Werner & Ingbar's The Thyroid: A Fundamental and Clinical Text [11 ed.] 2020009060, 9781975112967, 1975112962, 9781975112974, 1975112970

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Werner & Ingbar’s THE THYROID A FUNDAMENTAL AND CLINICAL TEXT ELEVENTH EDITION Editors Lewis E. Braverman, MD Professor of Medicine Section of Endocrinology, Diabetes, and Nutrition Boston University School of Medicine Boston, Massachusetts

David S. Cooper, MD, MACP Professor of Medicine and Radiology Division of Endocrinology, Diabetes, and Metabolism The Johns Hopkins University School of Medicine Professor of International Health The Bloomberg Johns Hopkins School of Public Health Baltimore, Maryland

Peter A. Kopp, MD Professor of Medicine Division of Endocrinology, Diabetes and Metabolism University of Lausanne Lausanne, Switzerland Adjunct Professor of Medicine Division of Endocrinology, Metabolism and Molecular Medicine Feinberg School of Medicine Northwestern University Chicago, Illinois

Acquisitions Editor: Nicole Dernoski Development Editor: Sean McGuire Editorial Coordinator: Tim Rinehart Marketing Manager: Phyllis Hitner Production Project Manager: Bridgett Dougherty Design Coordinator: Stephen Druding Manufacturing Coordinator: Beth Welsh Prepress Vendor: Aptara, Inc. 11th edition Copyright © 2021 Wolters Kluwer. Copyright © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business. Copyright © 2005, 2000 by Lippincott Williams & Wilkins. Copyright © 1996 by Lippincott-Raven Publishers. Copyright © 1991, 1986, by J. B. Lippincott Company. Copyright © 1978, 1971, 1962 by Harper & Row Publishers. Copyright © 1955 by Hoeber-Harper. All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at shop.lww.com (products and services). 9 8 7 6 5 4 3 2 1 Printed in China Library of Congress Cataloging-in-Publication Data Names: Braverman, Lewis E., 1929-2019, editor. | Cooper, David S. (Physician), editor. | Kopp, Peter A. (Peter Andreas), editor. Title: Werner & Ingbar’s the thyroid : a fundamental and clinical text / editors, Lewis E. Braverman, David S. Cooper, Peter A. Kopp. Other titles: Werner and Ingbar’s the thyroid Description: 11th edition. | Philadelphia, PA : Wolters Kluwer, [2021] | Includes bibliographical references and index. Identifiers: LCCN 2020009060 | ISBN 9781975112967 (hardback) Subjects: MESH: Thyroid Diseases | Thyroid Gland–physiology Classification: LCC RC655 | NLM WK 200 | DDC 616.4/4–dc23 LC record available at https://lccn.loc.gov/2020009060

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This edition is dedicated to Lewis E. Braverman, MD, MACE, MACP (March 30, 1929–June 10, 2019)

Dr. Lewis Braverman has served as co-editor of Werner & Ingbar’s The Thyroid: A Fundamental and Clinical Text for the fifth edition, published in 1986, to the present eleventh edition. Dr. Braverman was born in Quincy, MA and graduated cum laude from Milton Academy and Harvard College. He received his MD degree from Johns Hopkins University School of Medicine, followed by an internal medicine residency at Boston City Hospital and an Endocrinology Fellowship under the direction of Sidney H. Ingbar, MD, the editor of the third to fifth edition of The Thyroid, in the Thorndike Memorial Laboratory. Dr. Braverman was Chief of Endocrinology at St. Elizabeth’s Hospital in Boston, founding Director of Endocrinology and Chief of Nuclear Medicine at the University of Massachusetts Medical Center in Worcester, and Chief of Endocrinology, Diabetes and Nutrition at Boston Medical Center (BMC)/Boston University School of Medicine. He actively saw patients in the Endocrine clinic at BMC until January 2018 when he retired from clinical practice at the age of 88. Dr. Braverman was a prolific thyroid researcher with more than 600 publications. Among his many contributions to clinical and basic thyroidology, his seminal demonstration that thyroxine is converted to triiodothyronine in humans was of particular importance, and he was an internationally recognized expert in iodine metabolism. Dr. Braverman served as Secretary and President of the American Thyroid Association (ATA) and was the recipient of all of the ATA awards, including the Van Meter Award, the Distinguished Service Award, and the Paul Starr Memorial Award. He was the first Sidney Ingbar Memorial Lecturer and the first recipient of the Thyroid Pathophysiology Medal of the ATA. Among many other awards, he also received the Robert H. Williams Distinguished Leadership Award from the Endocrine Society and served as Editor-in-Chief of the society’s clinical journal, the Journal of Clinical Endocrinology and Metabolism.

Dr. Braverman has made a particular impact as an educator and mentor of fellows, residents, students and junior faculty. In honor of his legacy as mentor, the American Thyroid Association established the Lewis E. Braverman Distinguished Lectureship Award in 2011. This award “recognizes an individual who has demonstrated excellence and passion for mentoring fellows, students and junior faculty; has a long history of productive thyroid research; and is devoted to the ATA”. His legacy as mentor, clinician, researcher, and editor places him among the giants in thyroidology and endocrinology.

CONTRIBUTORS JUNG HWAN BAEK, MD, PhD Professor of Radiology Department of Radiology and Research Institute of Radiology University of Ulsan College of Medicine Asan Medical Center Seoul, Korea

ZUBAIR W. BALOCH, MD, PhD Professor of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Perelman School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

LUIGI BARTALENA, MD Full Professor of Endocrinology Department of Medicine and Surgery University of Insubria Chief, Endocrine Unit ASST dei Sette Laghi Varese, Italy

J. H. DUNCAN BASSETT, MA, BM, BCh, PhD Professor of Endocrinology Molecular Endocrinology Laboratory Department of Metabolism, Digestion and Reproduction Imperial College London Honorary Consultant in Endocrinology Honorary Clinical Research Fellow Imperial College Healthcare NHS Trust London, United Kingdom

PAOLO BECK-PECCOZ, MD Professor Emeritus University of Milan Milan, Italy

GREET VAN DEN BERGHE, MD, PhD Professor, Laboratory of Intensive Care Medicine Department of Cellular and Molecular Medicine Head of the Intensive Care Department University Hospital Leuven KU Leuven Leuven, Belgium

ANTONIO C. BIANCO, MD, PhD Professor of Medicine Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism Department of Medicine University of Chicago Chicago, Illinois

KEITH C. BIBLE, MD, PhD Professor of Oncology Department of Oncology Mayo Clinic Rochester, Minnesota

KRISTIEN BOELAERT, MD, PhD, FRCP Reader in Endocrinology Institute of Applied Health Research College of Medical and Dental Sciences University of Birmingham Consultant Endocrinologist University Hospitals Birmingham Birmingham, United Kingdom

ANITA BOELEN, PhD Head, Neonatal Screening Endocrine Laboratory, Department of Clinical Chemistry Amsterdam UMC location AMC University of Amsterdam Amsterdam, Netherlands

Steen Joop Bonnema, MD, PhD, DMSC Professor, Consultant Department of Endocrinology Odense University Hospital University of Southern Denmark Odense, Denmark

ALINA V. BRENNER, MD, PhD Senior Scientist Radiation Effects Research Foundation Department of Epidemiology

Hiroshima, Hiroshima Prefecture, Japan

GREGORY A. BRENT, MD Professor, UCLA Graduate Programs in Bioscience David Geffen School of Medicine at UCLA Vice Chair, VA Greater Los Angeles Healthcare System Los Angeles, California

HENRY B. BURCH, MD Professor of Medicine Division of Diabetes, Endocrinology and Metabolic Diseases National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Uniformed Services University of the Health Sciences Bethesda, Maryland

KENNETH D. BURMAN, MD Chief, Endocrine Section Medstar Washington Hospital Center Professor, Department of Medicine Georgetown University Washington, DC

Anne R. Cappola, MD, SCM Professor of Medicine Department of Medicine Division of Endocrinology, Diabetes and Metabolism Perelman School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

NANCY CARRASCO, MD Professor and Chair Molecular Physiology and Biophysics Vanderbilt University Nashville, Tennessee

Francesco S. Celi, MD, MHSC William G. Blackard Professor of Medicine Chair, Division of Endocrinology Diabetes and Metabolism Department of Internal Medicine Virginia Commonwealth University Richmond, Virginia

EMILY R. CHRISTISON-LAGAY, MD Assistant Professor of Surgery (Pediatrics) and Assistant Professor of Pediatrics Department of Surgery Yale School of Medicine

Attending in Pediatric Surgery Yale New Haven Children’s Hospital New Haven, Connecticut

RONALD N. COHEN, MD Associate Professor of Medicine Chief, Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism Department of Medicine University of Chicago Chicago, Illinois

DAVID S. COOPER, MD, MACP Professor of Medicine and Radiology Division of Endocrinology, Diabetes, and Metabolism The Johns Hopkins University School of Medicine Director, Thyroid Clinic The Johns Hopkins Hospital Baltimore, Maryland

SABINE COSTAGLIOLA, PhD Senior Research Associate IRIBHM deputy director Institute of Interdisciplinary Research in Molecular Human Biology (IRIBHM) Université Libre de Bruxelles Brussels, Belgium

TERRY F. DAVIES, MBBS, MD, FRCP, FACE Florence and Theodore Baumritter Professor of Medicine Icahn School of Medicine at Mount Sinai Director, Division of Endocrinology, Diabetes and Bone Diseases Mount Sinai Beth Israel Medical Center Attending Physician, The Mount Sinai Hospital Beth Israel Medical Center and The James J. Peters VA Medical Center New York, New York

JOHNNY DELADOëY, MD, PhD Associate Professor, Department of Pediatrics Researcher, Research Center of CHU Sainte-Justine University of Montreal Montreal, Quebec

CATHERINE A. DINAUER, MD Instructor of Clinical Surgery (Pediatrics) Department of Surgery Yale School of Medicine Attending in Pediatric Endocrinology Yale New Haven Children’s Hospital New Haven, Connecticut

HENNING DRALLE, MD Professor of Surgery Department of Visceral, Vascular and Endocrine Surgery Martin Luther University Halle-Wittenberg Halle (Saale), Germany University of Duisburg-Essen Essen, Germany

ALEXANDRA M. DUMITRESCU, MD, PhD Associate Professor Department of Medicine The University of Chicago Chicago, Illinois

JAMES A. FAGIN, MD Head, Division of Subspecialty Medicine Member, Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center Professor of Medicine Weill Cornell Medical College New York, New York

ALAN P. FARWELL, MD, FACE Chief, Section of Endocrinology, Diabetes, Nutrition and Weight Management Department of Medicine Boston University School of Medicine Director, Endocrine Clinics Boston Medical Center Boston, Massachusetts

VAHAB FATOURECHI, MD Consultant, Division of Endocrinology, Diabetes, Metabolism and Nutrition Professor of Medicine Mayo Clinic College of Medicine Rochester, Minnesota

Ulla Feldt-Rasmussen, MD, DMSC Professor, Chief of Medical Endocrinology Rigshospitalet, Copenhagen University Hospital Copenhagen, Denmark

SEBASTIANO FILETTI, MD Professor of Internal Medicine Department of Translational and Precision Medicine Sapienza University of Rome Rome, Italy

GARY L. FRANCIS, MD, PhD

Professor of Pediatrics, Pediatric Endocrinology Long School of Medicine Attending in Pediatric Endocrinology University of Texas Health Sciences Center San Antonio San Antonio, Texas

Bernard Freudenthal, MA (Cantab), MBBS, MSc Clinical Research Fellow, Molecular Endocrinology Laboratory Department of Metabolism, Digestion and Reproduction Imperial College London Honorary Clinical Research Fellow Imperial College Healthcare NHS Trust London, United Kingdom

DAGMAR FüHRER, MD, PhD Professor of Internal Medicine/Endocrinology Department of Endocrinology, Diabetes and Metabolism University Hospital EssenUniversity of Duisburg-Essen Essen, Germany

JEFFREY R. GARBER, MD Associate Professor of Medicine Harvard Medical School Chief, Endocrinology Atrius Health Boston, Massachusetts

THOMAS J. GIORDANO, MD, PhD Henry Clay Bryant Professor of Pathology Department of Pathology University of Michigan Ann Arbor, Michigan

David Halsall, PhD, FRCPath Consultant Clinical Scientist Cambridge University Hospitals Trust Cambridge, United Kingdom

BRYAN R. HAUGEN, MD Professor of Medicine and Pathology Head, Division of Endocrinology, Metabolism and Diabetes University of Colorado Cancer Center University of Colorado School of Medicine Aurora, Colorado

Laszlo Hegedüs, MD, DMSc Professor, Consultant Department of Endocrinology

Odense University Hospital University of Southern Denmark Odense, Denmark

JAMES V. HENNESSEY, MD Associate Professor of Medicine Harvard Medical School Director, Clinical Endocrinology Division of Endocrinology Department of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts

JEROME M. HERSHMAN, MD Distinguished Professor of Medicine Emeritus UCLA School of Medicine West Los Angeles VA Medical Center Los Angeles, California

ANTHONY HOLLENBERG, MD Professor and Sanford I. Weill Chair Department of Medicine at Weill Cornell Medicine Physician-in-Chief New York Presbyterian/Weill Corner Medical Center New York, New York

YASUHIRO ITO, MD, PhD Senior Surgeon Department of Surgery Kuma Hospital Kobe, Hyogo, Japan

JACQUELINE JONKLAAS, MD, PhD Professor Division of Endocrinology Georgetown University Washington, DC

BRIAN W. KIM, MD Associate Professor of Medicine Chief, Division of Endocrinology and Metabolism Rush University Medical Center Chicago, Illinois

CARI M. KITAHARA, PhD Investigator National Cancer Institute Division of Cancer Epidemiology and Genetics, Radiation Epidemiology Branch

Bethesda, Maryland

PETER A. KOPP, MD Professor of Medicine Division of Endocrinology, Diabetes and Metabolism University of Lausanne Lausanne, Switzerland Adjunct Professor of Medicine Division of Endocrinology, Metabolism and Molecular Medicine Feinberg School of Medicine Northwestern University Chicago, Illinois

TIM I.M. KOREVAAR, MD, PhD Postdoctoral Fellow, Department of Internal Medicine and Academic Center for Thyroid Diseases Erasmus University Medical Center Rotterdam, the Netherlands

MANASSAWEE KORWUTTHIKULRANGSRI, MD Assistant Professor Department of Pediatrics Mahidol University Faculty of Medicine Ramathibodi Hospital Mahidol University Bangkok, Thailand

PAUL W. LADENSON, MA (Oxon), MD John Eager Howard Professor of Endocrinology and Metabolism Professor of Medicine, Pathology, Oncology, and Radiology and Radiological Science University Distinguished Service Professor Johns Hopkins University School of Medicine Baltimore, Maryland

JILL E. LANGER, MD Professor of Radiology Ultrasound Section Chief Hospital of the University of Pennsylvania Perelman School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

LIES LANGOUCHE, MSc, PhD Associate Professor Laboratory of Intensive Care Medicine Department of Cellular and Molecular Medicine KU Leuven Leuven, Belgium

ANDREA LANIA, MD Associate Professor Humanitas University Head, Division of Endocrinology Istituto Clinico Humanitas Milan, Italy

Angela M. Leung, MD, MSc Associate Professor of Medicine Division of Endocrinology, Diabetes, and Metabolism Department of Medicine David Geffen School of Medicine at the University of California Los Angeles VA Greater Los Angeles Healthcare System Los Angeles, California

VIRGINIA A. LIVOLSI, MD Professor of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Perelman School of Medicine Philadelphia, Pennsylvania

ANDREAS MACHENS, MD Associate Professor, Medical Faculty, Department of Visceral, Vascular and Endocrine Surgery Martin Luther University Halle-Wittenberg Halle (Saale), Germany

SUSAN J. MANDEL, MD, MPH Professor of Medicine Chief, Division of Endocrinology, Diabetes and Metabolism Perleman School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

MICHAEL T. McDERMOTT, MD Professor of Medicine and Clinical Pharmacy University of Colorado School of Medicine Director of Endocrinology Practice University of Colorado Hospital Aurora, Colorado

DAVID G. McFADDEN, MD, PhD Assistant Professor Division of Endocrinology Department of Internal Medicine Department of Biochemistry UT Southwestern Medical Center Dallas, Texas

