTTGCACGCTGTCTGAACGGGGCCCTG 2 6 8

Coho 237

GCCGTGGGATGTGGTACGTTTGCCTGCCTGGAGAATTCTACCTGTGACAC 2 8 6

Eel

269

CAGGTGGGCTGCAGTGCATTTGCCTGTCTTGACAACTCCACCTGCAACAC 3 1 8

Coho 287

TGATGGCATGCATGATATCTGTCAACTGTTCTTCTTTCACACCGCAGCTACCT 3 3 6

Eel

CGACGGCATGCATGAAATCTGCAGGTCC&&~C&C&A&GGTGCTGCC-T

I l l

I I

I l l

I1

I l l I l l

1 1 1 1

I l l 1

IIIII

319

I I I I I

I I I I

I l l I l l

I

I I I I I I I I I I I I

I 1 I 1

I I t I

I I I I I I I I I I I I I I I I l I I l l l

II

IIIII

II

I I

I I I I I

I I I I I I I I IIIIIIII

I I I l l I l l 1 I

I I

I I I I I I I I I I

II

I

I

I I

I I l l

I l l

I l l 1 I l l 1

I l l

I1

I 1

1 1

I I I 1

I 1 I 1

II II

I I I I

I I I I I

I I

I I

I I

I I I I I l l 1

I I I I I

I I I I I I I

l l

l l I 1

I I

I I I I

I I

368

Coho 337

TTAACACACAGGGTAAGACATTTGTAAAGGAGAGTCTGAGGTGTATTGCC 3 8 6

Eel

&GACACACAGGGCAAGACTTTTGTGAAGGAGAGCCTGAAGT&&A&&

I 1

369

IIIIIIIIII I I I I I I I I I I

I I I I I I I I I I

1 1 I I I

IIIII

I I I I I I I I I I I I I I I I

IIII I I I I

I l l I l l

I I

I l l

418

Coho 387

AACGGTGTCACGTCTAAAGTCTTTCAGACCATCAGGCGCTGTGGAGTCTT 436

Eel

468 AATGGCATCACCTCCAAAGTGTTC~TTACCATCCGCCGC

I 1

419

II

II

Ill1 1 1 1 1

II

I1 II

I I I I I IIIII

II II

I

I I I I I I IIIIII

I I

I I I I I IIIII

I l l

I

Coho 437

CCAGAGAATGATTTCTGAGGTCCAGGAGGAGTGTTACAGTAGACTGGACA 486

Eel

ccAGAAGATG AT&

469

IIIII IIIII

I l l 1 1 I 1 I l l

I 1

I I I I I I I I I I

I I I I I I I I I I I I I I I I I I I I I I

I 1 I I

I I I I

I I

AG AGGTTCAGGAGGAGTGCPATAGCMCT

I l l I l l

I l l I l l

AGACC 5 1 8

Coho 407

TCTGTGGTGTGGCTCGCTCTAACCCTGAGGCCATTGGAGAGGTGGTGCAG 5 3 6

Eel

519

TCTGCTCTGTTGCC~AGAGCAACCCAGAGGCCATGGGGGAGGTGGCCAA~ 5 6 8

Coho 5 3 7

GTCCCTGCACACTTCCCCAACAGGTACTACAGCACTCTGCTCCAGTCCCT 5 8 6

Eel

569

GTG~~CAGCCAGTTTCCCAACAGGTACTACAGCACCCTGCTGCAGAGTCT 618

Coho 5 8 7

GCTAGCCTGTGATGAGGAGACAGTGGCTGTGGTCAGGGCAG~CTTGTTG6 3 6

Eel

T&GA~GTGTGATGAGGACACCGTGGAGCAGGTGAGGGCCGGGTTGGTGT

IIII

I1 I 1

I 1

619

I I I I I I I I I I

I I I I I I I I I I I

IIII

I I

I1 I 1

I

I I I 1

I I I I I I I I

I 1

I I

IIIIIIII

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I

I I I I I I I I I I I I I I I I I I I I I I

I 1 I I

I l l 1 I I I I

I l l I l l

I I I I I I I I I I I I I I

IIIII I I I I I

I I I I I IIIII

I l l

1 1 1 I l l

1 1 1 I l l

I I

1 1 I I

I 1 I 1

668

Fig. 3. Comparative nucleotide sequences of coho salmon and Australian eel preproSTC. T h e DNA sequence encoding eel preproSTC is derived from Butkus et al. (1987) and nucleotides are numbered as in the original article. T h e nucleotide sequence of salmon STC is numbered as in Fig. 2. On the basis of a 774-bp overlap (minus the stop codon and including a 6-bp gap in the salmon sequence) and after introducing two gaps in the eel sequence to maximize alignment, there was 66.8% sequence similarity between the two species. The initiator methionine, N-terminal phenylalanine of mature STC, and the termination codons are shown in boldface in both species. From Wagner st

(11.

(1992).

Salmon and eel STC share certain structural features that are common to the other characterized STCs as well. In all known species, for instance, phenylalanine occupies the N terminus of the mature hormone. There is also a single glycosylation consensus sequence at the same position in all known species: Asn,,-Sei-30-Thr:,, (Butkus et

282

GRAHAM F. \VAGNER

Coho 637

CTAGGCTGGGGCCAGACATGGAAACTCTCTTCCAGTTGCTGCAGAACAAA 686

Eel

~CCACCTGGAGCCAGAGATGGGGGTGCTCTTCCAGCTCCTCCAGACCAAG

I

669

Coho 687 Eel

719

1 1 1 1

I

I I I I

I I I I I I

1 1 - 1 1 1 1 1 1

I I I I I I

I I I I I I I I I

I I I I I I I I I

I I

I 1

I 1

I l l I l l

1 1 1 1 1 1 1 1

CACTGCCCCCAGGGTTCTAACCAGGGTCCTAACTCAGCCCCCGCTG.. I I I I I I I I I I I l I i I I I I I

I

I

I 1 I1

I

I 1

I I I I

I 1 I1

I

..

I

GCCTGCCCCCCAAGCG&GCCGGTGGCA&G&C&ATAGGGGCAGGAGG

7 18 732 768

Coho 733

..GCTGGCGCTGGCCAATGGGGTCGCCTCCTTCCTTCAAGATCCAGCCCA 780

Eel

769

CAGCTGGCGCTGCCCATGGG....GCCCCCCATGTTCAAGATCCAGCCCA 8 14

Coho 781

GCATGAGAGGAAGAGACCCCACCCACCTATTCGCTAGGAAACGCTCTGTG 830

815

ACCTGCGCTCCCGGGACCCCACCCACCTCTTTGCTAAGAAACGCTCGACC 864

I l l 1 1 1

I I I I I I I I I I

Eel

Coho 831 Eel

865

I I I I I I I I I I

I I

I1 I1

I I

I I

1 1 I 1

I l l 1 1 1

I I I I I I I I I I I J I I I I I I I I I I I I I I I I

I 1 1 1

1 1 I 1

GAGGCATTGGAGAGAGTGATGGAGTAG.... I I

1 1

I I

I I

I1 I 1

I l l l i l l l l l l l t l l l I I I I I I I I I I J I I I I I

1 1 1 1 1 1 1 1

I I I I I I I I I I I I I I I I I I

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

857

AGCTCCTAATTACCACCCACCAAGGCTAGCACTCATGGATTGTCCTTAR. 913

Fig. 3. Continued

u l . , 1987; Lafeber et al., l988a; Sundell et d., 1992; Wagner et al., 1986, 1988a, 1992). Whether or not nonglycosylated or more heavily glycosylated forms of the hormone occur in other fishes is unknown. However, it is interesting to note that STC from the bowfin (Arnia caluti) is incapable ofbinding to the plant lectin concanavalin A, distinguishing it from all other known species of the hormone (Marra et al., 1992). There are also numerous disulfide bonds in salmon and eel STC as a result ofthe large number ofhalf-cysteine residues (15in eel, 11in salmon), one of which must be unpaired and therefore involved in the formation of STC dimers. This has been conclusively proven in salmonids, where the native hormone is a dimer of identical subunits (Lafeber et al., 198811; Sundell et al., 1992; Wagner et ul., 1986,lYSSa) and is likely to be true in the eel as well, although definitive proof is lacking. The most salient differences between salmon and eel STC lie in their nucleic and amino acid sequences, particularly at the extreme ends ofthe molecule (Figs. 2 and 3 ) . In the prepro region for instance, encoded b y nucleotides 87-185 in salmon, the two species share only 49% identity in amino acid sequence and 51% identity at the nucleotide level. The eel hormone also has one less residue than s''I 1mon STC in this region. An even sharper divergence occiirs in the Cterminal region, STC171-223,encoded b y nucleotides 696-8.54 in salmon. Here there is 59% identity at the iiucleotide level (but only after introducing gaps in the sequences of both species to maximize alignment) and just 7.5% identity at the amino acid level. In comparison to the eel, salmon STC also has eight fewer residues on the C terminus. Yet there is reasonably high homology in the N-terminal

9.

REGULATION OF STANNIOCALCIN G E N E EXPRESSION

283

and midmolecule regions at both the nucleotide (72%)and amino acid level (78%);this region is STC,-,,, encoded by nucleotides 186-695 in salmon. Because this latter region is highly conserved, it likely contributes more than the C terminus does to STC receptor binding and subsequent biological activity. This has been born out in bioassays where N-terminal fragments of eel and salmon STC (STC,-,,,) are capable of inhibiting gill Ca2+transport (Milliken et al., 1990; Verbost et ul., 1993). However, these fragments are less potent on a molar basis than native STC. Hence, full biological activity clearly requires more than the first 20 amino acids of the hormone. Salmon and eel STC also share a dibasic pair (Arg,,-Arg,,) that may be used for bringing about posttranslational modifications to the hormone. There are two additional sites in salmon, one dibasic (Lys167-His168) and one tribasic (Arg,ll-Lys212-Arg213), which also appear accessible to proteolytic attack based on a hydropathy profile of the salmon hormone. There are nuinerous monobasic sites in both species that also could be cleaved. Posttranslational modifications to salmon STC do occur. Numerous truncated forms of the hormone are found in CS tissue (Wagner et d., 1988b, 1992) and the circulation (Wagner et al., 1991, 1993) and are released by CS cells in vitro (Wagner, 1993). Interestingly, there is no evidence of this occurring in the eel (Butkus et nl., 1987, 1989; Wagner et al., 1992).

V. STUDIES ON TISSUE-SPECIFIC EXPRESSION OF THE STANNIOCALCIN GENE Southern blot analysis reveals that there is more than one copy of the STC gene in salmonids (Fig. 4). This is not particularly surprising considering the tetraploid karyotype of salmonids; multiple genes have already been described for most salmonid hormones. But it has not been established that all gene copies are functionally expressed in CS tissue or whether they might also be expressed at other loci. As yet, there is little evidence for ectopic STC production in fish. However, the possibility always exists that one or more gene copies may be ectopically expressed and perhaps have novel functions at these loci, as in the case of placental growth hormone and decidual prolactin in primates. There are reports of STC immunoreactivity in fish brain and pituitary (Fraser et al., 1991), but they have not been characterized further. We have been unable to identify STC-immunoreactive cells in any tissue other than the corpuscles of Stannius. Northern blot and

A

S

C

T

S

B C

T

23

99

=4

-2

Fig. 4. Evidence that there is inore thaii one cop>-of the STC gene in salnionids. Swmples of DN.4 (15 pgilane) froin three salmonids-chinook salmon, Oiicorliyt~cl~us t.cliciiclyt.rclzcr (S), arctic char, S a l c e l i n u ~cilj~inus (C), and rainbow trout, 0. myki.c.s (T)-were digested with Pst 1 and subjected to Southern blot analysis. T h c blot w a s first prol)ed with a 100-bp fragment encoding the N terminus of salmon STC (panel A ) , then stripped and probed a second time (panel B) with a near full-length cDN.4 (1.7 kb). Panel A illustrdtes that the smaller cDNA probe hybridized to three 01-four fragments in each species following Pst 1 digestion. The arrows refer to fragnrents that 1iyl)ridized uniquely to this probe. Panel B illustrates the same blot probed instead \\it11 the 1.7-kb cDNA clone. The larger probe hybridized to many ofthe sanie fragments a\ the smaller probe, but additional fragments were revealed that hybridized uniquely to the larger probe (arrows and arrowheads). T h e key evidence for multiple gene copies is shown in panel A . Pst 1 digestion of salmon and trout DNA yielded four genornic fragments that hybridized to the 100-bp prohe. If there is only one copy of the gene in salmon and trout then it must contain tlirec. Pst 1 sites in the region encoded b, the probe. However, there is only o ~ i ePst 1 site in the entire e D N A sequence of salmon STC and it lies outside the region encoded b y the 100-lip probe. If there are three Pst 1 sites in the gene within the sanie 100-bp sti-etch encoded by the probe, this short region o f t h e gene would have to be interrupted I)>- three introns, each containing a Pst 1 site. This is highly improbable and argues for nriiltiple copies of the gene. From Wagner ct a / . (1992).

9. HEGULATION

OF STANNIOCALCIN G E N E EXPRESSION

285

in situ hybridization analyses of a wide range of salmon tissues (brain, pituitary, urophysis, pancreatic islets, thyroid, digestive tract, spleen, gonads, and heart) have likewise yielded negative results (Sterba et al., 1993; Wagner et al., 1992). And yet we still cannot rule out the possibility of the gene being expressed in other tissues, perhaps at some early stage in the life history of the fish. Future studies should probably approach this question with more sensitive methods of detection such as polymerase chain reaction technology. But until conclusive evidence is forthcoming, we should proceed on the assumption that the STC: gene is expressed exclusively in CS cells.

VI. LOCALIZATION OF STANNIOCALCIN mRNA IN CS CELLS BY I N SZTU HYBRIDIZATION An unusual histological feature of CS tissue is that the secretory activity of STC cells varies in different parts of the gland. In the CS of Colisa M i a , for example, successive rounds of depletion and subsequent repletion of stored hormone occur in select regions ofthe gland (Krishnamurthy, 1976). The availability of purified STC and specific antisera has allowed us to corroborate this phenomenon with greater precision using immunocytochemistry. We have found that it also occurs in winter flounder and sockeye salmon, for instance, and is manifested simply as low levels of immunoreactive hormone in specific regions of the gland (G. F. Wagner, unpublished observations). We have explored this phenomenon further by in situ hybridization using "S-labeled probes with interesting results (Sterba et al., 1993). First, there is always evidence of STC gene expression in every region of the gland. STC mRNA levels are often barely detectable, yet there is always a constitutive level of expression throughout the gland. Seeond, in some cases the level of' gene expression is equal throughout the gland (Figs. 5A and 5F),but typically it varies widely and is highest on the CS perimeter (Figs. 5C-5E), abutting either kidney tissue or the intraperitoneal cavity. There are statistically higher message levels in lobules of cells on the perimeter as compared to the center of the gland (Table I). Lastly, the level of STC gene expression is obviously a good indicator of STC synthesis. Throughout all regions of the gland, the levels of STC mRNA are closely correlated with the levels of immunoreactive hormone (Table I ) ; this is illustrated most convincingly in Fig. 6. It is apparent from these findings and those of Krishnamurthy (1976) that all CS cells are not in synchrony, synthesizing and secreting STC together at the same rate, and that this phenomenon is

Fig. 5. Dark-field illumination of sockeye salmon (Oncorhynchus nerku) CS following i n situ hybridization with "S-labeled STC cRNA probes. The positive hybridization signal appears as small silver grains, whereas the large white spots are pigment granules i n the kidney to which sense and antisense probes bound nonspecifically. (A) Silver grains are densely localized over CS tissue and much less evident over surrounding kidney tissue following the use of antisense probes. The level of STC gene expression is evenly distributed throughout the gland. (B) Specific hybridization is not evident o n a tissue section adjacent to A following the use of sense probes. (C) An example of variable STC gene expression in salmon CS tissue. Note the higher level of expression o n the perimeter ofthe gland adjacent to kidney tissue. (D)A second example ofvariable STC gene expression. In this case, the highest level ofexpression occurs on the perimeter

9.


287

Table I STC mRNA and Immunoreactive STC Levels in Sockeye Salmon CS Cells as Assessed by Morphometric Analysis"

CS region

STC mRNA (grains per cell)

Immunoreactive STC (optical density)