DONALD S.A. McLEOD, BSc, MBBS (Hon I), FRACP, MPH, PhD Senior Staff Specialist, Department of Endocrinology and Diabetes, Royal Brisbane and Women’s Hospital Honorary Senior Research Officer, Population Health Department, QIMR Berghofer Medical Research Institute Associate Professor University of Queensland Queensland, Australia

AKIRA MIYAUCHI, MD, PhD President and COO, Kuma Hospital Kobe, Hyogo, Japan

CARLA MORAN, MB BCh, BAO, PhD, MRCPI Consultant Endocrinologist Endocrine and Diabetes Department Beacon Hospital Dublin, Ireland

CAROLINE T. NGUYEN, MD Assistant Professor of Clinical Medicine and Obstetrics and Gynecology Division of Endocrinology, Diabetes, and Metabolism Department of Medicine and Obstetrics and Gynecology Keck School of Medicine University of Southern California Los Angeles, California

YURI E. NIKIFOROV, MD, PhD Professor of Pathology Vice Chair for Molecular Pathology Director, Division of Molecular and Genomic Pathology Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania

RALF PASCHKE, MD, PhD Chair, Provincial Endocrine Tumour Team Professor of Medicine, Endocrinology and Metabolism Departments of Medicine, Oncology, Pathology, and Biochemistry and Molecular Biology Arnie Charbonneau Cancer Institute Cumming School of Medicine University of Calgary Calgary, Alberta, Canada

Elizabeth N. Pearce, MD, MSc Professor of Medicine Boston University School of Medicine Section of Endocrinology, Diabetes, and Nutrition

Boston, Massachusetts

ROBIN P. PEETERS, MD, PhD Professor of Medicine Department of Internal Medicine, Academic Center for Thyroid Disease Erasmus MC, Rotterdam The Netherlands

LUCA PERSANI, MD, PhD Professor of Endocrinology, Department of Clinical Sciences and Community Health University of Milan Head, Department of Endocrine and Metabolic Diseases IRCCS Istituto Auxologico Italiano Milan, Italy

MICHEL POLAK, MD, PhD Full Professor, Université de Paris Head, Pediatric Endocrinology, Diabetology and Gynecology Hôpital Universitaire Necker Enfants Malades Assistance Publique-Hôpitaux de Paris, Head Regional Newborn Screening Program, INSERM U1016, institut IMAGINE Paris, France

DANIEL A. PRYMA, MD Associate Professor of Radiology and Radiation Oncology Chief, Division of Nuclear Medicine and Clinical Molecular Imaging Perelman School of Medicine University of Pennsylvania Attending Physician, Hospital of the University of Pennsylvania Philadelphia, Pennsylvania

SALMAN RAZVI, MBBS, MD, FRCP Senior Lecturer and Honorary Consultant Endocrinologist Institute of Genetic Medicine Newcastle University Newcastle upon Tyne, United Kingdom

SAMUEL REFETOFF, MD, CM Frederick H. Rawson Professor in Medicine Professor of Pediatrics and Committee on Genetics The University of Chicago Chicago, Illinois

SCOTT A. RIVKEES, MD Professor of Pediatrics University of Florida Department of Pediatrics University of Florida Gainesville, Florida

PATRICE RODIEN, MD, PhD Professor in Endocrinology diabetes and metabolic diseases Faculté de médecine, Université d’Angers Reference Center for rare disease of thyroid and hormone receptors Department of Endocrinology-Diabetes-Nutrition, Centre Hospitalo-universitaire d’Angers Angers, France

GILLES RUSS, MD Consultant Radiologist Thyroid and Endocrine Tumors Unit La Pitie Salpetriere Hospital Sorbonne University Paris, France

MARY H. SAMUELS, MD Professor of Medicine Division of Endocrinology, Diabetes and Clinical Nutrition Oregon Health and Science University Portland, Oregon

PILAR SANTISTEBAN, PhD Full Professor Institute of Biomedical Research “Alberto Sols” (IIBm) Spanish National Research Council (CSIC) Autonomous University of Madrid (UAM) Biomedical Research Center in Cancer Network (CIBERONC) Madrid, Spain

DAVID H. SARNE, MD Director, Endocrinology and Metabolism Clinic Department of Medicine University of Chicago Chicago, Illinois

ARTHUR B. SCHNEIDER, MD, PhD Professor of Medicine Emeritus University of Illinois at Chicago Department of Medicine Section of Endocrinology, Metabolism and Diabetes Chicago, Illinois

NADIA SCHOENMAKERS, MB BChir, PhD Honorary Consultant Endocrinologist University of Cambridge Metabolic Research Laboratories Wellcome Trust-Medical Research Council Institute of Metabolic Science Addenbrooke’s Hospital University of Cambridge

Cambridge, United Kingdom

KATHRYN G. SCHUFF, MD, MCR Professor of Medicine Division of Endocrinology, Diabetes and Clinical Nutrition Oregon Health and Science University Portland, Oregon

STEVEN I. SHERMAN, MD Professor, Department of Endocrine Neoplasia and Hormonal Disorders The University of Texas MD Anderson Cancer Center Houston, Texas

PETER A. SINGER, MD Professor of Clinical Medicine Division of Endocrinology, Diabetes, and Metabolism Department of Medicine Keck School of Medicine University of Southern California Los Angeles, California

ROBERT C. SMALLRIDGE, MD Emeritus Professor of Medicine Mayo Clinic Jacksonville, Florida

JULIE ANN SOSA, MD, MA, FACS Leon Goldman, MD Distinguished Professor of Surgery and Chair, Department of Surgery Professor, Department of Medicine Affiliated faculty, Philip R. Lee Institute for Health Policy Studies University of California San Francisco-UCSF San Francisco, California

DAVID L. STEWARD, MD Professor Director of Head and Neck Surgery, Endocrine Surgery, and Clinical Research Department of Otolaryngology-HNS University of Cincinnati, College of Medicine University of Cincinnati Medical Center Cincinnati, Ohio

GABOR SZINNAI, MD, PhD Senior Lecturer, Medical Faculty University of Basel Deputy-Head, Pediatric Endocrinology and Diabetology University Children’s Hospital Basel Basel, Switzerland

WEIPING TENG, MD Professor, Department of Endocrinology and Metabolism Professor, The First Hospital of China Medical University Shenyang, PR China

YARON TOMER, MD, FACP Anita and Jack Saltz Chair in Diabetes Research Professor and Chair Department of Medicine Albert Einstein College of Medicine Montefiore Medical Center New York, New York

R. MICHAEL TUTTLE, MD Professor of Endocrinology Department of Medicine, Endocrinology Service Memorial Sloan Kettering Cancer Center New York, New York

GUY VAN VLIET, MD Professor, Department of Pediatrics, University of Montreal Staff, Endocrinology and Diabetology Service CHU Sainte-Justine University of Montreal Montreal, Quebec

GILLBERT VASSART, MD, PhD Honorary professor, IRIBHM Université Libre de Bruxelles (ULB) Honorary Professor Department of Medical Genetics Hôpital Erasme (ULB) Brussels, Belgium

Julia Elisabeth von Oettingen, MD, PhD, MMSc Assistant Professor, Department of Pediatrics McGill University Clinician-Scientist McGill University Hospital Centre Research Institute Montreal, Quebec

LEONARD WARTOFSKY, MD, MACP Professor of Medicine Georgetown University School of Medicine Thyroid Cancer Research Unit MedStar Health Research Institute Washington, DC

Laura Watts, MA (Oxon), BM BCh Clinical Research Fellow, Molecular Endocrinology Laboratory Department of Metabolism, Digestion and Reproduction Imperial College London Honorary Clinical Research Fellow Imperial College Healthcare NHS Trust London, United Kingdom

Anthony P. Weetman, MD, DSc Emeritus Professor of Medicine The Medical School University of Sheffield Sheffield, United Kingdom

WILMAR M. WIERSINGA, MD, PhD, FRCP (Lond) Emeritus Professor of Endocrinology Amsterdam University Medical Center Amsterdam, The Netherlands

GRAHAM R. WILLIAMS, BSc, MBBS, PhD Professor of Endocrinology Molecular Endocrinology Laboratory Department of Metabolism, Digestion and Reproduction Imperial College London Honorary Consultant in Endocrinology Imperial College Healthcare NHS Trust London, United Kingdom

GRAHAM R. WILLIAMS, BSc, MBBS, PhD Professor of Endocrinology Molecular Endocrinology Laboratory Department of Metabolism, Digestion and Reproduction Imperial College London Honorary Clinical Research Fellow Imperial College Healthcare NHS Trust, London, United Kingdom

FREDRIC E. WONDISFORD, MD Henry Rutgers Professor and Chair Department of Medicine Robert Wood Johnson Medical School Medical Service Chair Robert Wood Johnson University Hospital Rutgers University New Brunswick, New Jersey

PAUL M. YEN, MD Professor, Cardiovascular and Metabolic Disorders Program

Duke-NUS Graduate Medical School Singapore Professor of Department of Medicine Duke Molecular Physiology Institute Duke University School of Medicine Durham, North Carolina

MICHAEL B. ZIMMERMANN, MD Professor Human Nutrition, ETH Zürich Swiss Federal Institute of Technology Zurich, Switzerland

RICCARDO ZUCCHI, MD, PhD Professor of Biochemistry Department of Pathology University of Pisa Pisa, Italy

PREFACE TO THE ELEVENTH EDITION The first edition of The Thyroid, conceived by Dr. Sidney Werner who believed, and we agree, that a comprehensive clinical and basic textbook devoted to the thyroid was needed, was first published in 1955 and again in 1962. Dr. Werner recruited Dr. Sidney Ingbar to co-edit the third and fourth editions in 1971 and 1978. Following Dr. Werner’s retirement, Dr. Ingbar asked his former endocrine fellow, colleague, and close friend, Dr. Lewis Braverman to co-edit the fifth edition, published in 1986. Dr. Ingbar’s untimely death in 1988 left a tremendous void in the scientific community. Fortunately, Dr. Robert Utiger, a renowned investigator, clinician, and deputy editor of The New England Journal of Medicine, joined Dr. Braverman to coedit the next four editions, including the ninth published in 2005. Dr. Utiger died in 2008 after a brief illness, and Dr. David Cooper agreed to co-edit the tenth edition with Dr. Braverman, which was published in 2013. Planning for this new edition, the eleventh, began in early 2018. When it became clear later that year that Dr. Braverman’s health was failing, we were fortunate to recruit Dr. Peter Kopp to be the third editor. Dr. Kopp has had a distinguished career as a basic scientist, clinician, medical educator, and as an outstanding Editor-in-Chief of Thyroid, the American Thyroid Association’s flagship journal. Sadly, Dr. Braverman passed away on June 10, 2019. The creation of this book was just one of Dr. Werner’s many contributions to thyroidology. Long interested in Graves disease, he carried out many of the early studies of the therapeutic efficacy and side effects of radioiodine therapy for Graves thyrotoxicosis, and was one of the first to question whether it was a pituitary disease, as many had thought. He was responsible for the first system for classifying the ocular manifestations of Graves

disease, and he carried out many studies of the pathogenesis and therapy of Graves ophthalmopathy. In his preface to the first edition, which is reprinted on the following pages, Dr. Werner commented that the book “is intended for those who must deal with the problems of thyroid function and thyroid disease in man.” That has been our intention as well. We want this book to be useful to anyone who is interested in any aspect of the thyroid in both health and disease. This includes not only clinicians and investigators, but also research and clinical fellows just entering the field, and perhaps even younger students at the time of their first exposure to thyroidology. This eleventh edition of The Thyroid owes much to its predecessors, but it has been changed, too. Authorship has become more international. There are new chapters, and some old ones are missing, their contents incorporated into related chapters, especially those related to the effects of thyroid dysfunction on other organ systems. As in the past, we have tried to minimize overlap among different chapters, but some overlap is inevitable and probably essential. For example, the topics of iodine metabolism and the effects of iodine deficiency and excess on thyroid function are so central to any understanding of the thyroid gland and its diseases, and yet so diverse, that they must be considered in many chapters. The same is true for the actions of thyroid hormone, the many different yet related aspects of thyroid autoimmunity, and the topics of thyroid nodules and thyroid cancer. Research into the thyroid gland and its disorders has flourished in recent years, as a result of application of a wide array of new research methods, from those of molecular biology to those of clinical trials and meta-analysis. We know much more about the mechanisms of action of thyroid hormone and the metabolism of thyroid hormones than just a few years ago. The genes for the transporters and enzymes involved in thyroid iodine metabolism and thyroid hormone production have been cloned. Studies of these and other previously cloned thyroid-related genes (e.g., those of the thyrotropin receptor and thyroglobulin) have provided much new knowledge about both normal and abnormal physiology. With respect to the common thyroid diseases, iodine deficiency is still the most common, and while much progress has been made to eradicate iodine deficiency, there is more to be done. We still know little about the pathogenesis of the thyroid autoimmune diseases or thyroid nodular disease, but even in these areas progress is being

made. There are many new concepts in disparate areas such as nonthyroidal illness, the effects of drugs on thyroid function, optimal management of hypothyroidism and thyrotoxicosis, and the treatment of thyroid dysfunction in pregnancy and the postpartum period. All of these areas are covered in extensively revised chapters in this new edition. The diagnosis and management of patients with thyroid nodules, including sonography, fine needle biopsy, cytopathologic classification, and molecular diagnostics have undergone a sea change over the last 7 years, reflected in greatly revised or entirely new chapters on these topics. Finally, over the past few years, much progress has been made in the understanding of the molecular genetics of thyroid cancer. And, tyrosine kinase inhibitors and other targeted therapies are now widely used in the treatment of advanced metastatic differentiated thyroid cancer, medullary thyroid cancer, and anaplastic cancer. These topics are covered in far greater detail in this edition. We wish to thank all the contributors to the book. Their charge was to ensure that their chapters are current and comprehensive, and to provide interpretation and guidance in areas of controversy. We have tried to guide but not dictate to them. We also wish to thank the entire editorial team at Wolters Kluwer for their tremendous encouragement and assistance in the production of the book. In this era of ready electronic access to many small packets of information, we still believe that there is a need for comprehensive review and evaluation of what is known and what is not known—in other words, a book, and it has been our goal to provide this review and evaluation. We hope this eleventh edition of The Thyroid will match up with the preceding ones, and that it will provide guidance and even inspiration to those who seek to increase their understanding of the thyroid gland and how to treat or prevent its diseases. DAVID S. COOPER PETER A. KOPP