All glandular cells Peripheral lobular cells Central lobular cells

27.8 5 11.1 74.7 2 14.2 11.5 2 10.9

0.038 ? 0.008 0.067 2 0.01 0.025 5 0.007

~~~~

"

N = 40 CS, data expressed as means

~

?

S.E.M. From Sterba et al. (1993)

widespread among fishes. How and why it occurs is uncertain, but it may be ficilitated by varying CS regional blood flow and is possibly a strategy for placing the burden of secretion on one region of the gland, while allowing the remainder of the gland to concentrate on renewed hormone synthesis. A disadvantage to using radioactive probes for in situ hybridization as illustrated in Figs. 5 and 6 is the lack of resolution inherent in autoradiography. As a consequence, it is difficult to pinpoint the exact cellular location of the mRNA using this technique. To circumvent this problem, we have turned to a nonisotopic method that uses digoxigenin-labeled cRNA probes for more precise localization of mRNA. The ability to localize STC mRNA within the cell has allowed us to visualize the sites of hormone production and furthered our understanding of structure-function relationships in CS cells. In sockeye salmon for instance, the CS glands are coniposed of individual lobules of concentrically arranged cells (Fig. 7). There is also a welldefined polarity to the organelles in these cells, whereby secretory granules are tightly packed on the lobule perimeter nearest the surrounding capillaries and cell nuclei are found at the opposite pole near the center of the lobule (Wagner et al., 1988b).The concentration of secretory granules on the lobule perimeter against the basolateral

of the gland facing the intraperitoneal cavity. (E) A third example of variable gene expression. In this case, the highest level of expression occurs in all the lobules on the perimeter of the gland. ( F ) As in panel A, the level of STC gene expression is evenly distributed throughout this particular gland. CS = corpuscle of Stannius; k = kidney; tfe = tissue-free environment or intraperitoneal cavity; calibration bar = 100 pm.From Sterba et al. (1993).

288

GRAHAM F. [L’AGSER

Fig. 6 . Correlative in situ hybridization (A) and immunocytocheniistry (B) in sockeye salmon corpuscles of Stannius. (A) Note the high levels of STC mRNA in half of the gland. Nonetheless, a low but discernable level of gene expression is evident i r i the other half as well. (B) Note that the levels of imniunoreactive STC are highest i n the region exhibiting the highest level of gene expression. CS = corpuscle of Stannius; k = kidney; calibration bar = 100 pni. Adapted from Sterba et u1. (1993).

9.


289

membrane ensures rapid release of STC into the circulation upon the appropriate stimulus. It now appears that STC mRNA also is polarized within the cell. The use of high-resolution, digoxigenin probes has revealed that STC mRNA is concentrated at the apical cell membrane nearest the center of the lobule, so that cell nuclei lie between the secretory granules at the one pole and STC mRNA at the other (Fig. 7). Ultrastructural studies on salmon CS cells have revealed that the apical pole is also rich in rough endoplasmic reticuluni (Carpenter and Heyl, 1974; Meats et nl., 1978).Therefore, it appears that newly synthesized message is preferentially released on the apical side of the nuclear envelope and becomes associated here with ribosomal RNA and the endoplasmic reticulum for the initiation of new hormone synthesis.

VII. CALCIUM REGULATION OF STANNIOCALCIN CELL ACTIVITY A. Regulation of Stanniocalcin Secretion by Calcium

The notion that STC cells might be responsive to calcium was first deduced on the basis of histological evidence. Beginning in the 1960s, it was commonly observed that transferring fish from fresh water to seawater altered the appearance of CS cells. Among the noted cytological changes were increased protein synthesis, nucleolar and nuclear hypertrophy, increased amounts of endoplasmic reticuluni and Golgi, cellular hypertrophy, glandular hypertrophy, and increased secretory activity of CS cells (Krishnamurthy, 1976, Wendelaar Bonga and Pang, 1986).The cause of these changes, in particular the secretory response, was subsequently identified as the calcium content of the water (Pang Fig. 7. I n situ localization of STC mRNA in sockeye salmon corpuscles of Stannius using a digoxigenin-labeled antisense cRNA probe. T h e black deposits throughout the tissue correspond to STC mRNA. Note how the cells are arranged into lobules that together form an individual corpuscle. Capillaries are found primarily in the clear regions between the lobules. T h e large arrow points to a cluster of three cell nuclei. T h e sinall arrows point to the lobule perimeter and site of the basolateral cell membrane, where secretory granules are concentrated and poised for release into the perivascular space. STC mRNA is localized for the most part at the opposite pole, against the apical cell membrane. Note that in most cases, CS cell nuclei lie between secretory granules and STC mRNA on the basolateral and apical cell poles, respectively. From T . Sterba and G . F. Wagner, unpublished.

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et al., 1973, 1974; Pang and Pang, 1974; Cohen et al., 1975),which is much higher in seawater (10 m M ) compared to fresh water (0.11.0 mM). It was concluded that the movement of calcium across the gills, gut, and integument after seawater transfer raised plasnia calcium levels to an extent that stimulated CS cells. It was subsequently shown that inducing hypercalcemia in 2jiz)o prompted a secretory response as well (Lopez et al., 1984), as did exposing glands in u i tr o to high calcium levels (Aida et al., 1980). From a physiological standpoint these findings made sense; cells that secreted a calcium-regulating hormone were in turn responsive to calcium levels in the extracellular compartment. Cytophysiological studies on CS cells were rendered obsolete with the purification of STC and the subsequent development ofimmunoassays (Gellersen et al., 1988; Mayer-Gostan et al., 1992; Wagner et al., 1Y9l), which now made it possible to quantify hormone release and assess the actions of reputed secretagogues (i.e., calcium). Both in c i t r o and in 2jiz;o model systems have since been used to explore the effects of calciuni on STC secretion, employing species such as rain1)ow trout (0.mykiss), coho salmon, Atlantic salmon (Salino salar), and, of course, the European eel. We have relied solely on primary cultured trout CS cells for our own in vitro studies because of their wide availability and ease of culture in a variety of media forniulations (Gellersen et al., 1988; Wagner et al., 1989). Our findings suggest that trout CS cells are extremely sensitive to changes in ionic calcium levels within the physiological range. Between 0.3 and 2.4 mM calcium, these cells undergo stepwise increases in STC secretion with each successive rise in calcium concentration (Fig. 8). The calcinmresponse curve is steepest around the physiological set point (- 1.2 mM Ca”), where CS cells are most responsive, and levels off’ at higher and lower calcium concentrations. This is precisely how these cells should respond given their role in preventing hypercalcemia and is wholly reminiscent of‘ calcium regulation of calcitonin secretion in mammals (Anast and Conway, 1972; Gage1 et ul., 1980). There is a l s o a temporal aspect to the secretory response as its magnitude increases with increasing length of exposure to calcium (Fig. 9A). These effects of calcium are not mimicked by magnesium (Fig. 9B), or by the principal monovalent ions in plasma, sodium and chloride (Wagner et al., 1989). Conflicting findings have been reported in the European eel, where cultured CS glands are reputedly unresponsive to changing calcium levels within the physiological range (Hanssen et aZ., lYYl), prompting the authors ofthis study to challenge the notion that calcium is a regulator of STC secretion. However, as their findings are com-

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m M IONIC CALCIUM Fig. 8. Stanniocalcin secretion by primary cultured rainbow trout CS cells is positively regulated by ionic calcium within the physiological range. Trout CS cells were maintained for 4 h r in serum-free RPMI media containing increasing concentrations of ionic calcium. Between 0.3 and 1.9 mM calcium, CS cells exhibited stepwise increases in stanniocalcin secretion. T h e secretory response was steepest around the set point (-1.2 m M ) and leveled off at higher and lower calcium concentrations. Each data point represents the mean ? S.E.M.ofthree replicate cultures (0.5 x 106cells/well).Redrawn from Wagner et al. (1989).

pletely at odds with previous work on this species (Fenwick and Brass e w , 1991; Lopez et al., 1984), an alternative explanation may lie in their use of a heterologous, salnionid STC immunoassay to quantify eel STC release. Salmon and trout CS cells respond similarly i n civo to elevations in plasma calcium levels, delivered via intraperitoneal or intra-arterial injections (Glowacki et al., 1990; Hanssen et al., 1991; Wagner et nl., 1991). There is a defined time course to the secretory response (Fig. 10). The response is dose-related in the sense that larger dosages of calcium result in more sustained elevations in hormone levels and magnesium has no effect on hormone release (Wagner et a,?., 1991). The similarities in the i n vivo and in citro responses to calcium support the notion that CS cells are finely tuned calcium sensors, capable of modulating secretory activity in accordance with changing levels of extracellular calcium. A rise in plasma calcium provokes a measured

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Fig. 9. (A) Stanniocalcin secretion by primary cultured CS cells increases with length of exposure to calcium. Rainbow trout CS cells were maintained for up to 4 hr in serum-free RPMI media containing 0.3 rnM (solid bars) or 1.6 nlk! ionic cakiunl (open bars). STC secretion increased 5-fold after a 30-min exposure to 1.6 tnM calcitini. T h e rate of STC secretion then rose progressively between 1 hr (&fold) and 2 hr (10fbld) and had leveled off by 4 hr (4-fold). Each data point represents the mean 2 S.E.M. of three replicate cultures (0.5 x loficellsiwell). (B) Magnesium has no effect on STC secretion. Trout CS cells were exposed for 4 hr to increasing amounts of magnesium. Each data point represents the mean t S.E.M. of three replicate cultures (0.5 x lo6 cellsiwell). From Wagner et ul. (1989).

increase in STC secretion, which causes a corresponding reduction in the rate of gill calcium transport. As plasma calcium levels decline, there is a gradual drop in the rate of STC secretion until norniocalcemia (or the set point) has been reestablished. The regulation of STC secretion b y calcium resembles that of mammalian calcitonin, which is also

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REGULATION OF STANNIOCALCIK GENE EXPKESSION

IS

Fig. 10. Plasma levels of stanniocalcin and total calcium in free-swimming, adult rainbow trout after an intra-arterial infusion of calcium (5 mgikg). Blood samples were withdrawn before and after infusing calcium chloride through a dorsal aorta cannula. Note the quick rise in plasma STC levels within 5 min of infusing calcium (sixfold) and the rapid restoration of nonnocalcemia. T h e reason for the comparatively slow restoration in plasma hormone levels is unknown. From Wagner et al. (1991).

positively regulated b y calcium (Anast and Conway, 1972; Gagel et al., 1980),but is in contrast with parathyroid hormone (PTH), which is negatively regulated b y calcium (Brown et d., 1987). B. Regulation of Stanniocalcin m K N A Levels b y Calcium Little is known about the regulation of STC synthesis at either the transcriptional or posttranscriptional level. In the case of PTH, calcium regulates the biosynthetic pathway at two different levels. Low levels of plasma calcium stimulate PTH gene transcription and discourage newly synthesized hormone from entering a degradative pathway. Meanwhile, low levels of plasma calcium are also a stimulus for PTH secretion. Hence, the regulation of' PTH secretion is tightly coordinated with renewed hormone synthesis, thereby ensuring that a constant supply of PTH is always available for release. In view of the

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regulatory effects of calcium on STC secretion, it would make sense for calcium to have a regulatory role in hormone biosynthesis as well. There is, in fact, one study in rainbow trout which has shou711 that administering repeated calcium injections over several days (presumably to deplete the glands of STC) has a significant effect (1.7-fold) on the rate of hormone synthesis (Flik et al., 1990). The notion that calcium stimulates STC biosynthesis is supported as well b y histological observations. For instance, the CS are more active in fish adapted to seawater or water that is simply high in calcium content. The CS cells in marine fishes have a more extensive endoplasmic reticulum and Golgi apparatus and a higher content of secretory granules, and generally have increased nuclear and cytoplasmic volumes in comparison to their freshwater counterparts (Krishnamurthy, 1976, Wendelaar Bonga and Pang, 1986, 1991). Stages in life history can also influence the activity of STC cells, especially ifthey involve changes in calcium metabolism. Reproduction in the Indian catfish ( M y s t u s uittatus), for instance, is correlated with large increases in both serum calcium and the mean nuclear diameter of CS cells (Ahmad and Swarup, 1990). The nuclear hypertrophy that occurs in CS cells may be indicative of increased STC gene expression to accommodate higher levels of hormone secretion. Although STC gene expression has not been nionitored in fish under different environmental conditions, calciiim does have direct effects on steady-state mRNA levels in primary cultured, rainbow trout CS cells. Moreover, as in the case of calcium-stimulated~ secretion, the effects ofcalciiirn on STC me ge levels are dependent ou both concentration and length of exposlire (Wagner and Jaworski, 1994). Short exposure times have only modest effects. For instance, exposing trout cells to calcium for 24 hr produces small, stepwise increases in STC mRNA levels between 0.7 and 1.9 mizl calcium and a maximum %fold induction in comparison to controls (Fig. 111. However, 3-day exposures produce steeper calciuni-response c u r \ ~ s (Fig. 12). There is also a greater induction of message levels following 3-day exposures to calcium (1.7-to 3-fold). Even longer exposure times, in this case 6 days, have the most pronounced effects on gene expression, resulting in steeper response curves and inducing message levels a s much as 14-fold in comparison to controls (Fig. 13).What is most interesting about these findings is that STC secretion and STC gene expression in trout CS cells are both subject to regulation over the same range of calcium concentrations, and that the maximum response in both cases occurs around 1.9-2.3 mM calcium (compare Figs. 8 and 12).That both STC secretion and mRNA levels are siinilarly regulated by calcium makes sense from a physiological standpoint. Above all,

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STCIActin, % of Control Fig. 11. STC mRNA levels in primary cultured rainbow trout CS cells following a 1-day exposure to calcium. STC mRNA levels were progressively stimulated between 0.7 and 1.9 mM calcium but were inhibited by higher calcium levels. Message levels were maximally induced 1.3- to 2-fold over controls (1.1 mM Ca”) in three separate experiments. Cell cultures were maintained in Leibovitz media (0.4x lo6 cells/well). Total RNA was harvested from each well of cells and subjected to Northern blot analysis as described in Fig. 1. The blot was then stripped and reprobed with a cDNA encoding carp beta actin. After X-ray film exposure, the STC and actin bands were quantified by densitometry and expressed as STC/actin mRNA ratios. For statistical analysis, all data were expressed as a percentage of controls and subjected to arcsine transformation. Each data point represents the mean rt S.E.M. ofthree replicates (*P< 0.05 in comparison to controls; two-tailed ANOVA and Dunnet’s test). Adapted from Wagner and Jaworski ( 1994).

it ensures a continuous supply of template for hormone synthesis, which would be especially important in high-calcium, marine environments where greater secretory demands are placed on CS cells (Glowacki et al., 1990; Mayer-Gostan et al., 1992). Oddly enough, STC mRNA levels in cells from seawater-adapted salmon are regulated

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Fig. 12. (A) STC mRNA levels in rainbow trout C 5 cells after a 3-dav exposuie to ~alciuiii Three-day exposure5 result in steeper response curves between 0 7 a i d 1 9 ink' cakiuni and more pronounced effects o n message levels STC mRNA le\el\ were maximally \timulated between 1 9 and 2.3 mM calciuin and inhibited I>\ higher calciiiin levels. Message levels were maximally induced 1.7- to 3-fold over controls (1 l inM Ca2+)in three separate experiments. Each data point represent\ the mean ? S.E.Xl of three replicate5 ( * P < 0.05, **P < 0 01 in comparison to controls, two-tailed ANOVA and Dunnet'\ te\t) Cell culture conditions, Northern blotting, arid data analv\i\ were a\ described in Fig 11 (B) Autoradiographs of STC and actin mHNA from replicate \veil\ of cell\ in the dewribed experinlent Adapted from Wagner and Tauorski (1994)

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Fig. 13. (A) STC niRNA levels in rainbow trout CS cells after a 6-day exposure to calcium. Six-day exposures produced the steepest response curves and the greatest induction of message levels. STC mRNA levels were inaximally stimulated between 1.9 and 2.3 m M calcium and again inhibited by higher calcium levels. Message levels were maximally induce 3-, 11-, and 14-fold over controls (1.1 m M Ca") in three separate experiments. Each data point represents the mean 2 S.E.M. of three replicates (**P < 0.01 in comparison to controls; two-tailed ANOVA and Dunnet's test). Cell culture conditions, Northern blotting, and data analysis were as described in Fig. 11. (B) Autoradiographs of STC and actin mRNA from replicate well ofcells in the described experiment. Adapted from Wagner and Jaworski (1994).

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over the same range as in trout (0.3-l.Y inM calcium) and stimulated

by calcium to roughly the same extent (1.6-fold; Fig. 14). One might expect CS cells from a marine fish to be more sensitive and exhibit greater responses to calcium, which may indeed be the case. However, any differences that do exist between marine and freshwater fishes may be apparent only under in cico conditions and may be lost in

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STCI 18s RNA; % of Control Fig. 14. STC m R S A levels in sea\vater-adaijted Atlantlc salnlon C S cells tollo\~~inq :i-day exposurc to calcium. The responses of salmon and trout CS cells were similar. There was a progressive rise iri STC mHNA levcls of salmon cclIs between 0.3 a ~ ~ d 1.0 mhf calciunr, after which message levels declined. Notice that 1.9 mA1 magncsiiuir ( i n the presence of 1.2 milf calcium) had no eftrct on message level\. Cell culture were a s described in Fig. 11, except contfitions, Northern blotting, and data anal) that a rabbit 18s probe was used in lieu of carp actin to normalize the data. Each data point represents the mean t S.E.RI. of three replicates (*P < 0.01 in comparison t o controls in 1.1 inM Cii” ; two-tailed ANOVA and Dunnet’s test). From C;. F. \\’agner, uii~mblished. ;i

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the primary cultured cell. Magnesium is not a regulator of STC gene expression in either species of salmonid (Figs. 14 and 15), which is expected because it also has no effects on secretion (Wagner et ul., 1989). It also strengthens the notion that of the major plasma electrolytes, calcium alone is a regulator of CS cell and STC gene activity.

1.2 m M Ca"

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STC/Actin, % of Control Fig. 15. Magnesium does not stimulate STC mRNA levels in rainbow trout CS cells. Cultured cells were exposed to 2.3 niM Ca" or 2.3 m M Mg'+/l.2 niM Ca" for 3 days and analyzed for STC mRNA content as described in Fig. 11. Calcium prompted a 1.8-fold induction of message levels over controls (1.2 m M Ca2+)whereas magnesium had no effkct. In additional experiments, &day exposures to magnesium were also without effect. Cell culture conditions, Northern blotting, and data analysis were as described in Fig. 11. Each data point represents the mean 2 S.E.M. of three replicates ( * P < 0.01 in comparison to controls in 1.2 m M Ca"; two-tailed ANOVA and Dunnet's test). Adapted from Wagner and Jaworski (1994).

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CS cells are unique in their ability to modulate STC mRNA levels bidirectionally in accordance with ambient calcium levels (Figs. 11-14). This is a quality that is not shared by PTH and calcitonin cells. The PTH gene, for example, is regulated in only one direction by calcium i n vioo and i n oitro (Brookman et al., 1987; Heinrich et al., 1983; Naveh-Many and Silver, 1990; Russel et al., 1983; Mouland and Hendy, 1991), whereas the calcitonin gene is not influenced by calcium at all (Naveh-Many et al., 1989, 1992). Therefore, primary cultured CS cells are exceptional by comparison, as STC mRNA levels are increased and decreased, respectively, by calcium concentrations about and below the physiological set point (-1.2 mM). Nevertheless, it is crucial that the regulation of STC gene expression is also characterized under in cico conditions. Whether or not the gene is siniilarly regulated b y calcium in the whole animal will be of particular interest. We have conducted only one i n vivo study to date, in this case on

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TIME AFTER INJECTION (hrs) Fig. 16. STC mRNA levels in calcium-challenged sockeye salmon as quantified by .situ hybridization. Juvenile, freshwater salmon (50 2 10 g) were given intraperitoneal injections of NaCl ( 0 )and CaC1, (V)equivalent to 30 mg/kg body weight of sodium and calcium. Five animals were sacrificed from each group at different times postinjection and CS glands were processed for in situ hybridization using 3sSS-labeledcRNA probes. All slides were developed at the same time and subjected to grain counting as previously described (Sterba et ul., 1993).Calcium prompted a rise in STC mRNA levels at the 5-hr mark that was statistically insignificant. Each data point represents the mean S.E.M.of five corpuscles ofStannius. From T. Sterbaand G. F. Wagner, unpublished. it1

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30 1

juvenile, freshwater-adapted sockeye salmon that were given intraperitoneal injections of sodiuni or calcium chloride (equivalent to 30 mg/ kg of Na’ or Ca”). The fish were sacrificed at different times postinjection (5, 24, and 48 hr) and because of the small size of the CS glands in these animals, STC mRNA levels were quantified by in situ hybridization and grain counting (Sterba et al., 1993). As expected, the calcium-injected salmon had elevated plasma STC levels at the 5-hr mark in comparison to sodium-injected controls. However, there were no statistically significant effects of calcium on STC mRNA levels at any time postinjection, though a small rise was apparent after 5 hr (Fig. 16). It is possible that minor hypercalcemic challenges do not require increased STC gene transcription and can be accommodated merely by increasing the rate of hormone synthesis from preexisting message. Increased rates of transcription may only be required in the event of a continuous calcium challenge such as that afforded by the marine environment.

VIII. CONCLUSIONS

The purpose of this chapter has been to provide a current perspective on the molecular biology of the corpuscles of Stannius and stanniocalcin. It should be readily apparent to the reader that the STC field, in spite of 30 years of progress, is still in its infancy in comparison to most other areas of endocrinology. This is true not only with respect to gene structure and function, but also as it applies to basic hormone physiology. Over the last 20 years, for instance, the only function definitively shown to be regulated by STC has been gill calcium transport. Only more recently have the intestinal transport of calcium (Sundell et al., 1992) and the renal handling of phosphate (Lu et al., 1994) been identified as being under the influence of stanniocalcin. Similarly, little is known ahout the regulation of STC secretion by other hormones, the nervous system, the life history of the fish, diet, or season, to name just a few factors ofpotential influence. In all fairness, part of the problem has to do with STC itself. The molecule is notoriously difficult to iodinate without causing irreparable damage to both its receptor binding and antibody binding properties. Consequently, there are grave difficulties inherent in identifying new targets and/or actions of STC and in studying the regulation of STC secretion using the traditional methods of radioreceptor assay and radioimmunoassay, respectively. Fortunately, these problems do not apply to studies on gene regulation, which are nonetheless still in the developmental

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stages. STC has been cloned and fully characterized in only two species of fish. However, several new clonings are currently in progress and the sequence information that will soon be forthcoming should broaden our perspective on the evolution of hormone structure and function even further. This is vitally important because unlike most other fish hormones, STC lacks an evolutionary perspective owing to its apparent absence in other vertebrates. The studies described here on calcium regulation of STC mRNA levels represent only the first in a series of steps by our laboratory to further our understanding of how this gene is regulated in salmon. Our next step is to determine the mechanisms by which calcium alters message levels; these include increased mRNA stability, an increased rate of gene transcription, or perhaps a combination of the two processes. Given the high level of induction that occurs after 6-day exposures to calcium (Fig. 13), it would appear that the gene is, in part, transcriptionally regulated by calcium. Beyond this, the next important objective will be to characterize the STC gene, which is uncharted territory at present. The exciting possibilities that the STC gene encodes more than one product, as in the case of the calcitonin-CGRP gene (Breimer et al., lYSS),and that novel transcription fixtors control the ontogeny and regulation of STC gene expression should keep us busy in the laboratory for years to come.

ACKNOWLEDGMENTS I am especially grateful to Henry G. Friesen, M.D., for giving me the opportunity of learning the basics of molecular biology. Salmon stariniocalcin was cloned and partially sequenced in his laboratory. I am also indebted to H . E. Ann MacPhail, l l S c . , for reviewing the manuscript. T h e contributions of my colleagues to the work discussed in this review are greatly appreciated as well. Grant and Scholarship support were provided by T h e Natural Sciences and Engineering Council of Canada and T h e Medical Research Council of Canada.

REFERENCES Ahmad, N., and Swarup, K. (1990).Seasonal changes in structure and behavior ofcorpuscles of Stannius in relation to the changes in serum calcium level and the reproductive cycle of a freshwater female catfish-Mystus oittatus (BLOCH). E u r . Arch. B i o l . 101, 285-294.