PREFACE TO THE FIRST EDITION This book is intended for those who must deal with the problems of thyroid function and thyroid disease in man. It is designed for use in the clinic and in the basic science laboratory connected with the clinic. The information made available has been brought together from widely diverse sources, and in some instances is reported here for the first time. Many subjects have been presented both in broad outline and in more comprehensive detail to meet differing requirements. It has been planned to provide sufficient documentation to satisfy most needs and, for more exhaustive requirements, to provide a bibliography adequate enough to initiate a search of the literature. The introduction of a book into a field of clinical medicine today requires considerable justification. In the thyroid field particularly, there already is a profusion of books including the almost classic works of Means in this country and of Joll in England, recently and capably revised by Rundle. Nevertheless, the recent growth of medical knowledge in general, and about the thyroid in particular, appears to have created need for a new volume constructed on a somewhat different basis from those of previous works. Barry Wood has compared the growth of medical information to that of bacteria. Bacteria show a lag at the beginning of growth and then multiply at a logarithmic rate. Wood considers the growth of current-day medicine to have reached the logarithmic phase. The accumulation of data about the thyroid provides a good example of this acceleration. One author of a recent review claims to have unearthed 3,000 new references pertaining to the gland and published during the single year before he wrote his article. The Quarterly Cumulative Index Medicus offers about 7,800 references to the thyroid in the past decade. More than this, the thyroid field is permeated by contributions from the cardiologist, neurologist, muscle physiologist, and

many others, bringing the highly unique techniques of their particular specialties to bear on the subject. It is evident that the ability of any one individual to follow progress in all directions at once has all but vanished. As a consequence, marked subspecialization of interest has developed and advances have come to depend upon the interchange of information among many specialists, each providing their own orientation. This trend has suggested that the information in a book about the healthy and diseased thyroid should also be subjected to the process of sifting and appraising through many eyes. The various specialists present material with which they have had direct experience, and the editor functions as the overseer to provide orientation and preserve the inherent orderliness of the entire subject. The total clinical and research experience made available in this way exceeds that of one person alone. Each topic can be subjected to the critique of a man who has worked intensively with the problem. Finally, a book of this sort can be readily kept current, because of the authors’ continued contact with investigation and the fact that there are no large sections to be rewritten by any one individual. Every effort has been made to make available sufficient basic and clinical knowledge to satisfy curiosity about either of these aspects. For example, sections on the fundamental properties of radioiodine that permit the use of the isotope and on the instrumentation that facilitates such use are presented as well as a discussion of the clinical application. Most basic sections are separated from the clinical material, but are incorporated with it where this has seemed reasonable. The fundamental aspects of thyroid function are man and the mechanisms which control the activity of the gland; the biochemistry of the hormone; and histology and comparative anatomy make up Part I. The mechanisms of action of the antithyroid drugs are included because of the intimate relationship of their effects to the problems of basic physiology. Part II presents the laboratory methods which supplement the clinical appraisal of thyroid secretory activity. The presentations of the basic principles involved in radioiodine usage and the instrumentation which is employed are included within the laboratory section and are available here for later reference when the therapeutic as well as diagnostic use of the isotope are considered. The diseases of the thyroid are considered in Part III. The disorders first

described are those in which the level of thyroid hormone in the circulation and tissues is within normal limits—euthyroidism. After this comes the derangements in which hormone levels are increased—toxic goiter or hyperthyroidism—or decreased—hypothyroidism or myxedema. The effects of hyperthyroidism and of hypothyroidism upon the individual body systems have been subjected to fairly detailed analysis. The plan to arrange disease by functional categories breaks down in relation to inflammation of the thyroid including the peculiar composite entity, chronic thyroiditis. Inflammations of the thyroid tend to inactivate the gland but chronic thyroiditis is almost as often associated with evidence of hyperthyroidism as with hypothyroidism. The inflammations have been placed under a separate heading on this account. Before the disease states are presented, several important preliminary subjects are considered in Part III. The normal and abnormal developments of the gland are described, together with the surgical anatomy and a method of physical examination that is an essential procedure because of the accessibility of the thyroid to this approach. The pathology is presented in its entirety in the introductory sections and is not dispersed among the various diseases. A concept of change in thyroid disease emerges in this way which could not otherwise become evident. A major goal throughout the volume has been to assess the validity of the facts on which current information or procedure is based. Corroborative information is often documented beyond reasonable doubt, but too often is based only on speculation or custom or is wanting altogether. The fact that a critical appraisal has been accomplished is a tribute to the contributors. The world today, as in the past, is threatened by prejudice, of which racial, social, and economic prejudices are but a few. Equally influential, but less well recognized, is the prejudice of “experience,” derived from uncritical or uncontrolled observation, from the word of an “authority,” or from emotional bias.1 Fortunately there are those who are willing to give time and effort to seek out and correct such distortions of the truth. Considerable aid has come to the editor from several sources. Dr. John Stanbury has been particularly helpful. The members of the Thyroid Clinic at the Presbyterian Hospital need recognition for their influence upon the formulation of many of the views presented herein. Credit must be given to the patience and forbearance of the many contributors who tolerated changes

in style and length of manuscript in the interest of creating an integrated volume out of a series of individual essays. The editor’s wife has acted as guardian of clarity, upon the thesis that even the layman should be able to read and understand a well-written article. Miss Anne Powell, of the librarian staff at P. & S., was extremely generous with her time. Finally, Mrs. R. Levine and Mrs. K. Sorenson were more than patient with the secretarial details. SIDNEY C. WERNER New York City 1“Conviction

is by no means devoid of emotion but is a disciplined and differentiated emotion, pointed to the removal of a realistic obstacle. By contrast, the emotion behind prejudice is diffused and overgeneralized, saturating unrelated objects.”—Gordon W. Allport: The Nature of Prejudice.

CONTENTS Contributors Preface to the Eleventh Edition Preface to the First Edition

section I The Normal Thyroid A.

History, Development, Anatomy

chapter 1

The heritage of the thyroid: A brief history Peter A. Kopp

chapter 2

Development and anatomy of the hypothalamic– pituitary–thyroid axis Pilar Santisteban and Sabine Costagliola

B.

Thyroid Hormone Synthesis and Secretion

chapter 3

Thyroid hormone synthesis: Thyroid iodide transport Nancy Carrasco

chapter 4

Thyroid hormone synthesis Peter A. Kopp

C.

Peripheral Thyroid Hormone Binding and Metabolism

chapter 5

Thyroid hormone transport proteins David Halsall and Carla Moran

chapter 6

Intracellular pathways of iodothyronine metabolism/implications of deiodination for thyroid hormone action

Antonio C. Bianco and Brian W. Kim

D.

Thyroid Hormone Action

chapter 7

Intracellular actions by thyroid hormones Paul M. Yen, Gregory A. Brent, and Anthony N. Hollenberg

chapter 8

Thyroid hormone structure–function relationships Riccardo Zucchi

E.

Factors That Control Thyroid Function

chapter 9

Chemistry and biosynthesis of thyrotropin Ronald N. Cohen, Anthony N. Hollenberg, and Fredric E. Wondisford

chapter 10

The thyrotropin receptor Gilbert Vassart and Patrice Rodien

chapter 11

Aging and the thyroid Anne R. Cappola

chapter 12

Effects of drugs on TSH secretion, thyroid hormones’ absorption, synthesis, metabolism, and action David H. Sarne

chapter 13

Thyroid disruptors Angela M. Leung

chapter 14

Nonthyroidal illness syndrome Anita Boelen, Lies Langouche, Wilmar Wiersinga, and Greet Van den Berghe

chapter 15

Iodine deficiency and excess, endemic cretinism Michael B. Zimmermann and Weiping Teng

section II Assessment of Thyroid Structure and Function chapter 16

Physical examination of the thyroid gland James V. Hennessey and Jeffrey R. Garber

chapter 17

Laboratory measurement of thyroid-related hormones, proteins, and autoantibodies in serum Ulla Feldt-Rasmussen

chapter 18

Nonisotopic thyroid imaging Gilles Russ and Jung Hwan Baek

chapter 19

Thyroid radionuclide uptake and imaging studies

Daniel A. Pryma

chapter 20

Cytopathology of the thyroid gland Zubair W. Baloch and Virginia A. Livolsi

chapter 21

Histopathology of the thyroid gland Zubair W. Baloch and Virginia A. Livolsi

section III Thyroid Diseases: Thyrotoxicosis A.

Causes of Thyrotoxicosis

chapter 22

Pathogenesis of Graves disease Terry F. Davies and Yaron Tomer

chapter 23

Thyrotropin-induced thyrotoxicosis Paolo Beck-Peccoz, Andrea Lania, and Luca Persani

chapter 24

Toxic adenoma and toxic multinodular goiter Ralf Paschke

chapter 25

Trophoblastic tumors Jerome M. Hershman

chapter 26

Sporadic painless, painful subacute and acute infectious thyroiditis Alan P. Farwell and Elizabeth N. Pearce

chapter 27

Thyrotoxicosis of extrathyroidal origin Angela M. Leung

B.

Organ System Manifestations

chapter 28

Overview of the clinical manifestations of thyrotoxicosis Henry B. Burch

chapter 29

Ophthalmopathy Luigi Bartalena

chapter 30

The cardiovascular system in thyrotoxicosis Salman Razvi

chapter 31

Thyroid hormones in intermediary metabolism, thermogenesis, and obesity Francesco S. Celi

chapter 32

The skeletal system in thyrotoxicosis

Bernard Freudenthal, Laura Watts, Graham R. Williams, and J. H. Duncan Bassett

chapter 33

Thyroid dermopathy and thyroid acropachy Vahab Fatourechi

chapter 34

Psychiatric and cognitive effects of thyrotoxicosis Mary H. Samuels and Kathryn G. Schuff

chapter 35

Thyrotoxic storm Leonard Wartofsky and Kenneth D. Burman

C.

Diagnosis and Management of Thyrotoxicosis

chapter 36

Diagnosis of thyrotoxicosis Paul W. Ladenson

chapter 37

Treatment of thyrotoxicosis David S. Cooper

chapter 38

Subclinical thyrotoxicosis Kristien Boelaert

section IV Thyroid Diseases: Hypothyroidism A.

Causes of Hypothyroidism

chapter 39

Chronic autoimmune thyroiditis Anthony P. Weetman

chapter 40

Genetic defects causing hypothyroidism N. Schoenmakers

chapter 41

Primary hypothyroidism due to other causes Caroline T. Nguyen and Peter A. Singer

chapter 42

Central hypothyroidism Luca Persani and Paolo Beck-Peccoz

B.

Organ System Manifestations of Hypothyroidism

chapter 43

Overview of the clinical manifestations of hypothyroidism Michael T. McDermott

chapter 44

Hypothyroidism and the heart Salman Razvi

chapter 45

The skeletal system in hypothyroidism Laura Watts, Bernard Freudenthal, J.H. Duncan Bassett, and Graham R. Williams

chapter 46

Psychiatric and cognitive effects of hypothyroidism Kathryn G. Schuff and Mary H. Samuels

chapter 47

Myxedema Coma Kenneth D. Burman and Leonard Wartofsky

C.

Management of Hypothyroidism

chapter 48

Diagnosis of hypothyroidism Paul W. Ladenson

chapter 49

Treatment of hypothyroidism Jacqueline Jonklaas

chapter 50

Subclinical hypothyroidism Robin P. Peeters

section V Thyroid Diseases: Nontoxic Diffuse and Multinodular Goiter chapter 51

Multinodular goiter: pathogenesis and management Steen Joop Bonnema and Laszlo Hegedüs

chapter 52

Clinical evaluation and management of thyroid nodules Susan J. Mandel and Jill E. Langer

section VI Thyroid Cancers chapter 53

Carcinomas of follicular epithelium: epidemiology and pathogenesis Arthur B. Schneider, Cari M. Kitahara, and Alina Brenner

chapter 54

Genomic landscapes of thyroid cancers of follicular cell derivation David G. McFadden, Thomas Giordano, James A. Fagin, and Yuri Nikiforov

chapter 55

Carcinoma of the follicular epithelium: surgical therapy

David L. Steward and Julie A. Sosa

chapter 56

Staging and prognosis of differentiated thyroid cancer Donald S.A. McLeod

chapter 57

Medical management of differentiated epithelial cell thyroid cancer Sebastiano Filetti, R. Michael Tuttle, and Steven I. Sherman

chapter 58

Papillary microcarcinomas Akira Miyauchi and Yasuhiro Ito

chapter 59

Medullary thyroid cancer Andreas Machens, Dagmar Führer, and Henning Dralle

chapter 60

Poorly differentiated thyroid cancer, anaplastic thyroid cancer, and miscellaneous tumors of the thyroid Bryan R. Haugen, Keith C. Bible, and Robert C. Smallridge

chapter 61

Thyroid cancer in children Catherine A. Dinauer, Emily R. Christison-Lagay, and Gary L. Francis

section VII The Thyroid in Infancy and Childhood chapter 62

The maturation of thyroid function in the fetus, in the perinatal period and during childhood Gabor Szinnai and Michel Polak

chapter 63

Hypothyroidism in infants and children Johnny Deladoëy, Julia von Oettingen, and Guy Van Vliet

chapter 64

Impaired sensitivity to thyroid hormone: defects of transport, metabolism, and action Alexandra M. Dumitrescu, Manassawee Korwutthikulrangsri, and Samuel Refetoff

chapter 65

Hyperthyroidism in the neonate, child, and adolescent Scott A. Rivkees

section VIII Thyroid Disorders in Pregnancy and Postpartum chapter 66

Thyroid disorders during preconception, pregnancy, and the postpartum period

Tim I.M. Korevaar and Elizabeth N. Pearce Index

SECTION

I

THE NORMAL THYROID

HISTORY, PART A DEVELOPMENT, ANATOMY

1 The heritage of the thyroid: A brief history Peter A. Kopp

Many view history narrowly as a chronologic narrative of events of the past, its participants dead, and its interpretation static. However, its purpose is ultimately a comprehension of the foundation of our current knowledge and concepts, which can be essential for developing visions for the future. The history of endocrinology and thyroidology has been summarized in comprehensive texts (1) and numerous biographical and topical vignettes (2), and the Thyroid History Timeline in the Clark T. Sawin History Resource Center of the American Thyroid Association provides a continuously updated overview of milestones in the history of the thyroid (3). This succinct overview is an expansion of the text published in previous versions of this textbook by Clark T. Sawin1, one of the distinguished historians of endocrinology and thyroidology (4).

GOITER, CRETINISM, AND THE THYROID GLAND The occurrence of goiter was widespread throughout the world and documented in numerous paintings, sculptures, and medical texts (Fig. 1-1) (5). It was, however, unknown that goiter was related to the thyroid gland and iodine deficiency, or that the thyroid gland existed (6). The ancient Greeks called the goitrous swelling in the neck a bronchocele (“tracheal outpouch”). A drawing by Leonardo da Vinci from around 1500 is thought to be the first illustration of a normal thyroid gland (Fig. 1-2) and Vesalius described it in 1543, although he used the term laryngeal glands for the entire organ that we

now know as the thyroid gland. The etymology of the word thyroid is derived from the Greek word thureos, which designates an oblong shield. It was introduced by Thomas Wharton in 1656 who named the thyroid gland after the Greek for “shield-shaped”—not by virtue of its own shape, but because of the shape of the nearby thyroid cartilage (7). Although Paracelsus (around 1529) and Platter (in 1562) speculated on a connection between goiter and cretinism (8), the function of the thyroid gland remained unknown until the 19th century. The most commonly held theory was that the thyroid regulates blood flow to the brain. Based on the concept that illness is an imbalance of the four humors (blood, phlegm, bile, and black bile), goiter was explained by an excess of phlegm.

GOITER AND IODINE Treatment of goiter was empiric and numerous remedies were proposed. Remarkably, some were complex mixtures of seaweed and marine sponge (6). These aquatic substances were well known in medieval Europe and had been used to treat goiter a thousand years earlier in China; some historians believe that the Europeans may have learned of these treatments from the Chinese indirectly via traders. In about 1812, Bernard Courtois discovered iodine in the residue of burnt seaweed (9). The concept that iodine is the active ingredient of the empiric therapies for goiter was then developed by Jean-François Coindet in Geneva, Switzerland (10). Around 1820, he treated patients with goiters with potassium iodide, after which their goiters shrank remarkably (11). To his chagrin, he also saw major toxic effects in some patients who took too much of the astonishing remedy, which consequently fell into disfavor. Still, iodine continued to be given for other disorders such as scrofula, syphilis, and tuberculosis (12). Coindet had in fact discovered iodine-induced thyrotoxicosis, presumably the earliest description of any form of thyrotoxicosis, although the true nature of the underlying pathophysiology was not recognized for several decades. However, despite the controversy over the use of iodine, the therapy represented a shift from an empiric, folk medicine to a rational treatment of a defined illness with a specific substance (10).

FIGURE 1-1. Large goiter in a woman from an area with a high rate of endemic goiter (Bern, Switzerland). The woman was a patient of Theodor Kocher (1841–1917) who was awarded the Nobel Prize in 1909 for his contributions to thyroid surgery and pathophysiology. (From Kocher T. Zur Pathologie and Therapie des Kropfes. Dtsch Z Chir 1874;4:417; with permission.)

FIGURE 1-2. Drawing of the thyroid gland by Leonardo da Vinci, ∼1500.

The use of iodine as a drug, even though it was effective at shrinking the goiter in many patients, by no means meant that practitioners knew that they were replacing a deficiency. For most of the 19th century, few accepted the idea that disease could be due to the lack of a nutritional substance. Although the use of iodine to prevent goiter was proposed in 1831—on the basis of observations in Colombia, South America (13), and later in 1850 by Chatin, a Parisian pharmacist, botanist, and physician (14)—these suggestions were not in tune with the times, were not accepted by the practitioners of that era, and were put aside. Most believed that goiter must be due to something in the water: a toxin, a bacterium, or a parasite. Only in 1851, Adolphe Chatin was

the first to formally hypothesize that the goitrous enlargement of the thyroid is caused by iodine deficiency. Initial attempts with iodine prophylaxis were then performed as early as 1869 in some department of France. However, the issue was not resolved until the early 20th century, when treatment with iodide was shown to prevent goiter development in children in seminal experiments by Marine and Kimball in the United States (Akron, Ohio) (15), as well as Hunziker, Eggenberger, and Bayard (Switzerland) (5,16). Iodine prophylaxis was then gradually introduced in these countries in the 1920s and more widely between 1970 and 1990, leading to elimination of endemic goiter and cretinism, and resulting in improved overall cognitive function in the population (16). In 1990, the United Nations World Summit for Children set the goal of eliminating iodine deficiency worldwide through programs of universal salt iodization (17). This global effort, which has been hugely successful, continues to be overseen by the Iodine Global Network (IGN; formerly International Council for Control of Iodine Deficiency Disorders [ICCIDD]) (Chapter 14) (18).

THYROTOXICOSIS The thyroid dysfunctions we know as thyrotoxicosis and hypothyroidism were not thought to be thyroid diseases when described in the 19th century. There was instead a slow accumulation of clinical and physiologic evidence that gradually defined these conditions as we know them today. Coindet was not the only one to describe thyrotoxicosis without realizing it. Caleb Parry, who saw spontaneous thyrotoxicosis before Coindet, but whose observations went unpublished until after his death (19) (and then in an obscure book published by one of his sons, rather than in a journal), saw a few patients with rapid heartbeat, goiter, and sometimes exophthalmos (exophthalmos was not described by Coindet who followed patients with iodine-induced thyrotoxicosis). Parry thought that this constellation of signs represented a form of heart disease. A few years later, Robert Graves described three women with similar complaints of goiter and palpitations. His published lectures included a fourth patient who also had exophthalmos (20). This additional patient had been mentioned to him by his student, friend, and

colleague, William Stokes. Both Graves and Stokes believed that the illness had a cardiac etiology. Graves description was not widely known on the European continent. Thus, when Carl von Basedow reported patients with the triad of goiter, tachycardia, and exophthalmos (Merseburger triad) in 1840 (21), he was thought to be the first to describe the illness. As a result, the term Basedow disease, rather than Graves disease, is still used widely on the European continent for what, in fact, should probably be called Parry disease. Even after Basedow’s report, however, the goiter itself was not considered of much importance. The belief that the syndrome was of cardiac origin receded by the 1860s, in part because of Charcot’s emphasis on the nervousness seen in most patients (22). The hypothesis of a neurologic etiology, rather than a thyroid disease, was then dominant for the rest of the 19th century. By the 1880s, surgeons were able to remove goiters, at least partially, in these nervous, hyperactive patients without fatal outcomes, a clear change from 20 years before, when this surgery did in fact kill the majority of patients who were operated upon (see below for a discussion on thyroidectomy). Interestingly, the nervousness often disappeared in the survivors. This, together with the observation in the 1890s that too much thyroid extract leads to similar nervousness and weight loss, brought a shift in thinking and led to the concept of thyrotoxicosis, an excess of thyroid hormone, as explanation of this peculiar syndrome. The term was applied to both the spontaneous disease and the disease induced by administration of desiccated thyroid. It is also noteworthy that the mechanism underlying thyrotoxicosis was thought to either consist of abnormal secretion or failure to inactivate a toxin, that is, a deleterious substance not found in a healthy person. Until the 1940s, subtotal thyroidectomy remained the standard of care for hyperthyroidism. This then changed with the introduction of radioactive 131iodine by Saul Hertz in 1941 (23,24) and the development of “antithyroid” thionamide drugs by Edwin Astwood (25) in 1943.

HYPOTHYROIDISM AND THE RECOGNITION OF THYROID FUNCTION

Hypothyroidism as a clinical syndrome was recognized even later than hyperthyroidism, and, at first, its cause was equally obscure. In 1850, Thomas Curling described a child and an infant with a cretinoid appearance and absence of the thyroid (26). A further substantial step toward the elucidation of the pathogenesis was the observation by William Gull who reported on a “cretinoid state supervening in adult life in women” in 1873 (27). A few years later, in 1878, William Ord confirmed this observation in a clinical report entitled “On myxoedema, a term proposed to be applied to an essential condition in the “cretinoid” affection occasionally observed in middle-aged women” (28). Importantly, Ord recognized that this phenotype is associated with an atrophy of the thyroid gland. While the two authors were unaware of the causal role of the thyroid in the pathophysiology of the myxedematous condition, they emphasized the phenotypic appearance and its resemblance with cretinism. The disorder was named myxedema because of the swollen skin (edema) and its excess content of mucin (myx-) (28). It was considered either a neurologic or a skin disease. There was no cure, and its course was inexorably progressive. There was only palliation with drugs such as pilocarpine, to be given in an attempt to reverse the patients’ decreased sweating. In 1882/83, the Swiss surgeons Jacques and Auguste Reverdin from Geneva reported on their experience with 22 total thyroidectomies (29,30). They described the clinical picture of postsurgical myxedema or “myxoedème opératoire” and accurately described the phenotype of severe hypothyroidism secondary to thyroidectomy. A few months later, in April of 1883, Theodor Kocher from Berne, Switzerland, reported results on 200 thyroidectomies performed by several surgeons in Switzerland and Germany at the Congress of the German Society for Surgery in Berlin. These findings were then summarized in Kocher’s most famous publication “Ueber Kropfexstirpation und ihre Folgen,” which translates into “Goiter extirpation and its consequences” (31). In this case series, Kocher meticulously described the sequelae of total thyroidectomy and he coined the name “cachexia strumipriva,” thus cachexia after goiter extirpation. Kocher was awarded the Nobel Prize in Physiology and Medicine for his impressive contributions to thyroid surgery and pathophysiology in 1909 (32). Later in 1883, Dr. Dawtrey Drewitt presented a patient with classical symptoms of hypothyroidism to the Clinical Society of London. Felix

Semon, a prominent Prussian laryngologist who had moved to Great Britain, and who had assisted Kocher’s presentation in Berlin, then commented “that there appeared to be three conditions closely allied to each other, and having in common either absence or probably complete degeneration of the thyroid body: namely, cretinism, myxoedema, and the state after total removal of the thyroid body.” Scoffed at, Semon persisted. The Society finally named a committee to investigate his observation. The committee also included Victor Horsley, a surgeon, who had performed thyroidectomies on animals; his experiments confirmed that removing the thyroid results in myxedema. After reviewing the available evidence during a period of 5 years, the committee concluded that the phenotype associated with myxedema, total thyroidectomy, and cretinism all result from “the annihilation of the function of the thyroid body,” findings that were published in the now famous Report on Myxedema from 1888 (33). Based on the conclusion in the Report on Myxedema and the experimental work from Horsley and Schiff (see below therapy of hypothyroidism), it became accepted that there is a causal relationship between cretinism, spontaneous and postsurgical myxedema, and the function of the thyroid. Yet, the mechanism remained obscure; the committee offered nothing in the way of therapy, and did not connect thyroid deficiency with any suggestion of thyroid replacement.

THERAPY OF HYPOTHYROIDISM In Geneva, Switzerland, the physiologist Maurice Schiff, revisiting his older experiments showing that animals of various species could not survive after removal of the thyroid gland, demonstrated in 1884 that thyroid grafts into the peritoneum reversed, though temporarily, the effects of thyroidectomy, and he suggested preparing “a thyroid paste” for repeated injections (34). In 1889, Brown-Séquard’s work in Paris on the supposed rejuvenating effects of testicular extracts of dogs and guinea pigs (35) led to the use of extracts of other tissues as treatments for many disorders. Most extracts eventually proved to be ineffective, but the idea led indirectly to the successful and remarkable cure of myxedema. In 1891, George Murray from Newcastle, England, reported that injected sheep thyroid extract was able to attenuate or

cure myxedema (Fig. 1-3) (36,37). A year later, the treatment was made even easier by simply eating, instead of injecting, ground or fried sheep thyroid, or tablets of dried thyroid tissue. This confirmation that Brown-Séquard’s organotherapy was effective in at least one serious illness was the origin of modern endocrinology. In fact, but not well known, Murray was preceded in his discovery of a successful therapy by two Portuguese physicians, Bettencourt Rodrigues and Serrano (38,39), who presented their findings in Portuguese and French in 1890. One should also acknowledge that the ingestion of animal thyroid–derived preparations had been propagated much earlier. It is said that Sun Simiao, a famous traditional Chinese physician of the Sui and Tang dynasty, who carries the title as China’s King of Medicine, reported in 652 AD that goiter occurred in mountainous regions and could be cured by prescribing both seaweeds and thyroid glands from deer and sheep. By the end of the 19th century, it became accepted that the thyroid contains an active principle exerting a broad spectrum of metabolic actions. This launched the quest for the isolation of this substance.

FIGURE 1-3. One of the patients with myxedema treated by George Murray with thyroid extracts at the end of the 19th century. (From Murray G. Diseases of the Thyroid Gland. Part I. Myxoedema and Cretinism. London: H. K. Lewis & Co. Ltd.; 1900. Figure 14.)

DISCOVERY OF THE ACTIVE PRINCIPLE AND THERAPY OF HYPOTHYROIDISM A vigorous search for the “active principle” then took place, but no one found anything helpful until 1895, when Baumann, to his great surprise, found iodine in the thyroid gland (40). Twenty years later, Kendall at the Mayo Clinic, using iodine as a marker for the isolation of the active substance, succeeded in crystalizing bioactive material on Christmas Day 1914, using 6,500 lb of hog thyroid glands as material for the extraction (41). He named it thyroxin, a contraction of thyroxindole, which was based on Kendall’s erroneous belief that the compound had an indole nucleus with three iodine atoms per molecule. He never changed his view, despite his repeated failure to synthesize an active molecule based on his presumed structure. By 1924, Kendall’s thyroxin had been shown to be one of the most important substances through which the thyroid exerts its control on basal metabolic rate. Kendall was much disappointed when, in 1927, an Englishman, Sir Charles Harington, not only found the correct structure, but found that it had four (not three) iodine atoms per molecule (42). Harington, to Kendall’s greater chagrin, then also successfully synthesized it (43). Harington also added an e to the name and called it thyroxine to fit the convention at the time for naming amino acid derivatives. His synthetic thyroxine (T4) was tested in two patients with myxedema and its effect in raising the basal metabolic rate was found to be quantitatively similar to that reported for that extracted from the thyroid. Kendall’s extracted thyroxine was patented and commercially licensed, but was far more expensive and not as effective as desiccated thyroid. In the 1920s, no one knew that thyroxine as the free acid is not well absorbed. Therefore, until the 1960s, the usual therapy for thyroid deficiency and goiter was the administration of desiccated thyroid. The successful synthesis of thyroxine in high yield in 1949 (44,45) (in the form of sodium L-thyroxine, which, in contrast to thyroxine itself, is quite well absorbed from the gut) made therapy with this form of thyroxine economically sensible. Although desiccated thyroid continued to be the treatment of choice for hypothyroidism well into the 1970s, this changed by the mid 1970s and L-thyroxine became the treatment of choice (Fig. 1-4) (46). Both Kendall and Harington suspected

that there might be another thyroid hormone other than thyroxine, but they were never able to find it. Kendall moved on to isolate the steroid hormones of the adrenal cortex, which won him a Nobel Prize in 1950 together with the Swiss chemist Tadeusz Reichstein and Mayo Clinic physician Philip S. Hench. Harington gave up the idea of a second thyroid hormone and became director of London’s National Institute for Medical Research. He was much surprised when his associate, Rosalind Pitt-Rivers, and her postdoctoral fellow, Jack Gross, found and synthesized triiodothyronine (T3) and then showed it to be more active than thyroxine in a bioassay. They suggested that T3 was the active form of the hormone and that T4 is the precursor (47–49). T3 was found almost simultaneously across the English Channel in Paris by Jean Roche, Serge Lissitzky, and Raymond Michel (50,51). Currently, levothyroxine is recommended as the preparation of choice for the treatment of hypothyroidism due to its efficacy in resolving the symptoms of hypothyroidism, long-term experience of its benefits, favorable side-effect profile, ease of administration, good intestinal absorption, long serum halflife, and low cost (52). However, there is ongoing controversy whether combination therapy with thyroxine and triiodothyronine may have advantages and result in a more physiologic method of thyroid hormone substitution (53,54).

FIGURE 1-4. Thyroid hormone prescriptions dispensed by retail pharmacies, United States, 1964–1988. National Prescription Audit. □ total; □ natural agents; ○ synthetic agents. (From Kaufman SC, Gross TP, Kennedy DL. Thyroid hormone use: trends in the United States from 1960 through 1988. Thyroid 1991;1[4]:285–291; with permission.)

SCREENING AND THERAPY OF CONGENITAL HYPOTHYROIDISM Congenital hypothyroidism affects about 1:3,000 newborns and, if left untreated, it leads to mental retardation and impaired development (Chapter 62). Treatment of children with congenital hypothyroidism had been undertaken in the early 20th century by Kendall with crystalline thyroxine. A report from 1957 by David Smith, Robert Blizzard, and Lawson Wilkins, the latter being one of the founders and pioneers of pediatric endocrinology, reported on the intellectual function of 128 infants and children with hypothyroidism, and emphasized that mental impairment was associated with the severity of hypothyroidism, the age of onset, and that early treatment with thyroid hormone was associated with better outcomes (55). However, one of the significant challenges in diagnosing congenital hypothyroidism in infancy was that clinical evaluation was the primary basis for deciding whether thyroid therapy was indicated. In the early 1970s, several groups confirmed that early treatment improved intellectual outcomes in children with congenital hypothyroidism, especially if therapy was started prior to 3 months of age (56,57). At the same time, Jean Dussault and Robert Volpé in Quebec City, Canada, developed radioimmunoassays for the measurement of thyroxine from the eluate of filter paper blood spots. Their abstract was not accepted by The Endocrine Society for the 1972 International Meeting, and was first presented at a Quebec Clinical Investigation Society meeting in 1972 (58,59). Supported by the Quebec Network for Genetic Medicine, Dussault and colleagues performed a pilot study on 30,000 samples, and detected four cases of congenital hypothyroidism. Their report on mass screening for congenital hypothyroidism was not accepted for publication by Clinical Chemistry or The New England Journal of Medicine (59), which judged the report to be irrelevant. Thus, the first report appeared in French in the journal of the Union Médicale du Canada in 1973 (60). In April 1974,

mass screening for congenital hypothyroidism was incorporated with newborn phenylketonuria and tyrosinemia screening for all newborns in the Province of Quebec (61). A landmark study was published in 1979 reporting on the screening of 1,046,362 infants (62). A total of 277 infants with congenital hypothyroidism were detected and seven were missed, resulting in a total of 284 affected infants in the screened population, and an overall incidence of 1 in 3,684 live births. Neonatal screening is now performed in all developed countries—40 years ago, in 1979, it was entirely novel and had initially been judged to be irrelevant by some established medical authorities (59). While populationbased screening has led to the disappearance of intellectual disability caused by congenital thyroid hormone deficiency in the developed world, it is also important to recognize that only about a third of newborns benefit from screening on a global scale (63).

DISCOVERY OF CURRENT CONCEPTS OF THYROID HORMONE SYNTHESIS, TRANSPORT, AND ACTION In 1948 Taurog and Chaikoff obtained strong evidence that most of the organic iodine in the circulation consisted of T4 reversibly bound to plasma proteins (64). Electrophoretic analyses then allowed them to identify thyroxine-binding globulin (TBG) as the major binding protein for thyroxine (65), eventually leading to the concept of protein-bound and free hormone fractions. The concept that T3 is the active thyroid hormone remained controversial until 1970, mainly because of difficulty demonstrating its presence in athyreotic humans who were injected with radiolabeled T4. In 1970, Lewis Braverman, Sidney Ingbar, and Kenneth Sterling (66), and Sterling et al. (67), were able to prove that T4 is indeed converted to T3 in vivo. This observation was preceded by experiments by Tata and Widnell who showed that T3 stimulates RNA and subsequent protein synthesis, indicating that it has a transcriptional effect and thus a nuclear action (68). In 1972, Oppenheimer et al. demonstrated the presence of a putative nuclear thyroid hormone receptor, and the receptor was found to have a much higher affinity for T3 than for T4 (69). Facilitated by the rapid progress in molecular

biology, the cloning of the thyroid hormone receptors α and β in 1986 by two independent groups was a major milestone that stimulated extensive research aimed at dissecting the mechanism of thyroid hormone action (Chapter 7) (70,71). The cloning of the thyroid hormone receptors was paralleled by the quest to identify the enzyme(s) that metabolize T4, and by the end of the 1990s, the complementary DNAs (cDNAs) of the three deiodinases, members of the selenoprotein family, had been cloned (Chapter 6) (72). Although there was substantial evidence that the entry of thyroid hormones into target cells is dependent on transporters, this concept was not widely accepted, and the dogma that they cross the plasma membrane by passive diffusion was embraced until the beginning of the 21st century (73). Since then, several transporters capable of transporting iodothyronines into target cells have been identified, and this has substantially refined the understanding of thyroid hormone action at the cellular level (Chapter 63) (74). The last decades have also led to the cloning and characterization of the genes and proteins involved in thyroid hormone synthesis, among them the sodium iodide symporter (NIS), which plays a pre-eminent role, since it is responsible for the uptake of iodide by thyroid follicular cells, and its function is essential for imaging and therapy with radioiodine (Chapters 3 and 4) (75).