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Aida, K., Nishioka, R. S., and Bern, H. A. (1980).Degranulation ofthe Stannius corpuscles of coho salmon (Oncorhynchus kisutch) in response to ionic changes in citro. Gen. C o m p . Endocrinol. 41, 305-313. Aliapoulios, M . A., Goldhaher, P., and Munson, P. L. (1966). Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science 151, 330-33 1. Anast, C. S., and Conway, H. H. (1972). Calcitonin. Cliiz. Orthop. 84, 207-262. Breimer, L. H., Maclntyre, I., and Zaidi, M. (1988). Peptides from the calcitonin genes: hlolecular genetics, structure and function. Biochern. J . 255, 377-390. Nicholson, L., O’Riordan, J. L. H., and Hendy, G . N. Brookman, J. J., Farrow, S. &I., (1987). Regulation b y calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J . Bone Miner. Res. 6, 529-537. Brown, E. M.,LeBoff, M. S., Oetting, M., Possilico, J. T., and Chen, C. (1987). Secretory control in normal and abnormal parathyroid tissue. Recent Prog. Horm. Res. 43, 337. Butkus, A,, Roche, P. J., Fernley, R. T., Haralambidis, J., Penschow, J. D., Ryan, G. B., Trahair, J. F., Tregear, C:. W., and Coughlin, J. P. (1987). Purification and cloning of a corpuscles of Stannius protein from Anguillu uustralis. Mol. Cell. Endocrinol. 54, 123-134. Butkus, A,, Yates, N. A,, Copp, D. H., Milliken, C., and McDougall, J. G. (1989). Processing and bioactivity of the corpuscles of‘ Stannilis protein of the australian eel. Fish Pliysiol. Biochem. 7, 359-365. Butler, D. G. (1969). Corpuscles of Stannius and renal physiology in the eel (Anguillu rostrutu).J . Fish. Res. Bourd Can. 26, 639-654. Carpenter, S. J . , and Heyl, H . L. (1974). Fine structure of the corpuscles of Stannius ofAtlantic salmon during the freshwater spawningjourney. Gen. C o m p . Endocrinol. 23, 212-223. Colien, R. S., Pang, P. K. T., and Clark, N. B. (197.5). Ultrastructure of the Stannius corpuscles ofthe killifish, Fundulus heteroclitus, and its relation to calcium regulation. Gen. Comp. Endocrinol. 27, 413-423. Fenwick, J. C. (1974). The corpuscles of Stannius and calcium regulation in the North American eel (Anguillu rostrutu LeSueur). Gen. C o m p . Endocrinol. 29, 127-135. Fenwick, J. C., and Brasseur, J. G. (1991). Effircts of stanniectomy and experimental hvpercalcemia on plasma calcium levels and calcium influx in American eels, Anguilla rostrutu, LeSueur. Gen. Comp. Endocrinol. 82, 459-465. Fenwick, J. C., and So, Y. P. (1974). A perfusion study of the effect ofstanniectoniy on the net influx of calcium-45 across an isolated eel gill.]. E x p . Zoo/. 188, 125-131. Flik, G., Labedz, T., Neelissen, J. A. hl., Hanssen, R. G. J. M.,Wendelaar Bonga, S . E., and Pang, P. K. T . (1990). Rainbow trout corpuscles of Stannius: Hypocalcin synthesis in citro. Am. J . Physiol. 258, R1157-1164. Fontaine, M. (1964). Corpuscules de Stannius et regulation ionique (Ca, K, et Na) du milieu interieur d’un poisson l’arrguille. C.R. Acud. Sci. Ser. D 529, 875-878. Fontaine, M., Delerue, N., Martelly, E., Marchelidon, J., and Milet, C. (1972). Role des corpuscule de Stannius dans les echanges d e calcium d’un poisson teleosteen (Anguille unguille L.) avec le milieu anibiant. C.R. Acud. Sci. Ser. D 275,1523-1528. Fraser, R. A., Kaneko, T., Pang, P. K. T., and Harvey, S. (1991). Hypo- and hypercalcemic peptides in fish pituitary glands. A m . J. P h y s i o l . 260, R622-626. Friedman, J., and Raisz, L. G. (1965). Thyrocalcitonin: Inhibitor of bone resorption in tissue culture. Science 150, 1465-1467. Gagel, R. F.. Zeytinoglu, F. N., Voelkel, E. F., and Tashijian, Jr., A. H. (1980). Establish-

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inent of a calcitonin-producing rat medullary thyroid carcinoma cell line. 11. Secretory studies of the tumor and cells in culture. Endocrinology (Baltimore) 107,

5 16-523. Garrett, F. D. (1942). The development and phylogeny of the corpuscles of Stannius in ganoid and teleostean fishes. J . Morphol., 70, 41-67. Gellersen, B., Wagner, G. F., Copp, D. H., and Friesen, H. G . (1988).Developnient of a primary culture system for rainbow trout corpuscles of Stannius and characterization of secreted teleocalcin. Endocrinology (Baltimore) 123, 913-921. Glowacki, J., Milhaud, G., Benson, A., Wagner, G., Cox, K., Fargher, R. C., and Copp, D. H . (1990). Effect of calcium challenge on secretion of stanniocalcin (teleocalcini hypocalcin) in adult seawater coho salmon: A preliminary study. I n “Calcium Regulation and Bone Metabolism” (D. V. Cohn, F. H. Glorieux, and T. J. Martin, eds.), pp. 74-79. Elsevier Science Publ., Amsterdam. Hanssen, H. G. J. M.,Aarden, E. M., van der Venne, W. P. H. G., Pang, P. K. T., and Wendelaar Bonga, S. E. (1991).Regulation of secretion of the teleost fish hormone stanniocalcin: Effects ofextracellular calcium. Gen. C o m p . Endocrinol. 84,155-163. Heinrich, G., Kronenburg, H. M., Potts, Jr., J . T., and Habener, J. F. (1983).Parathyroid hormone messenger ribonucleic acid: Effects of calcium on cellular regulation in citro. Endocrinology (Baltinzore) 112, 449-458. Hirano, T. (1989).The corpuscles of Stannius. In “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and X I . P. Schreibman, eds.), Vol. 3 , pp. 139-169. Academic Press, San Diego. Kaneko, T., Hasegawa, S., and Hirano, T. (1992). Embryonic origin and development of the corpuscles of Stannius in chum salmon (Oncorhynchus ketci). Cell Tissue Res. 268, 65-70. Krishnamurthy, V. G. (1976). Cytophysiology of corpuscles of Stannius. Znt. Rec. Cytol. 46, 177-249. Lafeber, F. P. J. G., Flik, G., Wendelaar Bonga, S. E., and Perry, S. F. (1988a).Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. A m . J . Physiol. 254, K891-R896. Choy, Y. M.,Flik, G., Hermann-Erlee, M.P. Lateher, F. P. J. G., Hanssen, R. G. J . M,, XI., Pang, P. K. T., and Wendelaar Bonga, S. E. (1988b). Identification of hypocalcin (teleocalcin) isolated from trout corpuscles of Stannius. Gen. Comp. Endocrinol. 69, 19-30. Lopez, E., Tisseran-Jochem, E. M., Eyquem, C., Milet, C., Hillyard, C., Lallier, F., Vidal, B., and MacIntyre, I. (1984). Immunocytochemical detection in eel corpuscles of Stannius of a mammalian parathyroid-like hormone. Gen. Comp. Endocrinol. 53, 28-36. Lu, X f . , Wagner, G. F., and Henfro, J. L. (1994). Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture. A m . J . Physiol. 267 (Regul. Integr. Comp. Physiol. 36), in press. hlarra, L. E., Youson, J. H., Butler, D. G., Friesen, H. G., and Wagner, C . F. (1992). Stanniocalcin-like immunoreactivity in the corpuscles of Stannis of the bowfin, Amia calva L. Cell Tissue Res. 267, 283-290. Xlayer-Gostan, N., Flik, G., and Pang, P. K. T. (1992).An enzyme-linked immunosorbent assay for stanniocalcin, a major hypocalcemic hormone in teleost. Gen. Comp. Endocririol. 86, 10-19. hleats, M., Ingleton, P. M., Chester-Jones, I., Garland, H. O., and Kenyon, C . J. (1978). Fine structure of the corpuscles of Stannius of the trout, Sulnlo gciirdneri: Structural changes in response to increased environmental salinity and calcium i o n s . Gen. Camp. Eiidocrinol. 36, 451-461.

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Milhaud, G., Perault, A.-M., and Moukhtar, M . S. (1965). Etude du mecanisme d e l'action hypocalcemiante d e la thyrocalcitonine. C . R .Hebd. Seances Acad. Sci. 261, 813-816. Milliken, C., Fargher, R. J . , Butkus, A,, McDonald, M., and Copp, D. H. (1990). Effects of synthetic peptide fragments of teleocalcin (hypocalcin) on calcium uptake in juvenile rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 77, 416-422. Mouland, A. J., and Hendy, G. H. (1991). Regulation of synthesis and secretion of chromogranin-A by calcium and 1,25-dihydroxycholecalciferolin cultured bovine parathyroid cells. Endocrinology (Baltimore) 128, 441-449. Naveh-Many, T.,and Silver, J. (1990). Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J . Clin. Inoest. 86, 1313- 1319. Naveh-Many, T., Friedlaender, M. M., Mayer, H., and Silver, J. (1989).Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in cioo in the rat. Dominant role ofl,25-dihydroxyvitaininD. Endocrinology (Baltimore) 125,275-280. Naveh-Many, T.,Raue, F., Grauer, A,, and Silver, J. (1992). Regulation of calcitonin gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J. Bone Miner. Res. 7, 1233-1237. Ogawa, M.(1967). Fine structure of the corpuscles of Stannius and the interrenal tissue in goldfish (Curussius auratus). Z . Zellerforsch. 81, 174-189. Pang, P. K.T., and Pang, R. K. (1974). Environmental calcium and hypocalcin activity in the Stannius corpuscles of the channel catfish, Ictulurus punctatus (Rafinisque). Gen. C o m p . Endocrinol. 26, 179-185. Pang, P. K.T., Pang, R. K., and Sawyer, W. H. (1973).Effect ofenvironmental calcium and replacement therapy on the killifish, Fundulus heteroclitus, after surgical removal of the corpuscles of Stannius. Endocrinology (Baltimore) 93, 705-710. Pang. P. K. T., Pang, H. K., and Sawyer, W. H.(1974). Environmental calcium and sensitivity of killifish (Fundulus heteroclitus) in bioassays for the hypocalcemic response to Stannius corpuscles from killifish and cod (Gadus rnorhuu).Endocrinology (Baltimore) 94,548-555. Russel, J., Lettieri, D., and Sherwood, L. M. (1983). Direct regulation by calcium of' cytoplasmic ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells. 1.Clin. Inaest. 72, 1851-1855. So, Y. P.,and Fenwick, J. C.(1977). Relationship between net "calcium influx across a perfused isolated eel gill and the development of post-stanniectomy hypercalcemia. J. Exp. Zool. 200, 259-264. So,Y.P.,and Fenwick, J. C.(1979).The in ciao and in citro effects ofStannitis corpuscles extract on the branchial uptake of "Ca in stanniectomized North American eel (Anguilla rostruta). Gen. Comp. Endocrinol. 37, 143-149. Stannius, H. (18.39). Ueber Nebenniere bei Knochenfischen. Arch. An&. Physiol. 6, 97- 101. Sterba, T.,Wagner, G. F., Schroedter, I. C., and Friesen, H.G. (1993). In situ detection arid distribution of stanniocalcin mRNA in the corpuscles of Stannius of sockeye salmon, Oncorhynchus nerka. Mol. Cell. Endocrinol. 90, 179-185. Sundell, K.,Bjornsson, B. Th., and Kawauchi, H.(1992). Chum salmon (Oncorhynchus keta) stanniocalcin inhibits in citro intestinal calcium uptake in Atlantic cod (Gadus morhua).1.Comp. Physiol. B 162, 489-495. Verbost, P. M., Butkus, A,, Atsma, W., Willems, P., Flik, G., and Wendelaar Bonga, S. E . (1993). Studies on stanniocalcin: Characterization of hioactive and antigenic domains of the hormone. Mol. Cell. Endocrinol. 93, 11-16.

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LVagner, G. F. (1993). Stanniocalcin: Structure, function and regulation. I n “Rioclicnristry and Molecular Biology of Fishes” (P. W. Hochachka and T. P. Moiiinisen, cds.), Vol. 2, Chap. 21, pp. 419-434. Elsevier Science Pirbl., Anisterdani. \Vagner, G. F., and Jaworski, E. (1994). Calcium regulates stanniocalcin niKNA levels in primary cultured rainbow trout corpuscles of Stannius. M o l . Cell. Encfocririol. 99,315-322. \Vagner, C. F., Hampong, M., Park, C. M., andCopp, D. H. (1986).Purification, characterization and bioassay of teleocalcin, a glycoprotein from saliiion corpuscles of Stanrrius. Cen. Comp. Endocrinol. 63, 481-491. \Vagner, G. F., Fenwick, J. C., Milliken, C., Park, C. XI., Copp, D. H., and Friesen, H. (2. (1988a). Comparative biochemistry and physiology of teleocalciii froni sockeye and coho salmon. Gen. Cornp. Endocrinol. 72, 237-246. Wagner, 6. F., Copp, D. H., and Friesen, H. G. (19881)). Imtiiurrological studics on teleocalcin and salmon corpuscles of Stannius. Erzdocririology (Baltimore) 122, 2064-2070. \Vagner, G. F., Gellersen, B., and Friesen, H. G. (1989). Primary culture of teleocalcin cells from rainbow trout corpuscles of Stannius; Regulation of teleocalcin secretioii b y calcium. M o l . Cell. Endocrinol. 62, 31-39. \\’agner, G. F., Milliken, C., Friesen, H. G., and Copp, D. H. (1991). Studies on tlic regulation and characterization of plasma stanniocalcin in rainbow trout. Mol. Cell. Endocrinol. 79, 129-138. Wagner, G. F., Di Mattia, G. E., Davie, J. R., Copp, D. H., and Friesen, H. G. (1992). Rfolecular cloning and cDNA sequence analysis of coho salmon stanniocalcin. M o l . Cell. Endocrinol. 90, 7-15. fi‘agiier, G. F., Fargher, R. C., Milliken, C., McKeown, B. A,, and Copp, U. H. (lYS3). The gill calcium transport cycle in rainbow trout is correlated with plasma levels ofbioactive, not immunoreactive, stanniocalcin. Mol. Cell. Endocrinol. 93,185- 191. \Vendelaar Bonga, S. E., and Pang, P. K. T., eds. (1986). Stannius corpuscles. Z t i “Vertebrate Endocrinology, Fundamentals and Biomedical Implications,” i’ol. 1, pp. 439-464. Academic Press, Orlando, Florida. \Vendelaar Bonga, S. E., and Pang, P. K. T. (1991). Control of calciuni regulating hormones in the vertebrates: Parathyroid hormone, calcitonin, prolactin, and stanniocalcin. I n t . Hec. Cytol. 128, 139-213. l’ouson, J. H., Butler, D. C., and Chan, A. T. C. (1976). Identification arid distri1)utioir of the adrenocortical homolog, chroniaffin tissue and the corpuscles of Stannius in Arnici ccilau L. Geri. Conip. Endocrinol. 29, 198-21 1 .

IV HORMONE REGULATION


10 COMPARATIVE ASPECTS OF PITUITARY D E V E L O P M E N T A N D Pit-1 F U N C T I O N SONALI MAJUMDAR AND HARRY P . ELSHOLTZ Department of Clinical Biochemistry and Banting %I Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada M5G 1L5

I. Introduction 11. Comparative Organization of the Pituitary Gland 111. Differentiation of Adenohypophysial Cell Types A. The Rat Adenohypophysis B. The Fish Adenohypophysis IV. Transcription Factor Pit-1 A. Role in Mammalian Pituitary Development B. Expression of Pit-I during Mammalian Pituitary Development C. DNA Binding and Target Gene Specificity D. POU Domain: Structure and Function E. N-Terminal Sequences: Multiple Isoforms F. Pit-1 Dimerization and Interaction with Other Proteins V. Comparison of Pit-1 in blanimals and Teleost Fish: Studies on the PRL Target Gene A. Conservation of Pit-1 POU Domain Function in Fish B. Species Differences in Alternative RNA Splicing C. N-Terminal Sequences of Rat and Salmon Pit-1 VI. Conclusion References

I. INTRODUCTION The pituitary gland or hypophysis is a critical endocrine regulator

of vertebrate growth, metabolism, reproduction, ion balance, and behavior, and accordingly it was once described as the “master gland.” Closely associated with the brain, the pituitary is under predominant neural control although multiple feedback signals from target organs 309 F I S l l I ’ l T l b l O L O G ~ 01. XI11

Copbiight 0 I994 In A c a d e m ~Pie\\ Inc A l l right, of Irprodnctmn i n arw form iewrved

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SONALI MAJUMDAR A h D €IARRY P ELSHOLTZ