AUTOIMMUNE ETIOLOGY OF GRAVES DISEASE AND HASHIMOTO THYROIDITIS The most common forms of hyper- and hypothyroidism, Graves disease and Hashimoto thyroiditis, have an autoimmune etiology (Chapters 22 and 38). The underlying pathophysiology is highly complex and involves interactions between genetic predisposition and environmental factors. First insights into an autoimmune etiology were provided by Hashimoto’s original description of the entity carrying his name in 1912 (76). He described, among other findings, an infiltration of the thyroid by lymphoid and plasma cells, formation of lymphoid follicles with germinal centers, and fibrosis, and confident that he had identified a new entity, he named it “struma lymphomatosa” (76). In 1956, Rose and Witebsky and Witebsky et al.

demonstrated that immunization of rabbits with extracts obtained from rabbit thyroid tissue led to histologic alterations in the recipients that resembled those described by Hashimoto (77,78). Deborah Doniach and Ivan Roitt and colleagues then purified antithyroglobulin antibodies from sera of patients with Hashimoto thyroiditis and proposed that it is an organ-specific autoimmune disease (79). In 1956, Duncan Adams and Herbert Purves from New Zealand first noticed that the serum of patients with Graves disease contained a “longacting thyroid stimulator” (LATS) that was distinct from TSH in terms of its duration of action (80,81). It was subsequently found to be an immunoglobulin G (IgG), and it became apparent that it stimulates adenyl cyclase activity through binding to specific receptors, although blocking antibodies could also be found in some sera (82). The discovery of the thyroid-stimulating hormone receptor (TSHR) in 1966 identified it as the target of these antibodies, thereby defining the cellular mechanism leading to cell proliferation and increased hormone production (Chapter 22) (82). Although the exact etiology of thyroid autoimmunity remains only partially understood, current concepts postulate a combination of multifactorial disorder in which genetic susceptibility and environmental factors that lead to breakdown of self-tolerance (83). Human and animal studies have identified a number of candidate genes and genetic loci associated with autoimmune thyroid disease; possible environmental triggers include stress, iodine, infection, and smoking (83).

THYROID CANCER By the early 20th century, a number of pathologists and surgeons had a strong interest in thyroid cancer, or “struma maligna.” For example, in a detailed study published in 1907, Kocher reported about 400 cases of thyroid malignancies, many of them with bone metastases, which suggests that these were frequently follicular carcinomas (84). This assumption is supported by the fact that follicular carcinomas are more common under conditions of iodine deficiency. Kocher also emphasized that the diagnosis is often made too late, and that better means for early diagnosis were needed. One of Kocher’s trainees, Carl Kaufmann, had recommended preoperative biopsy for

tissue diagnosis of thyroid tumors and better planning of surgery as early as 1879 (85). The histologic criteria were fundamentally different at the time and a discussion of the developments in the classification of thyroid malignancies is beyond the scope of this short review. However, as summarized elsewhere in this textbook (Chapters 50, 53, 58, 59), major developments in the understanding of the pathogenesis of thyroid nodules and carcinomas have taken place since the 1990s. They have culminated in the thorough characterization of the genomic landscape of thyroid carcinomas of various histologic subtypes (86–91), which now has a major impact on the preoperative diagnosis of thyroid lesions, as well as the treatment and prognosis of thyroid malignancies.

OUTLOOK New discoveries into the pathophysiology of thyroid disorders continue to emerge and novel therapies are being developed. It is humbling and inspiring to see how much effort by numerous inquisitive and creative minds has been dedicated to the study of the thyroid and its pathophysiology, and how this impacts the lives of countless patients. The knowledge and concepts summarized in this textbook form a strong foundation on which to build the future.

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1Dr.

Clark T. Sawin (May 23, 1934 to August 11, 2004) has authored the previous versions of this chapter.

2 Development and anatomy of the hypothalamic–pituitary–thyroid axis Pilar Santisteban and Sabine Costagliola

The thyroid gland is the most anterior organ that buds from the gut tube. It is composed mainly of endoderm-derived follicular cells, which are responsible for thyroid hormone production in all vertebrates. The main regulator of thyroid function is thyroid-stimulating hormone (TSH), a glycoprotein synthesized and secreted from the thyrotrope cells of the anterior pituitary gland, which in turn is under the control of thyrotropin-releasing hormone (TRH) secreted by the hypothalamus. Thyroid hormone secretion and serum concentrations of thyroxine (T4) and 3,5,3′-triiodothyronine (T3) are maintained by a negative feedback loop involving inhibition of TSH and TRH secretion by T4 and T3 (Fig. 2-1). Consequently, the regulation of thyroid function depends on the normal development of the hypothalamic– pituitary–thyroid axis, which occurs independently but coordinately during embryonic and neonatal life.

FIGURE 2-1. A: The hypothalamic–pituitary–thyroid axis. TRH is synthesized in specific hypothalamic neurons and secreted into the hypothalamic–pituitary portal venous system; it is carried through this system to the pituitary where it stimulates the synthesis and secretion of thyroid-stimulating hormone (TSH). This hormone binds to its receptor in the thyroid gland, stimulating the synthesis and secretion of T4 and T3. Precise control of the axis is maintained by the inhibitory actions of T4 and T3 on both TRH and TSH secretion. Somatostatin (SRIF) inhibits both TRH and TSH release. (Modified from Melmed S. The Pituitary. 3rd ed. Cambridge, MA: Blackwell Science, Inc; 2011.) B: Lateral view of the forebrain showing the hypothalamic divisions which are conserved throughout vertebrates. The hypothalamic nuclei are. ARC, arcuate nucleus; DMH, dorsomedial hypothalamus; LHA, lateral hypothalamic area; ME, median eminence; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; VMH, ventromedial hypothalamus. (Modified from Xie Y, Dorsky RI. Development of the hypothalamus: conservation, modification and innovation. Development 2017;144(9):1588–1599.) C: In the ventral hypothalamus, evaginates to give rise to multiple structures, including the posterior pituitary gland, the ME, and the pituitary stalk, which physically connects the hypothalamus and the pituitary gland.

This chapter will focus primarily on the formation of the thyroid gland

with comments and references on the development of the hypothalamic– pituitary axis. The role of several transcription factors expressed specifically in the hypothalamus–pituitary–thyroid system has helped to unravel the molecular events responsible for its development. Most of the recent advances in the understanding of the formation of this axis come from studies in animal models, and more recently with the use of embryonic stem (ES) cells and inducible pluripotent stem (iPS) cells. Nevertheless, wherever possible this information will be integrated with what is known in humans. Following standard nomenclature, human transcription factors and proteins will be depicted in capitals, and a capital followed by lowercase will be used for mice; italics will be used when referring to genes.

THE HYPOTHALAMIC–PITUITARY AXIS The vertebrate embryo is dependent on complex inductive interactions to orchestrate pattern formation, organogenesis and, ultimately, the development of integrated organ systems. The hypothalamic–pituitary axis is a prime example of such a system and it is considered as a unit that acts as a neuroendocrine transducer to mediate trafficking between the brain and the peripheral hormone-secreting target glands (Fig. 2-1A). The concept that the pituitary is centrally regulated by the hypothalamus required contributions from several disciplines, including neuroanatomy, biochemistry, and physiology. Summary information about the most important events in the development of the hypothalamus–pituitary axis will be given in this chapter (1–4). The hypothalamus has integral developmental, functional and physical connections with the pituitary gland, through which it controls endocrine hormone release (5). Experimental embryology and molecular genetic studies have yielded evidence that the determination, development, and differentiation of the hypothalamus and the pituitary gland are intimately coordinated. How both glands, with different embryonic origins, co-develop introduces a level of complexity that is still not well understood.

SIGNALING AND TRANSCRIPTIONAL MECHANISMS

INVOLVED IN HYPOTHALAMUS ORGANOGENESIS The hypothalamus has a key role in regulating the neighboring pituitary and peripheral endocrine organ functions and constitutes less than 1% of brain volume. It emerges during the sixth week of gestation in humans, and between embryonic day (E) E11 and E18 in rats. The developmental origin of distinct hypothalamic regions is currently a topic of considerable debate. The structure derives from the ventral-caudal neuroectoderm of the forebrain, the prosencephalon, a primary brain vesicle that divides to form two secondary brain vesicles—the telencephalon (endbrain, cortex) and the diencephalon (6). Historically, it has been described that the diencephalon ventrolateral wall generates the primordium of the hypothalamus; however, a “prosomeric model” (7), based on gene expression in the mouse suggests an overall different origin of the hypothalamus arising from the telencephalon. More comparative studies are necessary to clarify this fundamental anatomical question. The hypothalamus has a highly conserved anatomy throughout vertebrate species. The primitive hypothalamic region is initially localized at the most rostral (anterior) region of the neural tube, and later has a more caudal (posterior) and ventral position in the forebrain (Fig. 2-1B). The hypothalamus encompasses various nuclei or groups of neurons organized in a three-dimensional patchwork. The adult mammalian hypothalamus can be anatomically divided into four distinct rostrocaudal regions (preoptic, anterior, tuberal and mammillary) (Fig. 2-1B), and each region has distinct nuclei and associated functions. Thus, the preoptic region controls thermoregulation, reproduction and electrolyte balance. The anterior hypothalamus, including the supraoptic (SON), suprachiasmatic (SCN), paraventricular (PVN) and anterior periventricular (aPV) nuclei, regulates feeding, circadian rhythms and other homeostatic processes. The tuberal region, including the arcuate nucleus (ARC), median eminence (ME), ventromedial (VMH) and dorsomedial (DMH) hypothalamus, plays a role in energy balance, stress response and aggression. Finally, the mammillary hypothalamus, which includes the mammillary bodies, is involved in stress response as well as in spatial and episodic memory (8). The hypothalamic control of pituitary hormones secretion is regulated by

magnocellular and parvocellular neuroendocrine neurons. The former are located in the PVN and SCN, where they project into the posterior lobe of the pituitary (neurohypophysis) to directly release oxytocin (OXT) and arginine vasopressin (AVP). The latter, located in the tuberal, preoptic, ARC, aPV and PVN, project into the ME where, among other neuropeptides, they secrete TRH (for regulating the TSH-thyroid axis) or release-inhibiting hormones, into a small portal blood system connected to the anterior pituitary (adenohypophysis). These anatomical aspects are well conserved throughout evolution. Overall, these observations indicate that the hypothalamic hormones are produced in more than one nucleus and even a single nucleus may express several hormones. The ME, which connects the hypothalamus to the pituitary (1), mediates the transportation of these hormones (Fig. 2-1C).

Morphogenic Signals Involved in Early Steps of Hypothalamus Development The hypothalamic primordium is induced during neural plate formation and requires signaling from morphogens secreted by surrounding tissues that create a map of positional identity in the neural plate. The migration of a specific neuronal type into the hypothalamus, its settling in hypothalamic nuclei and its connectivity with a variety of target sites requires extrinsic signals such as growth factors, neuropeptides and their receptors. These signals act concomitantly with the intrinsic transcription factors to regulate the early steps of hypothalamus development. Nevertheless, although great progress has been made in identifying the extrinsic and intrinsic factors that control hypothalamic patterning and neurogenesis, much remains unknown (9). Extrinsic developmental factors such as Sonic hedgehog (SHH), Nodal, Bone Morphogenetic Factor (BMP) and Wingless (Wnt) are the main factors involved in hypothalamus development (10–14). SHH is crucial for the growth and axial patterning of the hypothalamus (15) and directly regulates the expression of its cognate receptor Patched 1, through which it likely signals to promote anterior-dorsal hypothalamic fate. SHH antagonizes Nodal activity in the development of the posterior-ventral hypothalamus, but

cooperates with Nodal in the maintenance of the anterior-dorsal hypothalamus, and with BMPs to drive hypothalamic dopaminergic neuronal specification. Nodal and BMP are two signaling pathway members of the transforming growth factor β (TGFβ) superfamily, and the crosstalk between SHH and TGFβ signaling is well acknowledged during hypothalamic development (review in [8]). The Wnt/β-catenin pathway plays an essential role in the formation of the anteroposterior axis. In addition, Wnt and its receptor Frizzled regulate the patterning, neurogenesis and differentiation of posterior hypothalamic cells in the zebrafish and of the ARC nucleus in the mouse. The role of other factors such as fibroblast growth factors (Fgfs) and retinoic acid (RA) in hypothalamus development are less clear. As the expression of the developmental factors SHH, Nodal, BMP and Wnt is maintained in the mature brain, it would be interesting to investigate whether these molecules are released in a synaptic manner, or whether they maintain their activity via a diffusion mechanism in the adult hypothalamus. Recent findings indicate that some neuropeptides are involved in the development of neural circuits in the developing hypothalamus. Thus, the PACAP type 1 (PAC1) receptor, which is the most specific receptor for pleiotropic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) (16), regulates the development of zebrafish dopaminergic and OXT neurons by controlling the rate of orthopedia homeobox protein (Opt) synthesis (see below) (17). Likewise, the anorexigenic peptide leptin, which regulates metabolic-related homeostatic functions in the hypothalamus through its cognate receptor, also directly regulates developmental neurite formation of ARC nucleus neurons, and the neural projections of both orexigenic neuropeptide Y and anorexigenic pro-opiomelanocortin neurons.

Transcription Factors Controlling Hypothalamus Development Although more and more transcription factors regulating the development of the vertebrate hypothalamus are being identified, the gene regulatory networks that orchestrate this development remain poorly understood. The distinct anatomical regions in the hypothalamus need several neuronal cell

populations to develop connectivity. This raises many questions regarding the mechanisms that underlie the development of hypothalamic brain nuclei and the specification of the neuronal populations. Due to its complexity and the increasing number of transcription factors so far identified, we here discuss only the best characterized factors (Reviewed in [9]). The Lim homeobox (Lhx) family of transcription factors, which contain a unique cysteine-rich, zinc-binding domain, have key roles in hypothalamic induction and patterning. Specifically, the Lhx gene Islet-1 (Isl1) plays crucial roles in the development of multiple ARC neurons (18). Further, the homeodomain transcription factor Opt, which is well conserved across species, is expressed in hypothalamic domains (19) where it plays an important role in the differentiation of several neurohormone-secreting nuclei including aPV, PVN, SON, the arcute and the ventromedial nuclei (20). Otppositive neurons are diencephalic in origin as Otp instructs dopaminergic identity. Beyond its role as a developmental regulator of several neuroendocrine lineages, Otp is also an important regulator of migratory processes of other neuronal populations; for example, in zebrafish, Otp cooperates with the transcription factor Sim1 (see below) to regulate the expression of TRH and somatostatin, among others. Single-minded homologous 1 (Sim1) and Aryl hydrocarbon-receptor nuclear translocator (Arn2) are two PAS (PER-Arnt-Sim)-containing transcription factors belonging to the large basic helix-loop-helix family (bHLH). Both form a heterodimeric protein complex to activate or repress their target genes and activate hypothalamic differentiation in zebrafish. Sim1 and Arnt2 null mice die shortly after birth and analysis of newborn brains indicates the absence of the hypothalamic aPV, PVN, and SON nuclei, phenocopying the Otp null phenotype, although the latter mice show a dramatic decrease (almost 30%) in brain size. Moreover, Sim1 and Arnt2 null mice display developmental impairments that correlate with deficits in neuronal migration and differentiation (21,22). Sim1, Arnt2 and Otp function along parallel pathways, as they are all required for Sim2 expression in the PVN for the differentiation of neurons that secrete TRH; and in the aPV, for the differentiation of neurons that secrete somatostatin. These transcription factors are required in the PVN and SON nuclei to maintain Brn2 expression, a POU domain transcription factor necessary for the development of OXT-, AVP-, and corticotropin-releasing hormone (CRH)-producing neurons.