can play equally important regulatory roles. The pituitary displays remarkable morphological variation, not only among different vertebrate classes but even within the same species (e.g., different breeds of dogs). Yet the secretory cell types and polypeptide hormone products first identified in eutherian mammals have also been observed in distantly related vertebrates, suggesting that common mechanisms may dictate pituitary organogenesis. Inimunohistological, biochemical, and molecular studies have provided new insights into the mechanisms of pituitary differentiation (Voss and Rosenfeld, 1992). Factors required for the regulation of pituitary-specific genes have been identified or cloned and their fiinctional domains characterized. This chapter focuses on the transcription factor Pit-1, which plays a pivotal role in the differentiation of specific pituitary cell lineages and in the activation of a subset of endocrine genes. Structural and functional comparisons of mammalian and fish Pit-1 are discussed.

11. COMPARATIVE ORGANIZATION OF THE PITUITARY GLAND The pituitary can be divided into two principal structures, the adenohypophysis or anterior pituitary and the neurohypophysis or posterior pituitary. During embryogenesis the pituitary derives from two different sources. The adenohypophysis develops from the ectoderma1 cells growing out from the roof of the oral cavity, an embryonic structure called Rathke’s pouch. This pharyngeal evagination ultimately separates to associate with an outpouching of the diencephalon, partitioned from the oral cavity by the sphenoid bone of the skull. In teleost fish the adenohypophysis is further organized into rostra1 and proximal portions that are distinguishable on the basis of specific endocrine cell types (described in the following). The vertebrate neurohypophysis, which is also of ectodermal origin, develops from the downward outgrowth of the diencephalon and contains both neural and glial cell types. Cells of the embryonic adenohypophysis that contact the neurohypophysis give rise to a third pituitary structure, the intermediate lobe (pars intermedia), which separates the anterior and posterior lobes of the pituitary. This lobe may he either well defined (e.g., rodents, reptiles, teleost fish), poorly defined (e.g., primates, birds), or completely absent (e.g.,whales) depending on the species. Another pituitary structure subject to species differences is the pars tuberalis, which arises from lateral extensions of Rathke’s pouch. In some species, particularly in birds, it forms a prominent collar around the u p p e r

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311

pituitary stalk, whereas in others, including teleost fish and a number of mammals, it is well defined during development but difficult to observe in the mature organism. In most tetrapods stimulatory or inhibitory factors from the hypothalamus that regulate pituitary secretory function are transported by the hypothalamohypophysial portal plexus. In teleosts a similar portal vascular system is lacking and arterial blood passes directly to the pituitary; blood vessels within the neurohypophysis are associated with neurosecretory fibers from the hypothalamus and transport neural regulatory factors to the anterior lobe. Aminergic fibers from the hypothalamus (and in some cases peptidergic fibers) can also interact directly with cells of the adenohypophysis. In some cyclostomes such as the hagfish, the pituitary is a loosely organized tissue in which hypothalamic fibers project to the neurohypophysis and regulatory factors reach the cells of the anterior lobe by simple diffusion (Holmes and Ball, 1974; Batten and Ingleton, 1987).

111. DIFFERENTIATION OF ADENOHYPOPHYSIAL CELL TYPES The mature anterior pituitary contains five major endocrine cell types characterized by their polypeptide hormone product (Chetelain et al., 1979; Watanabe and Daikoku 1979; Hoeffler et al., 1985). These horniones are critical to homeostatic regulation, growth, and reproduction. Adrenocorticotropin (ACTH), synthesized by the corticotrophs, regulates steroid hormone production by the adrenal cortex; thyroidstimulating hormone (TSH) from the thyrotrophs promotes synthesis and release of T3 and T4 from the thyroid; luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the gonadotrophs regulate ovarian or testicular function; and growth hormone (GH) from somatotrophs enhances physical growth. Prolactin (PRL) is the most functionally diverse of the adenohypophysial hormones, regulating milk production and lactation in mammalian species, osmoregulation in teleost fish, and reproductive and behavioral functions in certain birds and mammals. A. The Rat Adenohypophysis In the rat embryo the a-subunit ofthe glycoprotein hormones (FSH, LH, TSH) serves as the earliest marker for anterior pituitary development, detectable by the eleventh day in ectodermal cells beneath the neural tube (Simmons et al., 1990). Corticotrophs are observed by

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Day 13 or 14 and, based on organ culture studies, appear to arise independently of exogenous cues (Begeot et al., 1982). By contrast, cell types arising at later stages of pituitary development are more dependent on paracrine or endocrine factors. Thyrotrophs are detectable by about embryonic (e) Day 14 in the rostral tip of the anterior lobe and are followed by gonadotrophs (Day e l 6 to e17) and somatotrophs (Day e l 7 to e18).Somatotrophs are located caudally and proliferate dorsally around Day e18. Prolactin-producing lactotrophs are only weakly detectable by Day el7 to el8 but undergo a dramatic expansion in cell number during the early postnatal period. Unlike the endocrine cells of the fish anterior pituitary (see the following), mature rat adenohypophysial cells are distributed in a random manner, a characteristic that may be determined by local migratory factors or intercellular recognition signals (Voss and Rosenfeld, 1992). In rodents the appearance of lactotrophs is largely dependent on the differentiation of the somatotroph lineage. Transgenic studies have demonstrated a dramatic reduction in lactotroph number in animals carrying a toxin gene specifically targeted to embryonic somatotrophs (Behringer et al., 1988; Borrelli et al., 1989). These data suggest that all lactotrophs or at least the majority (Behringer et al., 1988) derive from a somatotroph stem cell. In the mature animal the anterior pituitary retains a population of cells that coexpresses growth hormone and PRL; the ratio of growth hormone to PRL produced b y these cells may be altered depending on physiological requirements (Frawley and Boockfor, 1991). B. The Fish Adenohypophysis The pattern of pituitary cell development in teleost fish is distinct from that ofmammals. Even within the same family (e.g., chum salmon vs. coho salmon) the chronological appearance of distinct cell types varies. In chum salmon, 5 weeks postfertilization and prior to hatching the dorsal half of the adenohypophysis contains columnar cells packed tightly, whereas the ventral portion contains cells that are more randomly and loosely arranged. Prolactin-producing cells are the first cell type to appear and are located in the rostroventral portion o f t h e adenohypophysis as follicular structures. Somatotrophs also appear early followed by corticotrophs and thyrotrophs. Whereas soniatotrophs are centrally positioned, corticotrophs are observed dorsally from the rostral to the caudal region, and thyrotrophs are interspersed with somatotrophs. The gonadotrophs appear late in development, becoming detectable at 3 weeks after hatching (Naito et nl., 1993). In

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the coho salmon, prolactin-producing cells are again the first cell type to appear but are followed by thyrotrophs and then corticotrophs. Somatotrophs in this species develop between 5 and 6 weeks postfertilization, that is, within a week of hatching. As in chum salmon, gonadotrophs appear last-gonadotrophin (GTH) I cells at about 2 weeks after hatching and GTH I1 cells shortly before gametogenesis (Ma1 et az., 1989). The early appearance of PRL-secreting cells is not restricted to salmonid species but is also observed in the euryhaline fishes such as tilapia (Hwang, 1990) and certain marine species. In a report of pituitary development in sea bream (Power and Canario, 1992),somatotrophs are first detectable on Day 1 after hatching. PRL-producing cells are detectable on Day 4. The number of PRL-producing cells increases in the rostral pars distalis up to Day 12. Gonadotrophs are observed by the sixth day progressively up to Day 12, at which time they project into the pars intermedia. GTH-producing cells in the sea bream occupy almost 30%of total pituitary volume. Two populations of ACTH-producing cells are observed on Day 8, one in an anterior location in the pars distalis and a second in a posterior location in the pars intermedia (Power and Canario, 1992). The early appearance of PRL cells in a number of teleost fish is an interesting distinction from the pattern of lactotroph development in rodents. Whether developmental differences are in any way related to phylogenetic changes in PRL function has not been determined. Furthermore, in certain fish species PRL cells appear late during pituitary ontogeny. In the sea bass, for example, the predominant cells on Day 1 after hatching are corticotrophs, with lesser numbers of somatotrophs and thyrotrophs detectable in the rostral portions of the pituitary. PRL-producing cells are observed on Day 9 posthatching, whereas gonadotrophs are not detected until after Day 26 (Cambr6 et az.,

1990).

IV. TRANSCRIPTION FACTOR PIT-1 A. Role in Mammalian Pituitary Development

The coordination of developmental processes that determine the appearance of specific cell types is dependent on the interaction of transcriptional regulators with specific target genes. One critical class of developmental regulators is encoded by the homeobox genes, originally identified in invertebrate species and characterized by genetic

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SONALI h l A J U h l D A R AhLD IIAHRY P. ELSHOLTZ

analyses (Akam, 1987; Gehring, 1987). Multiple homeobox genes have also been identified in vertebrate species (Kessel and Gruss, 1990; McGinnis and Krumlauf, 1992) and targeted knock-out approaches have revealed their developmental functions in several cases (McGinnis and Krumlauf, 1992 and references therein; Joyner et al., 1991; Mouellic et al., 1992; Ramirez-Solis et ul., 1993). In the manimalian pituitary a homeodomain-containing factor, Pit-1 or GHF-1 (Ingraham et al., 1988,Bodner et al., 1988),plays a pivotal role in the development of specific adenohypophysial cell lineages. Although Pit-1 was first described in biochemical experiments as a pituitary-specific DNAbinding protein capable of activating the growth hormone (Bodner and Karin, 1987, Nelson et al., 1988) and PRL (Nelson et nl., 1988) gene promoters, genetic evidence has now established the importance of Pit-1 in vivo during embryogenesis. Dwarf mouse strains (Snell, Jackson, Ames) that underexpress the pit-1 gene (Li et al., 1990) share a common phenotype, that is, hypoplastic pituitaries deficient in three specific cell types-somatotrophs, lactotrophs, and thyrotrophs. Serum levels of GH, PRL, and TSH are essentially undetectable. In the case ofthe Jackson and Snell mutants the pit-1 gene is rearranged or pointmutated, respectively, whereas in the Ames mouse the pit-1 gene is apparently normal but underexpressed because of a defect at a separate chromosomal locus. A variety of mutations in the human p i t - l gene have been reported (Tatsumi et ul., 1992; Radovick e t al., 1992; Pfiiffle et al., 1992; Ohta et ul., 1992) and linked to combined pituitary hormone deficiency (Winter et al., 1974; Kogol and Kahn, 1976). The endocrine abnormalities, which included cretinism and dwarfism, vary in severity depending on the position and nature of the mutations in the p i t - l gene. Examples of such mutations are discussed in Section IV,D. B. Expression of Pit-1 during Mamm,a 1.ian Pituitary Development The earliest detectable expression ofpit-l RNA transcripts is found in the neural tube of the embryonic mouse on Day 10 (He et ul., 1989, Sininions et al., l99O), although the absence of neural tube defects in Pit-1 deficient dwarf mice may question the developmental significance ofthis event. In the anterior pituitary, expression of p i t - 1 transcripts and protein precedes the appearance of GH- and PRL-secreting cells. Both pit-l niRNA and protein are observed by Days e l 5 to e l 6 (Simmons et ul., 1990; Dolle et al., l990), and levels of'immunoreactive Pit-1 increase gradually until Day 10 after birth (Day p10). This in-

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315

crease in the level of Pit-1 protein probably reflects the expansion of cell populations (e.g., lactotrophs) that express p i t - 1 . Pit-1 protein is not detectable in pituitary corticotrophs or gonadotrophs, being restricted to somatotroph, lactotroph, and thyrotroph cell types (i.e., the cell types absent in Pit-1-deficient animals). The early appearance of‘ the thyrotroph cell lineage on Day e l 4 (see earlier) prior to expression of pit-1 would appear to suggest a maintenance function for Pit-1 in this cell type. Other evidence, however, indicates that the developing pituitary may produce two populations of thyrotrophs-an early transient and Pit-l-independent population that is subsequently replaced by a permanent population of Pit-l-dependent cells (Lin e t d . , 1994). Although in rodents expression of pit-1 protein is restricted to cells producing GH, PRL, or TSH, the level of pit-1 mRNA was found to be similar in all five endocrine cell types (Simmons et uZ., 1990). These data suggest that a translational mechanism may determine the distribution of Pit-1 in the anterior pituitary. Similar observations have not yet been reported for other mammalian species. We have shown in human pituitary adenomas and nontumorous pituitary tissue that the pattern of Pit-1 protein expression correlates well with that of Pit1niRNA (Asa e t al., 1993).The human data, therefore, are more readily explained by a “pretranslational” regulatory model for Pit-1 express ion.

C. DNA Binding and Target Gene Specificity Pit-1 binds with high affinity to specific regulatory elements of pituitary target genes to activate transcription. Although Pit-1 likely regulates multiple genes in somatotroph, lactotroph, and thyrotroph cells, the PKL and GH genes have been studied most extensively. In the rat, both the PRL and GH gene contain proximal Pit-1 sites within 250 base pairs 5‘ to the transcription start site. The PRL gene contains an additional Pit-1 binding enhancer region located 1.5 to 1.8 kb upstream of the proximal region. The sequence of this distal enhancer appears to be about 80% identical among mammalian PRL genes (e.g., rat, human, cow) and the proximal region nearly 90% identical. In the case of the rat PRL gene, deletional analysis demonstrated that the conserved proximal and distal regions are required for activation in pituitary cells (GH4, GC) but lack a stimulatory cis-activity in heterologous cells, such as fibroblasts or HeLa cells. In cultured pituitary cells, the relative activity of the distal enhancer and proximal region can be variable (Nelson e t ul., 1986, 1988; Lufkin and Bancroft, 1987), due

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in large part to the particular cell line used (Jones and Catanzaro, 1991). Based on transgenic mouse studies, however, both the proximal and distal Pit-1 binding regions are required for optimal high-level expression of the PRL transgene in pituitary cells (Crenshaw et al., 1989). Interestingly, PRL genomic sequences between the two Pit1 binding regions appear to be necessary to prevent inappropriate expression of the transgene in Pit-l-containing thyrotrophs. Alignment of multiple DNase I footprints from GH and PRL genes has revealed a consensus binding site for Pit-1-(A/T),TATNCAT (Nelson et al., 1988). The specificity of this consensus sequence has been examined in the most proximal site of the rat PRL promoter using a series of clustered mutations or point transversions in the TATNCAT core and flanking bases (Elsholtz et al., 1990). Individual mutations at most positions in the core reduce Pit-1 binding by >70%. Flanking mutations 5' of the core also reduce the binding of Pit-1 albeit to a lesser degree, whereas mutations 3' to the core have little effect. Interestingly, phosphorylation of Pit-1 by protein kinase A or C can decrease the ability of Pit-1 to bind to certain DNA sites by a mechanism dependent on nucleotides immediately upstream of the TATNCAT box (Kapiloff et al., 1991). These nucleotides could therefore determine the efficiency of Pit-1 sites to function as hormone response elements.

D. POU Domain: Structure and Function The cloning of Pit-1 (Ingraham et al., 1988; Bodner et al., 1988) and two other mammalian transcription factors, Oct-1 and Oct-2 (KO et al., 1988; Muller et al., 1988; Scheidereit et al., 1988; Sturni et ul., 1988), revealed a novel conserved sequence N-terminal to the homeodomain that was also present in the product of a Caenorhabditis elegans developmental gene, unc-86 (Finney et al., 1988). This sequence was about 80 amino acids in length and was separated from the homeodomain by a nonconserved linker sequence of 15 to 25 residues. The bipartite sequence was named the POU domain and is composed of an N-terminal POU-specific domain (POU,) and a Cterminal POU homeodomain (POU,,) (Fig. 1). Many POU domain proteins have been identified over the past few years in both vertebrates and invertebrates (Rosenfeld, 199 1;Verrijzer and Van der Vliet, 1993). The POU protein family appears to be of particular importance in the development ofthe CNS (Treacy and Rosenfeld, 1992). Classification of POU proteins into groups POU-I to POU-VI (Rosenfeld, 1991; Okamoto et al., 1993, Johansen et al., 1993) is based on overall

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POU-specific domain-

89% High affinity DNA binding Enhanced site specificity Protein/protein interaction

317

LPOU-homeodomain71%

85% Low affinity DNA binding Relaxed site specificity

Fig. 1. Schematic representation of the bipartite POU domain indicating the POUspecific and POU homeodomains. Boxes depict the predicted a-helices. Positions of basic ( + ) and acidic ( - ) amino acid residues, and the percent conservation in the two domains between salmon and rat, are indicated at bottom.

sequence similarity, especially in the basic amino acid cluster at the N terminus of the POUHDand in the spacer region separating the POU, and POUHD. The Pit-1 POUHI,is about 20-30% identical to the classic homeodomains encoded by the Drosophila developmental genes such as Antennapedia and Ultrabithorax (Gehring, 1987; Scott and Carroll, 1987). Of the nine amino acid residues invariant among Drosophila homeodomains, seven are conserved in Pit-1. The POU,, contains three ahelices, ofwhich the the third helix (also called the recognition helix) is most highly conserved among POU proteins. Based on crystallography studies of the Drosophila engrailed homeodomain (Kissinger et al., 1990), the POUHDrecognition helix, KXV(V/I)RVWFCN(R/Q)RQ (K/ R)KR, is likely to form base contacts within the major groove of the DNA site. The functional importance of the Trp(W) residue in the Pit1 POUIiDis well demonstrated by the Snell mouse, whose pit-l gene contains a single-nucleotide change that causes a Trp to Cys substitution in the recognition helix. The mutation abolishes Pit-1 binding to DNA, resulting in a dwarf phenotype. Pit-1 binding to DNA is also abolished by substitution of a Gly residue at a conserved Arg position (i.e., W F C N G R Q ) in helix 3 (Ingraham et al., 1990). Interestingly, conversion of the highly conserved Cys(C) residue to a Gln(Q) (found in many “classic” homeodomains) does not impair Pit-1 binding but reduces by three- to fourfold the ability of Pit-1 to activate the P R L promoter. This demonstrates that the Pit-1 POU,, functions not only in DNA recognition but also in transactivation. A similar loss-of-function mutation has been reported in a patient with combined pituitary hormone deficiency (Radovick et al., 1992); in this case disruption of a basic amino acid near the C terminus of the Pit-1 POUHD (Arg to Trp substitution) yielded a dominant negative Pit-1 mutant that bound DNA but failed to activate transcription.

318

SONALI MAJUMIIAH AND HAKHY P. ELSWOLTZ

In experimental Pit-1 mutants that lack the POUS domain, the POUF1, is sufficient for low-affinity interactions with AIT-rich sequences (Ingraham e t al., 1990). Studies with the POU protein Oct-1 indicate that the POUs domain may also be capable of autonomous low-affinity DNA binding (Verrijzer et al., 1992). Together, however, the POU, and POU,, cooperate to facilitate recognition ofthe specific consensus element and enable high-affinity interactions with the D N A site (Ingraham e t al., 1990; Sturm and Herr, 1988; Verrijzer et al., 1990). Although the POUS domain is critical for POU protein function and its sequence is highly conserved, its structure has only recently been characterized. Using nuclear magnetic resonance analysis, AssaMunt et al. (1993) and Dekker et a1. (1993) have determined the solution structure ofthe POU, domain of Oct-1. The POUS consists of four a-helices packed around a core of hydrophobic residues. Helices two and three form a helix-turn-helix structure with striking similarity to the DNA-binding doniains of certain prokaryotic proteins, including bacteriophage A repressor and 434 Cro. Helix 3 contributes numerous contacts within the DNA major groove and is the most highly conserved sequence of the POU, domain. Accordingly, inversion of a short peptide sequence within the third helix of the Pit-1 POUSdomain disrupts Pit-1IDNA interactions (Ingraham e t al., 1990). Introduction of two Pro residues into POUS helix 2, or rearrangement of the amino acid sequence in POU, helix 1, also interferes strongly with Pit-1 binding to DNA. It is noteworthy that POU, helix 2 may have a transactivating function in addition to its role in DNA binding. An interesting clinical case was reported in which the a-helical structure of the Pit-1 POUS helix 2 was perturbed by an Ala to Pro substitution; the mutant protein in this patient retained DNA-binding activity but failed to activate the PRL or GH promoter (Pfaffle et ul., 1992), as in the case of certain POUIIDmutations discussed earlier.

E. N-Terminal Sequences: Multiple Isoforms

The major transactivating function of Pit-1 has been localized to sequences N-terminal to the POU domain (Theill et al., 1989; Ingraham et al., 1990). Deletion of aniino acids 8-80 resulted in an 85% decrease in reporter gene activation without a concomitant loss in DNA-binding activity. Furthermore, when fused to the DNA-binding domain of the E . coli repressor, LexA, N-terminal pit-1 sequences strongly activate promoters containing LexA binding sites. The Nterminal region of pit-1 is rich in serine and threonine residues that are likely to be important for the transactivating function. A similar

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ASPECTS OF PITUITARY DEVELOPhlENT AND Pit-1 FUNCTION

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content of hydroxylated amino acids is found in the transactivation domains of certain other POU proteins, such as Oct-2. As a result of alternative RNA processing and dual translational start sites, rat Pit-1 occurs in a number of isoforms having unique Nterminal sequences. Voss et al. (1991b) have demonstrated that translational use of an internal methionine residue (position 26) produces the characteristic doublet (33 and 31 kDa) observed on Western blots of purified Pit-1 protein. Functional differences or differential regulation of these two variants has not yet been established. Alternative processing of the rat Pit-1 primary transcript results in an isoform referred to as Pit-lp (Konzak and Moore, 1992), GHF-2 (Theill et al., 1992), or Pit-la (Morris et al., 1992). Pit-lp contains an insertion of26 amino acids resulting from use of an alternate RNA splice acceptor in the first intron. Although the ratio of the Pit-la to Pit-lp isoforms in rat pituitary is 7:1, Pit-lp has been shown in some transfection studies to be a more potent activator of the rat GH promoter than Pit-la (Konzak and Moore, 1992; Theill et al., 1992; Morris et al., 1992). In contrast, the PRL promoter is preferentially activated by the Pit-la isoform. Another isoform of Pit-1 has been reported that utilizes an unusual "AT" splice acceptor in the first p i t - l intron between the aand /3-specific "AG" acceptor sites; this variant, called Pit-lT, contains 14 C-terminal amino acids of the @insert. The expression of Pit-1T appears to be restricted to pituitary thyrotrophs and transfection studies have demonstrated the ability of this variant to activate the TSHp promoter (Haugen et al., 1993).

F. Pit-1 Dimerization and Interaction with Other Proteins In addition to its DNA-binding functions, the POU domain of Pit1mediates protein/protein interactions required for activation of transcription. Gel mobility shift and protein cross-linking experiments support a model in which Pit-1 binds to DNA as a dinier although it exists as a monomer in solution (Ingraham et wl., 1990). A qualitative difference in Pit-1 dimerization has been proposed as the basis for inefficient transactivation by the human Pit-1 (A1a'"Pro) mutant (PfAffle et al., 1992; described in Section IV,D), in which the a-helical structure of' the POUS domain was disrupted. Heterodimerization of normal and mutant Pit-1 has also been proposed as one possible mechanism for the dominant negative effect of' the human Pit-1 Arg'"Trp mutant (Radovick et ul., 1992; described in Section IV,D). Because the products of both the mutant and normal allele bound DNA with

320

SONALI MAJUMDAR A N D HARRY P. E L S H O L T Z

high affinity, the severe pituitary deficiency phenotype in this case is consistent with an effective block of normal Pit-1 function by the mutant at the level of the target gene. The function of Pit-1 is also determined by interactions with other proteins. These include potential interactions with other POU proteins such as Oct-1. The native rat PRL promoter and a promoter construct containing an individual Pit-1 binding site are each activated more efficiently by cotransfection of Pit-1 and Oct-1 than by transfection of Pit-1 alone (Voss et al., 1991a). Protein binding studies support a model in which Pit-1 and Oct-1 interact synergistically by formation of heterodimers. Pit-1 can also interact with structurally unrelated transcription factors. The distal enhancer ofthe PRL gene, for example, contains an estrogen response element adjacent to one of the Pit-1 sites. In nonpituitary cells, expression of an estrogen receptor construct alone has little effect on basal activity of the PRL promoter, but when coexpressed with Pit-1, the estrogen receptor becomes strongly stimulatory (Day et al., 1990; Simmons et al., 1990). In the case of the GH promoter, cooperative interactions between Pit-1 and the thyroid hormone receptor have been observed (Schaufele et al., 1992). A novel zinc finger protein Zn15 has been identified that binds a conserved “Z-box” sequence in the rat GH promoter between positions - 94 and - 113 (Lipkin et al., 1993); this binding site is located between two proximal Pit-1 binding sites. Mutations in the Z-box resulted in >loofold decrease in GH promoter function when assessed in transgenic mice, demonstrating that the two Pit-1 sites were insufficient for full function of this pituitary-specific promoter. In cultured cells, cotransfection of Zn15 and Pit-1 indicated a strong synergism between these two factors in activation of the GH promoter. Lastly, Pit-1 has also been shown to interact with heterologous factors in the regulation of‘ its own gene. Binding of Pit-1 at a distal enhancer element appears to be critical for retinoic acid responsiveness ofthe pit-1 gene (Rhodes et al., 1993).