Distal-less homeobox-1 (Dlx1) and its homolog Dlx2 are homeodomain transcription factors that play an important and redundant role in the fate specification of GABA and tyrosine hydroxylase (TH) neurons in the forebrain (23). It has recently been demonstrated that these factors are robustly expressed in the embryonic ARC, pointing to a role in the fate specification or differentiation of arcuate neurons. Interestingly, Otp and Dlx1/2 are expressed in alternating domains in the embryonic hypothalamus and both are required for the specification of growth hormone–releasing hormone (GHRH)- and Agouti-related protein (AgRP)-neurons, respectively. These data provide evidence that GHRH- and AgRP-neuronal fates are interconnected and suggest that the Dlx1/2-Otp axis plays a role in producing a balanced ratio of AgRP- to GHRH-neurons in the hypothalamus. The Hmx homeobox genes Hmx2 and Hmx3 seem to be important for the differentiation of GHRH-secreting neurons in the ARC, and are subsequently expressed in the ventral hypothalamus from E10.5. While the overall number of neurons present in the ARC does not appear to change, Hmx2, Hmx3 null mice exhibit postnatal dwarfism and have a reduced number of GHRHsecreting neurons (24). Nuclear receptor subfamily 5 group A member 1 (Nr5a1), which encodes the Sf1 protein (25) and has been classically associated with normal gonadal and adrenal development, is also important for VMH development. Nr5a1 mutant mice lack normal survival and migration of VMH neuronal precursors from the ventricular zone. Nr5a1 and Nkx2-1 expression is nonoverlapping, and it appears that Sf1 represses Nkx2-1 expression in vitro. It is important to note that the homeodomain transcription factor Titf1/Nkx2-1 (hereafter termed Nkx2-1, see also later in this chapter) is induced in early central nervous system development. Nkx2-1 mutant mice die at birth and present severe abnormalities in the ventral hypothalamus, including agenesis of the ARC and ventromedial nucleus. In the context of the hypothalamic–pituitary–thyroid axis, Nkx2-1 is expressed in the embryonic ventral forebrain and in specific hypothalamic areas, such as the median ganglionic eminence that gives rise to the pallidal component of the basal ganglia at E10.5. Consistent with this pattern of expression, Nkx2-1 null mice do not form the pallidal structures because of a ventral-to-dorsal transformation of the pallidal primordium into a striatum-like structure

(26,27). Interestingly, Nkx2-1 has been shown to play key roles in the developing telencephalon where it regulates the identity of progenitor cells in the medial ganglionic eminence. In addition, Nkx2-1 controls cell-fate determination and regulates neuronal migration by direct transcriptional regulation of guidance receptors in postmitotic cells (28). This is important, as it has been demonstrated that NKX2-1 mutations in humans cause the socalled Brain–Lung–Thyroid syndrome, which in addition to causing hypothyroidism and pulmonary dysfunction, is also associated with benign chorea due to problems with the migration of the basal ganglia (29,30) as well as pulmonary problems with respiratory distress (31). Besides Nkx2-1, other transcription factors such as Sox3 and Lhx2, expressed in the developing diencephalon and infundibulum at E10.5, but not in the pituitary itself, are required for proper diencephalon development and secondarily affect pituitary formation. Overall, these data support the importance of signal exchange between the diencephalon and the forming pituitary for the development of both tissues. The role of the pituitary primordium in the development of the hypothalamus has been less extensively studied but contact between the two structures is important for proper formation of the ventral hypothalamus and the ME, and also for differentiation and proliferation of the different types of pituitary cells.

SIGNALING AND TRANSCRIPTIONAL MECHANISMS INVOLVED IN PITUITARY ORGANOGENESIS Anatomically, the pituitary gland is located in the sella turcica (pituitary fossa) of the sphenoid bone and is covered superiorly by the diaphragm sellae (dura), laterally by the wall of the cavernous sinus, and anteroinferiorly by the posterior wall of the sphenoid sinus. In anterosuperior position, the pituitary lies near the optic chiasm. As explained earlier, in the ventral hypothalamus, the infundibulum evaginates to give rise to multiple structures, including the posterior pituitary gland, the ME, and the pituitary stalk, which physically connects the hypothalamus and the pituitary gland (Fig. 2-1C). The endocrine part of the pituitary gland derives from the most anterior segment of surface ectoderm. When head development begins and the neuroepithelium expands to form the brain, the anterior neural ridge is

displaced ventrally and ultimately occupies the lower facial and oral area. The midline portion of the oral ectoderm invaginates to give rise to the pituitary anlage, the so-called Rathke pouch, which appears at around day E8.5 in mice and detaches from the ectoderm by E12.5 (1–3). In humans, pituitary organogenesis begins at week 4 of fetal development and it ultimately separates altogether from the oral epithelium at week 6 to 8. Thus, the pituitary is an organ of dual origin: the anterior lobe (adenohypophysis) is derived from the oral ectoderm and is epithelial in origin, whereas the posterior lobe (neurohypophysis) derives from the neural ectoderm (32,33). The complex nature of the pituitary requires that the neural and oral ectoderm interact physically. Precise spatial and temporal coordination and regulation of signals from both structures is critical for pituitary formation and for the differentiation of the various hormone-producing cell types in the anterior lobe. Thus, corticotropes secrete adrenocorticotropic hormone (ACTH), thyrotropes secrete TSH, somatotrophs secrete growth hormone (GH), lactotrophs secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and melanotropes secrete melanocyte-stimulating hormone (MSH) (3,4,34). All these cells arise from progenitors in Rathke pouch in a precisely orchestrated spatial and temporal fashion during pituitary development. Accordingly, multiple signals regulate progenitor cell proliferation, positional determination, lineage commitment, cell-fate specification, and terminal differentiation (Fig. 2-2A). Indeed, the development of the pituitary provides an instructive model system for elucidating the molecular mechanisms by which different cell types arise from a common progenitor lineage in response to multiple extrinsic and intrinsic signals. These pathways are specific and secrete proteins that control differentiation and function of recipient cells in a paracrine and/or autocrine manner. Tight and concerted actions of these signals lead to precise cell fates and to the formation of distinct cell populations in the developing pituitary, while in the adult pituitary they regulate proliferation and hormonal secretion of pituitary cells.

Morphogenic Signals Involved in Early Steps of Pituitary Development and Cell Specification

The initiation of pituitary organogenesis is a multistep process initiated by formation of Rathke pouch as an invagination of the oral ectoderm, and a subsequent evagination of the ventral neuroectoderm, which are dependent on morphogenetic signals from the prechordal plate/notochord such as the Notch pathway. The signals that control pattern formation in the anterior pituitary primordium comprise a complex of morphogenetic factors originating in the ventral diencephalon/infundibulum and in Rathke pouch itself. These signals are essential for the stratification of Rathke pouch, cell migration, proliferation and specification, and for the spatial and temporal organization of endocrine cell types. In this respect, the most studied morphogenic signals are BMPs, Fgf, SHH and Wnt factors (35–37).

Notch Pathway The Notch signaling pathway is an evolutionary conserved cell signaling mechanism in development. Ligands and receptors of Notch signaling are cell surface proteins that mediate cell–cell communication (38,39). Briefly, the membrane-bound ligands, Delta-like or Jagged, activate the transmembrane receptor Notch on neighboring cells. Binding of the ligand to the Notch receptor triggers the cleavage of the Notch intracellular domain, which translocates to the nucleus and displaces transcriptional repressors when the pathway is inactive. This permits the transcriptional activation of targets such as the Hairy enhancer of split (Hes), a member of the bHLH family of transcription factors (40). Thus, an imbalance between secretion of the ligand and activation of the receptor causes a cell selection process. The Notch pathway is required in the embryo in different contexts and its dysregulation is associated not only with inherited and degenerative diseases, but also with cancers in humans.

FIGURE 2-2. Schematic diagram of the development of the hypothalamic– pituitary axis showing the main signals (A) and transcription factors (B) involved in pituitary organogenesis in mice. A: At embryonic day E7.0, the anterior neural ridge (ANR), the primordial endocrine hypothalamus, and the primordial anterior pituitary are present. The signals originating on E9.5 in the ventral diencephalon include Wnt5a, BMP4, and Fgf-8/10/18. At E10.5, the mesenchyme makes direct contact with the oral ectoderm and induces the formation of Rathke pouch (RP). The ventral signal Shh and the opposing dorsal Fgf and ventral BMP2 gradients express proliferative and positional cues by regulating combinatorial patterns of transcription factor gene expression. All these signals control the expression of specific transcription factors involved in the commitment of cell lineages at E11.5. Cell types later arise in a temporally and spatially controlled fashion starting on E12.5 (corticotrophs [C], at E13.5 rostral thyrotropes [Tr]), and subsequently from E15.5 to E17.5 (somatotrophs [S], lactotrophs [L], thyrotropes [T], gonadotropes [G], and melanotropes [M]). B: Temporal expression of the main transcription factors controlling specification of pituitary cell types and organogenesis. The functions of a number of signaling molecules, transcription factors, and cofactors regulating lineage commitment and terminal differentiation of distinct cell types are delineated in a genetic pathway. (Modified from Zhu X, Wang J, Ju BG, et al. Signaling and epigenetic regulation of pituitary development. Curr Opin Cell Biol 2007;19[6]:605–611.)

The role of the Notch pathway in early ventral diencephalon formation is not fully understood, but it is required for infundibular morphogenesis and for maintaining pituitary progenitors. In addition, Hes1 is expressed throughout the ventral diencephalon and Hes null mutants display reduced evagination and loss of the pituitary posterior lobe. Several members of the Notch pathway are expressed in Rathke pouch becoming largely restricted to progenitor cells during the progress of gland development. For example, the Notch pathway directly activates the expression of the transcription factor Prop1, which is essential for the emergence of Pit1 precursor cells (see later). This signal also plays a role in specifying the intermediate and posterior lobes of the pituitary gland. The upregulation of Notch pathway components results in a global impairment of endocrine differentiation. Accordingly, Notch signaling appears to be required early to prevent differentiation, ensuring the generation of enough progenitors, and later to promote the emergence of the Pit1 lineage. Thus, the attenuation of Notch signaling in Pit1 precursor cells is necessary for the emergence of distinct Pit1 lineages and for ensuring proper terminal differentiation (38,40). Overexpression of Notch in thyrotropes and gonadotropes leads to defects in their differentiation (41).

Bone Morphogenetic Proteins BMPs are secreted ligands that, upon binding to receptor serine–threonine kinases, initiate an intracellular signaling cascade resulting in activation of SMAD signaling and the regulation of gene transcription. BMPs are involved in multiple events in the embryo and dysregulation of the pathway is linked to several diseases. BMP4 is the earliest signaling molecule known to be extrinsically required for Rathke pouch invagination and development (42). It is expressed in the overlying ventral diencephalon at E8.5–9 and subsequently in the infundibulum where its levels ultimately decline (Fig. 22A). Blockade of BMP4 signaling results in the arrest of pituitary gland development and in the loss of all pituitary endocrine cell types (35,36). Studies in different mouse models have demonstrated that BMP4 signaling is critical for the continued progression of pituitary development because it controls the expression of different pituitary transcription factors. BMP2 controls the expression of ventrally expressed transcription factors

and the expression of the gene for the α-subunit of the glycoprotein hormones, which is an early marker of cells destined to become thyrotropes and gonadotropes. These data suggest that BMP2 signaling specifies the progenitors that will later give rise to the ventral pituitary cell types, providing a ventralizing positional cue for pituitary development (35).

Fibroblast Growth Factors Fgfs are secreted molecules that bind to receptor tyrosine kinases resulting in activation of multiple signaling cascades through MAPK, PI3K and phospholipase C, affecting cell proliferation, survival and motility, respectively. During Rathke pouch development, Fgf8, -10, and -18 are present in the ventral diencephalon and in the posterior lobe of the pituitary, appearing later than BMP4—from E9.5 until at least E14.5. Fgf8 and Fgf10 control pituitary proliferation and positional-restricted determination of pituitary cell lineage (35,43). Deletion of Fgf10 or its receptor Fgfr2IIIb results in the formation of a small apoptotic pouch and agenesis of the anterior pituitary, which suggests that Fgf10 signaling, is essential for cell survival. Inactivation of Fgf8 causes early lethality at E8.5 before pituitary development whereas its overexpression leads to increased Lhx3 expression and pituitary hyperplasia. The function of Fgf8 is to maintain proliferation and to oppose the ventral BMP2 differentiation signal. The requirement for Fgf8 has been further suggested by studies in mice lacking Nkx2-1, in which the pituitary is completely absent at birth (26). In Nkx2-1 mutant mice, BMP4 expression is maintained in the diencephalon, but Fgf8 expression is undetectable. These data suggest that Rathke pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of BMP4 homozygous null mutant embryos. Second, Fgf8 is necessary for activation of the key regulatory genes Lhx3 and Rpx (Rathke pouch–specific transcription factor), leading to the progression of the rudimentary pouch to a definitive pouch (42).

Sonic Hedgehog SHH is a secreted factor that has been described as a morphogen in the

embryo and as a mitogen, in particular for adult stem cells. Functionally, SHH binds to its receptor Patched, which results in the derepression of the co-receptor smoothened (Smo) and in the subsequent activation of intracellular glioma-associated transcription factors (Gli), which bind to SHH target genes and switch them on. In the absence of SHH, Patched inhibits Smo, and Glis are converted to repressors to silence target gene expression. Dysregulation of SHH signaling results in holoprosencephaly, a developmental anomaly of the forebrain characterized by severe midline defects. In addition, Shh ablation results in the absence of Rathke pouch. However, this effect seems to be a consequence of a general defect of midline structures including the diencephalon/infundibulum rather than a primary defect in the anterior pituitary primordium itself. During mouse pituitary development, Shh is expressed in the ventral diencephalon and the oral ectoderm but not in the region that forms Rathke pouch (44). Within the anteroventral domain, Shh is required for maintenance of the prospective hypothalamus. In the absence of Shh in the ventral diencephalon, the infundibular BMP4 and Fgf10 patterns are expanded ventrally, resulting in an anterior shift and in the duplication of Rathke pouch. Conversely, when the Shh domain is expanded dorsally, Fgf8 and BMP4 are still expressed while Fgf10 is downregulated in the infundibulum where more proliferation is observed. Moreover, evagination fails and consequently Rathke pouch does not progress beyond E10.5. Overall, these studies suggest an antagonism between BMP and Shh signaling in the ventral diencephalon that is required for infundibular morphogenesis and hence Rathke pouch positioning and development. Shh may also act in a signaling cascade with BMP2 in the determination of ventral pituitary cell lineages (Fig. 2-2A). The Shh downstream transcription factors Gli1, Gli2, and Gli3 are expressed in the ventral diencephalon and developing Rathke pouch. Gli1 and Gli2 inactivation results in loss of the pituitary.

Wnt Pathway The secreted extracellular Wnt molecules have important signaling roles in the control of embryonic patterning, cell fate determination and homeostasis. In the adult, the Wnt pathway regulates the proliferation of different stem cell

populations and, not surprisingly, its dysregulation causes many diseases including cancer. The canonical Wnt pathway is characterized by the central role of its intracellular mediator, β-catenin. Activation of the canonical Wnt/ β-catenin pathway stabilizes β-catenin protein, which subsequently translocates to the nucleus and functions as a co-activator of DNA-binding transcription factors, such as Lef/Tcf, by displacing HDAC and transducinglike enhancer protein (TLE/groucho) co-repressor complexes and recruiting p300/CBP and Brahma-related gene 1 (Brg1), which stimulate the expression of target genes (45,46). A second Wnt-activated pathway regulates planar cell polarity affecting cell morphology and movement, and a third pathway is the related noncanonical Wnt pathway, which is associated with intracellular calcium release. The Wnt/β-catenin pathway is required for Pit1 lineage determination and pituitary gland growth. Accordingly, the Wnt/β-catenin pathway is active between E11.5 and E15.5 in the pituitary as illustrated by the expression Axin2, a direct downstream target gene. Temporal control by Wnt/β-catenin signaling is essential for correct pituitary development, as premature activation of β-catenin leads to Hesx1 repression (see later) and in consequence, pituitary gland agenesis at E13.5 (47). Other components of the Wnt/β-catenin signaling pathway, including frizzled-2 (Fzd2), Lef1, Tcf3, and Tcf4, are expressed during pituitary development. Fzd2 is detectable at E12.5 in Rathke pouch and the infundibulum; Tcf3 is detectable between E9.0 to E14.5 and is restricted to the Pit1-expressing caudomedial region of the gland; Tcf4 is detectable in the early pituitary in ventral diencephalon and Rathke pouch as well as in surrounding tissues, and is markedly decreased at E13.5; Lef1 has a biphasic expression—transiently at E9.0 in Rathke pouch, and later reappearing in the anterior and intermediate lobes of the gland at E13.5. Inactivation of Tcf4 results in hyperplasia of the anterior lobe, whereas inactivation of Lef1 leads to elevated expression of Pit1 as well as GH and TSH-β, indicating a role for Lef1 in inhibiting Prop1/β-catenin–mediated Pit1 activation (47,48). The Wnt family member Wnt4a is expressed transiently and exclusively in the developing pituitary on E12.5 and is specifically associated with the early development of the anterior pituitary, illustrated by its expression in Rathke pouch until E14.5. Mice lacking Wnt4 have a hypocellular pituitary gland that shows normal differentiation but incomplete ventral expansion (47), causing

a modest reduction in the expression of Pit1, which is required for the appearance of somatotrophs, lactotrophs, and thyrotrophs. Expression of noncanonical Wnt5a is present throughout the ventral diencephalon and Rathke pouch from E9.5 to E12.5 and is necessary for correct patterning and indirectly affects shaping of Rathke pouch. In the absence of Wnt5a, infundibular signaling is ectopically expanded ventrally, resulting in a greater amount of oral ectoderm recruited to form Rathke pouch and the appearance of extrabifurcations. Moreover, its ablation results in a similar phenotype as that seen in Tcf4 deficient mice. Both proteins are likely independently involved in patterning within the ventral diencephalon and hence influence the positioning of Rathke pouch. Wnt5 and BMP4 induce the expression of the α-subunit gene of glycoprotein hormones, suggesting that both signals may act in synergy to expand pituitary cell lineages of the αsubunit gene and induce cell determination (49,50). All morphogens and growth factors that contribute to the development of the hypothalamus–pituitary axis are important for thyroid development and function, since pituitary TSH is the main regulator of thyroid gland function.

Transcription Factors Controlling Pituitary Development and Cell-Type Specification The appropriate expression of several transcription factors is essential for the proper development of Rathke pouch: some are also present in the ventral diencephalon where they are necessary for infundibular morphogenesis and therefore indirectly for Rathke pouch morphogenesis. These transcription factors are expressed in a hierarchical order (Fig. 2-2B).

Pituitary Homeobox Transcription Factors (Pitx) The paired homoeodomain Pitx proteins are important morphogenetic regulators for different organs. They are also recognized for their role in left– right asymmetry. Three related Pitx transcription factors have been identified: Pitx3 is critical for the proper development and survival of mesodiencephalic dopaminergic neurons in mammals, whereas Pitx1 and Pitx2 have overlapping, but distinct expression patterns during pituitary development (2,4,51). Both Pitx1 and Pitx2 are required for cell proliferation, survival and

differentiation in a dosage-sensitive manner. Pitx2 plays a more prominent role than Pitx1 because Rathke pouch development is absent in Pitx2 null mutants, but normal in Pitx1 null mutants. Both factors function redundantly and are required for maintenance of early Rathke pouch progenitors and later for the maintenance of differentiated endocrine cells. In addition, both control the expression of Lhx3, which is required for progenitor maintenance. Pitx1 interacts with the aminoterminal domain of the pituitary-specific POUdomain protein Pit1 (see later) and is expressed in the earliest stages of pituitary organogenesis in the anterior region of the neural plate, the oral ectoderm. Targeted disruption of Pitx1 leads to the decreased expression of terminal differentiation markers for gonadotropes and thyrotropes (52). PITX2 mutations are associated with Axenfeld–Rieger syndrome, mainly characterized by eye defects and also pituitary abnormalities (53). Both Pitx1 and 2 are expressed in most pituitary cell types, from early to late pituitary ontogeny, and also in other developing organs. Pitx2 is a target of Wnt signaling and induces progenitor proliferation through direct transcriptional activation of cyclins. Proper Pitx gene dosage is required throughout development for the generation and/or maintenance of endocrine cell numbers. These transcription factors bind within the promoters of the αsubunit of glycoproteins TSHβ, LHβ, FSHβ, GnRHR, PRL, and GH. Furthermore, Pitx1 and Pitx2 are also redundantly required postnatal for thyrotrophic function (54). In conclusion, Pitx1 and Pitx2 are required for pituitary morphogenesis and for endocrine function, reflecting their many interacting partners and, in consequence, target genes.

LIM Homeodomain Proteins The LIM homeodomain (LIM-HD) proteins have two tandemly repeated LIM domains located between the N-terminus and the DNA-binding homeodomain. The LIM domain provides a protein–protein interaction interface that recruits cofactors mediating LIM-HD functions (55). Multiple members of the LIM-HD family of transcription factors are expressed in Rathke pouch, including Lhx2, Lhx3, Lhx4, and Isl1. Lhx2 is expressed throughout the ventral diencephalon during Rathke pouch development and is required for the infundibulum and, consequently, for posterior lobe formation. Lhx3 is specifically expressed in Rathke pouch at E9.5 in mice

and, together with Lhx4, plays a decisive role in the earliest phases of pituitary organogenesis. Lhx4 is subsequently restricted to the future anterior lobe and then downregulated at E15.5, while Lhx3 is maintained throughout in the adult gland. The two proteins initially function redundantly, as shown by gene deletion studies where increased apoptosis is a major consequence. However, whereas Lhx4 null mice have hypoplastic pituitaries containing all endocrine cell types, Lhx3 null mutants are deficient in all cell lineages. This reflects the requirement for Lhx3 in the activation of several pituitary hormones, hypothalamic peptide receptors and transcription factors (for review see [56]). Additionally, expression of Notch2 is absent in Lhx3 mutants and this could underlie some of the differentiation defects seen in these mice. Therefore, Lhx3 and Lhx4 are initially required for progenitor maintenance rather than proliferation—and later Lhx3 is required also for proper endocrine differentiation. The similarity of the phenotypes of Pitx2 and Lhx3 null mice suggests that these two classes of homeodomain factors collaborate to regulate the same pituitary-specific genes. Indeed, both factors act synergistically to activate the expression of the α-glycoprotein subunit gene (57). In summary, the induction of Lhx3 expression in response to infundibular Fgf signals is a critical step in the selection of the oral ectoderm for development into the pituitary gland, and it acts synergistically with Pitx2 to direct pituitaryspecific gene expression. Decreased expression of the bHLH transcription factors Mash1 and Mash3 accounts for some of the defects induced by ectopic Notch activation (39). Mash1 executes roles in the terminal differentiation of thyrotropes, gonadotropes, corticotropes, and melanotropes. The expression of Mash3 in the pituitary is regulated by Pit1, begins at E13.5, is maintained in adulthood, and regulates the response to GHRH. Insulin gene enhancer protein (Islet 1) is a LIM-HD transcription factor essential for cell fate decisions in different embryonic and stem cell contexts. Isl1 is initially expressed in the oral ectoderm at E8.5 and then in Rathke’s pouch at E9.5/E10.5 is restricted to the most ventral region of the pouch where it co-localizes with the α-subunit of glycoproteins in the rostral thyrotrope cells. In the adult gland, Isl1 expression is maintained in gonadotropes, where it is proposed to participate in Lhx3 activation together with Pitx1 (58). The expression of Isl1 is activated by BMP4 and BMP2 and is repressed by Fgf8/Fgf2, two opposing ventral/dorsal signals that are critical

for pituitary development. Isl1 null mice die at E10 with multiple defects in the heart, pancreas, and motor neurons. Moreover, the primitive pouch forms in Isl1 null mice at E9.5, but with an aberrant epithelium, suggesting that Isl1 is necessary for proliferation and differentiation of pituitary progenitors.

Homeobox Expressed in Embryonic Stem Cells 1 (Hesx1) Hesx1 is a member of the paired-like class of homeodomain genes. It is first expressed in the prechordal plate precursor and later becomes restricted to the ventral diencephalon and Rathke’s pouch at E9.5 (2,4). Hesx1 is a transcriptional repressor with two repressor domains in the N-terminal and in the homeodomain regions. The homeodomain recruits the NcoR/Sin3/HDAC corepressor complex and the N-terminal eh1 motif interacts with the Groucho-related TLE corepressor. This association is critical for Hesx1 function, as temporal regulation of the Hesx1/Groucho (TLE) complex is fundamental for pituitary organogenesis (59). Expression in the developing pituitary persists until E12, when Hesx1 is downregulated in a spatial and temporal manner that coincides with the increase of Prop1 transcripts and progressive differentiation of pituitary-specific cell types. Hesx1 expression is regulated by Lhx-3 and Prop1. Genetic ablation of Hesx1 leads to postnatal lethality caused by central nervous system defects, retarded prosencephalon development and pituitary dysplasia. Loss of Hesx1 in the anterior neuroectoderm results in posteriorization due to lack of repression of the Wnt/β-catenin pathway. Despite these defects, endocrine cell differentiation occurs normally in Hesx1 mutants, but with an increase in cell numbers. Hesx1 is therefore an important regulator of early pituitary development and its interaction with Prop1 regulates Rathke pouch development. Human HESX1 mutations are associated with septo-optic dysplasia, a rare congenital syndrome characterized by variable central nervous system midline defects and hypopituitarism. The Rathke pouch factor Rpx is a homologue of Hesx1. Its expression is restricted to the oral ectoderm and to Rathke pouch. The attenuation of Rpx/Hesx1 expression by different corepressors coincides with the appearance of terminal differentiation markers for anterior pituitary cell types, suggesting that downregulation of this gene is required for progression of pituitary development. Rpx can dimerize with Prop1 and inhibit its

activity, suggesting that Rpx/Hesx1 acts to antagonize Prop1 function (60).

Paired-Like Homeobox 1 (Prop1) Prop1 is a transcription factor essential for pituitary development and is the earliest exclusive marker of pituitary identity. It was first identified by positional cloning in Ames dwarf mice and was named Prophet of Pit1 (Prop1). The protein has a C-terminal transactivator domain and an Nterminal repressor domain (4,43,51), suggesting that Prop1 function is both an activator and a repressor. In agreement with this notion is the finding that the Prop1/β-catenin complex acts as a transcriptional activator of Pit1 and as a transcriptional repressor of Hesx1, depending on the associated cofactors. The onset of Prop1 expression coincides with the closure of Rathke pouch at E10.5. Prop1 expression peaks at E12.5 followed by its downregulation at E15.5 to E16.5, which is the time of terminal differentiation of pituitaryspecific cells. Notch signaling is required for maintaining high levels of Prop1 expression at E12.5. Prop1-deficient mice do not express Pit1, and consequently lack somatotrophs, lactotrophs, and thyrotropes. Prop1 is also required for the generation of gonadotropes and corticotrophs, indicating that it is important for the expansion of all anterior pituitary cell lineages. Mutations in human PROP1 are the cause of combined pituitary hormone deficiency (reviewed in [56]).

Sine Oculis Homeobox (Six) Sine oculis homeobox transcription factors are a family of homeodomain proteins encoded by the genes Six1, Six3, Six4, and Six6 and are expressed in the developing pituitary. They are characterized by two evolutionary conserved domains: the SIX domain is involved in protein–protein interactions and the HD is involved in DNA-binding. They can positively or negatively regulate transcription by interacting with the Eye-absent (Eya) transcriptional activators or the Groucho/TLE and Dach co-repressors, respectively (61). Six1 and Six4 are co-expressed in many embryonic primordium structures including Rathke pouch, although pituitary development is not impaired in Six1 or Six4 null embryos. Six3 promotes cell proliferation by repressing Wnt and BMP signaling and specifying anterior identity. As regulators of proliferation, they are also involved in

tumorigenesis. Deletion of Six3 is deleterious and mutants arrest before pituitary development is initiated. Because of this, later functions of the gene are not known. Six6 is expressed in a dorsoventrally gradient in the early stages of pituitary development, but its expression decreases at E13.5. Six6 null mice have pituitary and retina hypoplasia with a reduced number of terminally differentiated cells due to defects in precursor cell proliferation. It has been suggested that Six6 promotes proliferation of progenitors by repressing the cell cycle negative regulator p27Kip1, and such an interaction has been demonstrated to underlie the Six6 null retinal phenotype.

Paired-Domain Factor Pax6 Paired-domain factor pax6 is important in the early development of Rathke pouch. Pax6 is transiently expressed in the dorsal part of the pouch and is downregulated when cell-type differentiation starts. Thus, Pax6 is expressed early in the anterior neural plate that will become the telencephalon, diencephalon, eyes, and pituitary. During pituitary development, Pax6 is expressed in the oral ectoderm at E9.0 but is excluded from the Shhexpressing region. It is detected in Rathke pouch at E10-E12 with an apparent dorsal/ventral gradient and is downregulated at E13.5, disappearing when cells reach terminal differentiation at E17.5. In Pax6 null mice, the pituitary gland presents an expansion of the ventral cell types that express αglycoprotein subunits, predominantly thyrotropes, with a corresponding loss of the more dorsal somatotrophs and lactotrophs. Thus, Pax6 is required for delineating the dorsal/ventral boundaries between the thyrotrope/gonadotrope and the somatotroph/lactotroph progenitor regions of the pituitary gland (62,63) and reviewed in (3).

Retina and Anterior Neural Fold Homeobox Protein (Rx/Rax) Rx/Rax is a paired-like homoeodomain protein expressed early in the anterior neural plate that plays a significant role in eye morphogenesis. Rx/Rax null mutants lack part of the forebrain. In addition, Rx/Rax is expressed in ventral diencephalon and its deletion causes abnormal morphogenesis, with the absence of infundibular evagination and downregulation of Fgf10 expression from E10.5. Accordingly, Rathke pouch fails to develop beyond this stage and remains fused to the oral ectoderm (64). It is also important for later

hypothalamic morphogenesis (65).

Sox Transcription Factors (Sox2 and Sox3) The Sox family (Sry-related HMG box) of transcription factors contain a conserved high-mobility group (HMG) box domain and show high sequence similarity to the sex-determining-region on the Y-chromosome gene (SRY), the founding member of the Sox gene family. Sox2 and Sox3 are expressed in central nervous system progenitors, where they promote an undifferentiated proliferative state (66). Sox2 is also required for pluripotent cell types in the early embryo, and homozygous Sox2 null mouse embryos die around implantation. In humans, heterozygous SOX2 loss-of-function mutations are associated with anophthalmia (67) and hypogonadotrophic hypogonadism, whereas deletions and duplications of SOX3 are linked to mild hypopituitarism. In the ventral diencephalon these two factors activate the same targets—Shh, Six3 and Six6 (68). In the absence of Sox3, the infundibulum does not evaginate fully and cells proliferate less, Fgf8 and BMP4 domains are expanded anteriorly, and Rathke pouch is bifurcated, as explained earlier. Sox3 and Sox2 null mutant mice are affected by mild hypopituitarism and the gland is hypoplastic. In the case of Sox3, this reflects its role in the hypothalamus where its expression is maintained, while Sox2 is additionally required in Rathke pouch. Loss of Sox2 also significantly affects the number of gonadotropin-releasing hormone (GnRH) neurons; therefore, the hypogonadotropic hypogonadism found in patients with SOX2 mutations is likely to be of hypothalamic origin (69). Sox2 is present in Rathke pouch from E9.5 and its expression is maintained with another Sox member, Sox9. Deletion of Sox2 in Rathke pouch at E12.5 results in the reduction of proliferation and, as a consequence, endocrine cell deficits in particular somatotrophs. Furthermore, expression of the transcription factors Prop1 and Pit1 is downregulated in Sox2 mutant embryos.