V. COMPARISON OF PIT-1 IN MAMMALS AND TELEOST FISH: STUDIES ON THE PRL TARGET GENE In marked contrast to its lactogenic role in mammals, PRL in teleost fish regulates transport of ions across the gill epithelia. Despite functional differences among distantly related vertebrates, the PRL gene is conserved within coding regions and at all intronlexon splice junctions

10.


321

(Xiong et al., 1992). Interestingly, sequence conservation in the 5’flanking regions of mammalian and salmon PRL genes is observed only in the TATA box. Sequences similar to the mammalian proximal activating region and distal enhancer appear to be absent in the salmon PRL gene. Accordingly, we examined the ability of divergent salmon PRL 5’ sequences ( - 2.4 kb) to direct gene expression in heterologous and PRL-secreting cell lines of mammalian or fish origin. Similar to the rat PRL gene, 5’ sequences of the salmon PRL gene were unable to activate expression of a CAT reporter in nonpituitary cells. In PRLsecreting rat GH4 cells, 5’ sequences of the salmon PRL gene activated transcription significantly, although at levels 90-fold lower than similar constructs containing rat PRL 5’-flanking sequences (3.0 kb) (Elsholtz et al., 1992). These data indicated that pituitary-specific factors are required for salmon PRL gene activation, but suggested also that species-specific differences in pituitary cell function may impede efficient use of the salmon PRL gene promoter. The species specificity of transcriptional regulation was further examined in salmonid primary pituitary cells. Interestingly, in these cells the salmon PRL/CAT constructs were expressed at very high levels, whereas rat PRL/CAT constructs were only weakly active (Elsholtz et al., 1992). These studies supported the argument that both the rat and salmon PRL promoters are species specific, requiring a homologous pituitary system for optimal expression. The restricted expression of the salmon PRL promoter in rat GH4 cells strongly suggested that rat Pit-1 might be involved in promoter activation. Moreover, the species differences observed in transfected GH4 cells and salmonid pituitary cells further suggested that a teleost fish Pit-1 may activate the salmon PRL promoter more efficiently than rat Pit-1. To perform a functional comparison of rat and salmon Pit-1 we first used a combination of polymerase chain reaction (PCR) and cDNA library screening (chinook salmon pituitary) to isolate the salmon homolog of rat Pit-1. Homology was confirmed in three fulllength clones on the basis of sequence similarity to mammalian Pit-1’s and the pituitary-specific expression of salmon p i t - l RNA transcripts. The chinook salmon pit-1 cDNA contains an open reading frame of 1074 nucleotides encoding a protein of 358 amino acids. In the Cterminal half of salmon p i t - l the POU domain exhibits 87% identity with mammalian pit-l POU domains. The highest conservation is observed in the N-terminal part ofthe POUSdomain and in the third helix of the homeodomain. Most amino acid substitutions were localized to the C-terminal portion of the POUS domain and to helix 1 and 2 of the POU,,. In the salmon POU domain there are 14 amino acid substi-

322

SONALI MAJUMDAR AND HARRY 1'. ELSHOLTZ

tutions at positions highly conserved among mammalian Pit-1's. Nterminal to the POU domain, sequences of salmon Pit-1 are less than 60% identical to those of mammalian Pit-1's and contain numerous amino acid insertions, deletions, and nonconservative substitutions (Fig. 2). A p i t - l cDNA has been cloned from a second salmonid species, chum salmon (Oncorhynchus keta) (Ono and Takayama, 1992). Chinook and chum salmon Pit-1 sequences are highly conserved, although an insertion (or deletion) of four amino acids has occurred in the N terminus and a deletion of seven base pairs in the chum salmon 3' untranslated region extends the pit-1 open reading frame by 11codons, relative to chinook salmon and mammalian pit-1 ' s .

A. Conservation of Pit-1 POU Domain Function in Fish Because the POU domains of sahnon and rat p i t - l contain several amino acid differences, we tested whether these might contribute to functional differences in PRL gene activation. A chimeric pit-1 was constructed in which most ofthe salmon pit-1 POU domain was substituted for the rat pit-1 POU domain in a rat p i t - l c D N A expression vector. Transactivation b y the rat/salmon chimeric Pit-1 was then compared to wild-type rat Pit-1 in HeLa cells cotransfected with rat PRL

salmon rat

salmon

rat

salmon rat

gL:Ni'

'

QEMLSASISQTRILQT~SVPHPNMVNGANTL

143

______..______ ___----107

L 291 I 249

359 318

Fig. 2. Amino acid sequence comparison between salmon and rat Pit-1. The I~lack l ~ o s e srepresent residues conserved between salmon and rat Pit-1. Abbreviations for the aiiiino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H , His; I , I l r ; K, Lys; I,, Leu; M, Met; N, Asn; P, Pro; 0, Gln; R, Arg; S, Ser; T, Tlir; Y,\'id; \\'. Trp; :und Y, T y .

10.

ASPECTS O F PITUITARY DEVELOPXIENT AND Pit-1 FUNCTION

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or salmon PRL promoters. Over a range of vector concentrations, the chimeric rat/salmon Pit-1 transactivated the rat and salmon PRL promoters with comparable efficiency to wild-type rat Pit-1 (Elsholtz et al., 1992). These data suggested that any structural differences in the POU,,, of salmon p i t - 1 that result from amino acid substitutions do not significantly change its ability to regulate the PRL target gene. B. Species Differences in Alternative RNA Splicing Chinook salmon p i t - 1 encodes a protein of 358 amino acids in contrast to the rat pit-la, which encodes a protein of 291 amino acids. The greater length of the salmon Pit-1 polypeptide is due primarily to insertions of' 26 amino acids at a position corresponding to the junction of exon I and I1 (i.e., the /?-insert describe earlier) and also to a 33-amino-acid sequence (which we refer to as the y-insert) positioned at the junction of Pit-1 exons I1 and 111. Interestingly, although a similar y-insert has not been reported in mammalian Pit-l's, a sequence 76% identical to the salmon Pit-1 y-insert is present in a turkey pit-1 cDNA (Wong et al., 1992). In salmon the predominant form of Pit-1 contains both the p- and y-inserts. The a form of Pit-1, which lacks the /?-insert and is the major Pit-1 isoform in the rat, appears to be completely absent in chinook salmon. Even with a combination of PCR (exon I- and exon 111-specific primers) and Southern analysis, an a-specific Pit-1 splice was not detected in total salmon pituitary cDNA (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Sequence analysis indicates that in the salmon, the /?-specific splice site may be used by default, because the consensus splice acceptor dinucleotide AG used for a-specific splicing of mammalian Pit-1 pre-mKNA is replaced by CG in this teleost species. To determine whether the 33-amino-acid segment in salmonid Pit1 represents a novel alternatively spliced product of the p i t - 1 gene, we isolated a pit-1 genomic clone from an EMBL3 salmon genomic library. In contrast to the /?-insert described here, the y-insert is encoded by a distinct exon flanked by a consensus splice acceptor, polypyrimidine tract, and branch point at its 5' end, and by an intron splice donor consensus at its 3' end. Using degenerate PCR primers, designed to match conserved y-insert sequences in salmonid and turkey p i t - l , we have been unable to isolate related sequences from genomic DNA of'three divergent mammalian species (human, rat, cow). Furthermore, using PCR and Southern analysis, primers specific for rat Pit-1 in exon I and exon I11 did not amplify specific p i t - 1 fragments of

324

SONALI MAJUMDAR AND HARRY P. ELSHOLTZ

greater mass than Pit-lp (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Our data suggest, therefore, that the Pit-1 y-insert may be restricted in its phylogenetic distribution to nonmammalian classes of vertebrates.

C. N-Terminal Sequences of Rat and Salmon Pit-1 In spite of structural differences in the N-terminal sequences of rat and salmon p i t - 1 , rat Pit-1 efficiently activates reporter constructs containing the salmon PRL promoter and 5'-flanking region (Elsholtz et al., 1992).To determine whether N-terminal sequences of salmon Pit-1 (which contain the p- and y-inserts) could activate the rat PRL promoter as efficiently as N-terminal sequences of rat Pit-1, a chimeric cDNA was constructed with N-terminal salmon pit-1 sequences fused to a rat pit-1 POU region. Rat Pit-1, salmon Pit-1, and the chimeric salmon/rat Pit-1 were functionally compared in several heterologous mammalian cell lines, including HeLa (cervical carcinoma), HepG2 (hepatoma),and Ltk- (fibrosarcoma), and in a salmonid hepatoma cell line, RTH. Although minor variations were observed among the different cell lines, each of the Pit-1 constructs strongly stimulated expression of the rat PRL/CAT construct (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Control reporter constructs indicated that activation was specific for the PRL gene. Therefore, phylogenetic changes in the structure of the Pit-1 N-terminal region do not prevent cross-species activation of PRL genes by Pit-1. Our data suggest that the dramatic species differences in PRL promoter function, observed using rat or salmonid pituitary cells (Elsholtz et al., 1992), are likely to depend on pituitary factors other than Pit-1. VI. CONCLUSION

In mammals the POU transcription factor Pit-1 has a critical role in anterior pituitary development and endocrine gene activation. Although the p i t - l gene is structurally conserved in teleost fish, both the DNA-binding POU domain and the N-terminal transactivation region have undergone a number of structural changes in these vertebrates. The rapid divergence of PRL gene 5' regulatory sequences and the species-specific pattern of PRL gene expression in mammalian and fish pituitary cells suggest that changes in Pit-1 function resulting from phylogenetic divergence might contribute to species-specific ex-

10.

ASPECTS OF PITUITARY DEVELOPMENT A N D Pit-1 FUNCTION

325

pression of the PRL gene. Our studies suggest, however, that basal transcription of the PRL gene is activated with similar efficiency by Pit-1’s of distantly related vertebrates. Because these studies were performed in heterologous cell types, experiments are now needed to assess the impact of Pit-1 evolution on PRL gene regulation by other transcription factors in pituitary cells, including members of the steroid receptor family. An obligatory role for Pit-1 in teleost pituitary development has not been established. The early appearance of PRL cells during differentiation of Rathke’s pouch suggests that expression of the pit-1 gene may occur at the onset of pituitary organogenesis in certain families of fish. Lastly, transgenic approaches with targeted ablation of teleost pituitary cells will be necessary to establish whether a common Pit1-expressing progenitor cell can give rise to more than one cell type, as demonstrated in mammalian species.

ACKNOWLEDGMENTS We wish to thank Valdine Sundmark for proofreading the manuscript and Dr. Vladimir Lhotak for help with computer analysis of salmon and rat Pit-1. We would also like to thank Dr. C. L. Hew and members of his lab for providing the chinook salmon pituitary cDNA library, salmon genomic library, and salmon PRL/CAT constructs.

REFERENCES Akam, M. (1987).The molecular basis for metameric patterns in the Drosophilu embryo. Deoelopment (Cambridge, U K ) 101, 1-22. Asa, S. L., Puy, L. A., Lew, A. M., Sundmark, V. C., and Elsholtz, H. P. (1993). Celltype specific expression of‘ the pituitary transcription activator Pit-1 in the human pituitary and pituitary adenoma. 1.Clin. Endocrinol. Metub. 77, 1275-1280. Assa-Munt, N., Mortishire-Smith, R. J., Aurora, R., Herr, W., and Wright, P. E. (1993). The solution structure of the Oct-l POU-specific domain reveals a striking similarity to the bacteriophage A repressor DNA binding domain. Cell (Cumbridge, Muss.) 73, 193-205. Batten, T. F. C . , and Ingleton, P. M. (1987). The hypothalamus and pituitary gland. I n “Fundamentals of Comparative Vertebrate Endocrinology” ( I . Chester-Jones, P. M. Ingleton, and J. G . Phillips, eds), pp. 285-409. Plenum Press, N.Y. and London. Begeot, M., Dubois, M. P., and Dubois, P. (1982). Comparative studies in t h o and in Gitro of the differentiation of immunoreactive corticotropic cells in fetal rat anterior pituitary. Neuroendocrinology 35, 255-264.

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Behringer, R. R., Mathews, L. S., Palmiter, R. D., and Brinster, K. L. (1988). Dwarf mice produced by genetic ablation of growth hormone-expressing cells. Genes Dec. 2,453-461. Bodner, M., and Karin, 11. (1987). A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells. Cell (Cambridge, Mass.) 50, 267-275. Bodner, M.,Castrillo, J. L., Theill, L. E., Deerinck, T., Elisman, M., and Karin, hl. (1988).T h e pituitary-specific transcription factor GHF-1 is a honieobox-contaitiing protein. Cell (Cunrbridge, Moss.) 55, 505-518. Borrelli, H. R., Arias, P. E., Sawchenko, P. E., and Evans, R. (1989). Transgenic mice with inducible dwarfism. Nature ( L o n d o n )339, 538-541. Canihre, M., Mareels, G., Corneillie, S., Moons, L., Ollevier, F., and Vandesande, F. (1990). Chronological appearance of the different hypophysial hormones in the pituitary of sea bass larvae (Dicentrurchus lahrux) diiring their early development: An immuirocytochemical demonstration, Gen. Comp. Endocrinol. 77, 408-415. Chetelain, A., Dupuoy, J. P., and Dubois, M . P. (1979). Ontology of' cells producing polypeptide hormones in the fetal hypophysis ofthe rats: Influence ofthe hypothalaniiis. Cell Tissue Res. 196, 409-427. (:rcnshaw 111, E. B., Kalla, K., Simmons, D. hl., Swanson, L. W., and Kosenfelcl, 11. G. (1989).Cell-specific expression ofthe prolactin gene in transgenic mice is controlled b y synergistic interactions between promoter and enhancer elements. Genes D e c . 3, 959-972. Day, K. S . , Kioke, S., Sakai, M., Muramatsu, hl., and hlaurer, I

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