Transcription Factors Controlling Specification of Different Pituitary Cell Types As discussed earlier, Pit1 is a member of an evolutionary conserved family of POU domain-containing transcription factors consisting of an N-terminal

POU-specific domain and a C-terminal POU-homeodomain separated by a short linker. Both subdomains contain helix-turn-helix motifs with a high DNA-binding affinity. Also referred to as GHF1, Pit1 was originally identified through analysis of the nuclear proteins regulating the transcription of GH and prolactin (PRL). Later, Pit1 was found to be required for the generation and cell-type specification of three pituitary cell lineages: somatotrophs, lactotrophs and thyrotropes, as well as for repression of gonadotropes. Pit1 binds to the promoter region of the genes encoding GH, PRL, the β-subunit of TSH, GHRH receptor, the type 1 somatostatin receptor, and the TRH receptor, where it interacts with other transcription factors to form functionally active heterodimers. Pit1 also interacts with various members of the nuclear-receptor family including thyroid hormone receptors and retinoic acid receptors. The early activation of Pit1 at E13.5 requires the actions of Prop1 and Wnt/β-catenin signaling. In humans, mutations in PIT are associated with congenital defects (56). The Pit1-dependent cell lineage, the thyrotropes, share some common features with the non–Pit1-dependent gonadotropes. Both arise from the ventral portion of the gland and secrete heterodimeric hormones containing the common α-glycoprotein subunit and specific β-subunits. These two cell lineages diverge at an early stage of development. Their plasticity is dependent on Pit1 as has been demonstrated in several genetic models where gonadotropes can be converted into thyrotropes and vice versa. This stresses the critical function of Pit1 in lineage choice. The zinc finger transcription factor GATA2, whose expression is under the control of the ventral-to-dorsal BMP2 gradient, interacts with Pit1, with this interaction critical for the development of thyrotropes and for the activation of thyrotrope-specific genes such as the gene encoding the β-subunit of TSH, which ultimately regulates thyroid function. In gonadotropes, Pit1 inhibits GATA2 binding to promoters not containing an adjacent Pit1 site. In Snell dwarf mice, which have a mutated Pit1 gene, Pit1 and GATA2 do not interact, and the thyrotropes assume the fate of gonadotropes (reviewed in [3,4]). Taken together, these studies indicate that there are extrinsic and intrinsic signaling mechanisms that govern the early and late aspects of the development of the hypothalamus and the pituitary gland (Fig. 2-2). Coordination between signaling molecules and between transcription factors

is necessary for the early patterning, proliferation, and positional determination of pituitary cell types, including the thyrotropes.

THE THYROID GLAND In mammals, the thyroid gland emerges from a diverticulum of the primitive pharynx. It is located in the neck region and consists of two lobes connected by an isthmus with the shape of a butterfly rather than a shield, as its name, which derives from the Greek word shield or “thureos,” would suggest. The thyroid synthesizes and secretes thyroid hormone from the epithelial or follicular cells and calcitonin from the parafollicular or C cells. For many years, it was accepted that these two thyroid cell types derived from endoderm and neural crest, respectively (70,71). However, it is currently accepted that both are endodermal in origin (72). Accordingly, the C-cell precursors arise from the ultimobranchial bodies, which derive from the ventral recess of the fourth pharyngeal pouch (72,73) and constitute a minority cell population that is neuroendocrine in nature. By contrast, the epithelial cells arise from a midline anlage in the pharyngeal floor and are the most numerous cell population. The developed thyroid is rich in capillaries surrounding the follicles that provide systemic delivery of secreted hormones. The stroma, which encapsulates and separates the tissue, is formed from mesenchyme fibroblasts derived from the neural crest. Finally, other interstitial cells can be found, such as macrophages and mast cells, which play functional roles in thyroid cancer (reviewed in [74]).

FIGURE 2-3. Frontal view of the stages of thyroid organogenesis: Its correlation with the timing of relevant events in humans and mice development. The anlage first appears as a group of cells located on the floor of the primitive pharynx (upper panel), which then forms as a visible bud on E20–22 in humans and E8–8.5 in mice. The bud proliferates ventrally, and then expands laterally, forming the characteristic bilobed structure of the thyroid gland. Caudal migration occurs between E24 to E45–50 in humans and from E9.5 to E13.5 in mice, reaching the thyroid its final position. The association between the medial and lateral (C cells) thyroid anlage with an isthmus connecting the two lateral lobes appears on E70 in humans and E15.5 in mice. At that time begins the folliculogenesis. (Modified from Van Vliet G. Development of the thyroid gland: lessons from congenitally hypothyroid mice and men. Clin Genet 2003;63:445.)

In this section, we will focus on the epithelial cells that form the thyroid

follicles, spherical structures that are essential for thyroid hormone synthesis and serve as storage units for thyroid hormones which allow the controlled release of hormones. Thyroid epithelial cells phylogenetically derive from the primitive iodide-concentrating gastroenteric cells in an area named the endostyle, which during evolution migrated and specialized in the uptake and storage of iodine in follicular cellular structures. This enabled organisms from an iodine-rich sea environment to adapt to an iodine-deficient land environment (75).

THYROID BUD FORMATION The thyroid gland in mammals is the most anterior organ developing from the foregut endoderm, emerging as a thickening in the ventral wall on the primitive pharynx floor (Figs. 2-3 and 2-4). It is the first endocrine structure that becomes recognizable in humans. This thickening primordium, termed anlage in embryology, forms a placode in the midline of the pharyngeal floor in the embryonic mouth cavity in its posterior part, just behind the prospective tongue, between the first and second branchial arches (Fig. 24A). The anlage emerges as a visible bud at E20–22 in humans and at E8–8.5 in mice, in a process termed specification or determination that in developmental biology occurs in parallel to morphologic and biochemical changes that make these cells clearly different from their neighboring cells. Thickening in a restricted region of the embryonic cell layer is a common event in the initiation of organogenesis and is essential for the generation of signals required for this process to continue. The initial thickening expands and gives rise to a small pit, the thyroid bud, at E8.5–9 in mice; this is followed by outpouching from the endoderm adjacent to the newly differentiating myocardium (Fig. 2-4A).

FIGURE 2-4. Sections of human embryos showing thyroid development. A: Section through a 2-somite embryo (×150). B: Section through the thyroid anlage of a 10-somite embryo (×15).

Surprisingly little is known about the factors that are necessary to trigger thyroid lineage development in the foregut endoderm in vivo. In contrast to the requirements of other foregut derivatives, retinoic acid (RA) is not necessary for early thyroid specification and development (76). As the ventral pharyngeal endoderm lies in close apposition to the cardiogenic mesoderm, it has been proposed that signals originating in the heart primordium may be responsible for thyroid cell specification. The existence of cardiogenic signals has been shown for other endoderm-derived organs—such as liver and pancreas (77). In addition, defects in the foregut secondary to defects in heart organogenesis have been described (78), and cardiac malformations represent the most frequent birth defects associated with congenital hypothyroidism (79). Altogether, these observations suggested a role for neighboring cells in thyroid specification, with the primitive cardiac cells being the most likely candidates. Recently, some of the factors involved in thyroid specification have been identified through in vitro studies of mouse embryonic stem cells (mESCs) and iPS cells (See below in this chapter “mESCs and iPS”). Among these factors, Fgfs and bone morphogenic proteins (BMPs) are inductive signals of thyroid fate. Indeed, patterns of thyroid differentiation have been observed after treating foregut explants with Fgf2 and, in zebrafish, Fgf1, Fgf2 and Fgf8 redundantly rescue thyroid development in mutants lacking thyroid (80). Additionally, Fgf2 and BMP4 are necessary and sufficient to induce differentiation of mouse and hESCs and human iPSCs into functional thyroid cells (81), whereas inhibition of either Fgf or BMP signaling blocks thyroid development.

These data are in overall agreement with previous observations that mice deficient for Fgfr2b (82) or Fgf10 (83) present athyreosis, although the putative role of Fgf10 in thyroid specification has been recently questioned since Fgf10 knockout mice have a unilateral thyroid rudiment (84) or display symmetric hypoplasia (85). Other data concerning the key role for Fgfs in initiating thyroid fate come from ex vivo studies of mouse endoderm, focusing on the role of cardiogenic mesoderm in lung specification (86). All these data suggest that cardiac mesoderm is the most important source of thyroid inductive signals and are in accord with the previous notions that thyroid specification and consequently budding formation is TSHindependent (87). Clearly, there are likely additional factors that are important for the specification process that remain to be identified. The Notch pathway seems to be involved in early thyroid development, as its disruption appears to limit the number of progenitor cells forming the anlage. It is important to note that morphogenic signals such as Wnt are not involved, as thyroid development is not impaired in mice deprived of Wnt2, Wnt2b, or β-catenin (88). Similarly, Shh is not involved in the specification process, as Shh−/− mice present a phenotype with thyroid hypoplasia and hemiagenesis (89).

THYROID MIGRATION The formation of the thyroid anlage is first evidenced close to the base of the tongue. Then, appears the thyroid bud close to the apical pole of the aortic sac. Later, the thyroid follicular progenitor cells proliferate and start to migrate downward (caudally) from the thyroid primordium to their pretracheal position at E24 in humans and at E9.5 in mice. The first signs of proliferation are observed within the primordium between E10.5 and 11.5. At this stage, the embryonic thyroid is surrounded by mesenchyme; however, in contrast to the important role of mesenchyme-derived signals in other endoderm organs, the potential influence of mesoderm on thyroid proliferation at this developmental stage is largely unknown. Interestingly, the expression of the transcription factor Tbx1 in a population of mesoderm cells close to the migrating thyroid suggests that Tbx1, through Fgf8 or other soluble factors, might stimulate the propagation of thyroid progenitors also

after leaving the endoderm (90). The fact that the hypoplastic thyroid phenotype in Shh−/− mice is similar to that of Tbx1 mutant embryos suggests that Shh gradients produced by pharyngeal endoderm exert distant effects that directly or indirectly promote later proliferation of thyroid progenitors (reviewed in [74]). Subsequently, the thyroid bud expands ventrally as a diverticulum, with a rapid proliferation of the cells at its distal end (Fig. 2-3), but it remains attached to the pharyngeal floor by a tubular stalk called the thyroglossal duct (Fig. 2-3). This structure fragments and degenerates between E30–40 in humans and at E11.5 in mice, and subsequently the primitive thyroid loses its connection with the floor of the pharynx. The remnant of the anlage in the floor of the primitive pharynx is termed the foramen cecum, a small hole located at the base of the tongue at the site of the thyroid’s origin in the pharyngeal floor. During migration, thyroid cells proliferate laterally, which leads to the process of bifurcation in which the thyroid tissue spreads out bilaterally along the pharyngeal arch arteries. In vertebrates, thyroid bilobation also involves fusion with the ultimobranchial body, which merges with each of the lateral thyroid lobes. The thyroid reaches its definitive position in the trachea and migration is completed at E45–50 in humans and at E13.5 in mice (Fig. 2-3). Once the thyroid reaches its final position, the thyroid lobes expand and the gland obtains its definitive form, with a narrow isthmus connecting the two lateral lobes (Fig. 2-3). The mechanism leading to cell proliferation during thyroid lobe expansion and to the formation of symmetrical thyroid lobes remains unknown. TSH signaling, the major thyroid proliferation stimulus for adult thyroid cells, appears not to be involved as the TSHR is not yet expressed (87,91) (and reviewed in [74,92]). It is anticipated that more information about the origin of these signals will be obtained through the generation of new animal models. The mechanism leading to thyroid migration is complex and still not well understood. It has been suggested that the thyroid progenitor cells themselves have an important role in this mechanism (93), suggesting an active rather than passive migration. However, this concept has been questioned by the observation that thyroid precursor cells express classical epithelial differentiation markers, such as cadherins, rather than markers related to epithelial mesenchymal transition (EMT) (94), which is necessary for cell

migration in other systems. Currently, the underlying concept is that thyroid migration is due both to an active process, by a mechanism not yet identified, and to mechanical traction through the close association of thyroid and heart. Thus, the thyroid might be viewed as being pulled caudally by the heart during its descent. The development and migration of the medial and lateral thyroid anlage should be viewed in conjunction with the development and migration of other structures of the head and neck, such as the parathyroid glands and the thymus. The thyroid anlage and the ultimobranchial bodies migrate from their respective sites of origin and ultimately merge in the definitive thyroid gland at E60 in humans and E14 in mice. In this process, the complete structures are lost and become dispersed cells. The cells that originate from the anlage will give rise to the thyroid follicles, whereas the C cells scatter within the interfollicular space. In the mature thyroid gland, C cells occur either singly or in small groups, and their contribution to the total thyroid mass is small (∼10%). They are more concentrated in the central parts of the lobes, but are found throughout the gland (95,96).

THYROID FOLLICULOGENESIS Thyroid folliculogenesis is a complex process that begins with the proliferation of irregularly arranged cell cords derived from the endoderm. The first evidence of follicle formation is at E70 in humans and at E15.5 in mice, when small follicles appear disseminated within the gland. Although the signals and transcription factors that regulate the process of folliculogenesis are increasingly known, mainly by the use of in vitro mESC and iPSC models (see later in section In Vitro Generation of Thyroid Cells From Pluripotent Stem Cells), much remains to be learned about this complex mechanism. A question that has remained unanswered for a long time is whether follicle formation solely depends on intrinsic properties of thyroid-committed cells or whether there are extrinsic factors, systemic or locally produced, which contribute to this formation. The concept that intrinsic factors contribute to follicle formation originally derived from the observation that dissociated thyroid cells in suspension self-assemble into cysts and, if

embedded into an inner matrix of type 1 collagen, form stable follicles without further requirements (97). The existence of intrinsic factors involved in follicle formation derived from the seminal work of Antonica et al. (98), who demonstrated that mESCs transiently over-expressing Nkx2-1 and Pax8 were able to form functional follicles when grown in 3D culture. Between these two factors, Pax8 plays a critical role in the formation and maintenance of the follicular structures, as Pax8−/− mice lack follicles (99). The mechanism through which Pax8 controls this process is mediated by its transcriptional target cadherin-16 (Cdh16), which is expressed from the early stages of thyroid morphogenesis. In the absence of Pax8, the levels of Cdh16 decrease and, consequently, the acquisition of apical-basal membrane polarity is inhibited and lumen formation is impaired (100). Further intrinsic factors such as Smad1/5 have been described to be involved in thyroid follicle development (101). Both in the case of Pax8 and Smad1/5, the mechanism of folliculogenesis is mediated by laminins α1, β1, γ1. The existence of extrinsic factors derives from early studies showing that follicular cells explanted from a developing chick thyroid required fibroblasts obtained from the capsule surrounding the thyroid gland to organize into a correct histologic pattern. This suggested that a mesenchymal component is necessary for follicles to develop (102). In addition, mutations of genes expressed in neighboring tissues of the thyroid bud impair correct folliculogenesis (reviewed in [92,103]). Other studies using ex vivo mouse thyroid primordia have shown that microvessels contribute to folliculogenesis, involving vascular endothelial growth factor A (Vegfa) (104). Proper follicle development in the mouse embryo requires the formation of an epithelial basement membrane, which is governed by epithelial-endothelial crosstalk mediated by BMP through Smad signaling and laminins, as explained earlier (101). TSH has no role in de novo follicle formation since a follicular orthotopic gland develops correctly in both Tsh and Tshr knockdown mice (87). However, in 3D in vitro culturing studies forcing Nkx2-1 and Pax8 expression in mESCs, TSH is required to generate follicles (81,98). The terminal differentiation program of the thyroid only takes place when the migration process is completed and the follicles are formed. At this time, thyroid cells express a series of proteins essential for thyroid hormone biosynthesis. The fact that the onset of thyroid function occurs only after final

migration points to a temporal and spatial control. However, at odds with this concept is the observation that patients with a sublingual thyroid produce low amounts of thyroid hormone. In addition, mice null for the transcription factor Foxe1 (see later) with a sublingual thyroid express Tg (93). These observations suggest that the final location of thyroid cells and thyroid location in the trachea are not requirements for the onset of thyroid function. By contrast, the timing of expression of thyroid-specific genes is necessary for thyroid hormone biosynthesis. At this stage, genes start to be expressed according to a strict temporal pattern: Tg, Tpo, and Tshr are expressed at E14.5 in mice (105) and Nis and Duox1/2 at E15.5 (106,107). Thyroid hormone T4 is detected at E16.5 in mice (87) and TG has been detected in human thyroid tissue as early as the fifth gestational week, when thyroid morphogenesis is still occurring. Iodide uptake and T4 production occur by the 10th to the 14th week, during a period that corresponds to the final stages of formation of the lumen of the follicles.

FIGURE 2-5. Schematic diagram of a thyroid follicular cell showing the major intracellular structures and the main proteins involved in the biosynthesis and secretion of the thyroid hormones T3 and T4. Iodide ions (I−) are concentrated by follicular cells from the circulation by the NIS symporter located in the basolateral membrane and then rapidly transported into the follicular lumen by Pendrin and other channels in the apical membrane. Amino acids (tyrosine and others) and sugars are assembled into thyroglobulin (Tg) in apical vesicles that are released into the follicular

lumen. Iodination of tyrosine residues occurs within Tg, after the I− is oxidized by thyroid peroxidase (TPO), using H2O2 generated by two oxidases DUOX1 and 2. The iodinated Tg is taken up by endocytosis of colloid, and the colloid droplets fuse with lysosomes, after which Tg is enzymatically cleaved with the release of MIT, DIT, T4, and T3. MIT and DIT are deiodinated by an iodotyrosine dehalogenase, whereas the T4 and T3 are released into the extracellular fluid through the MCT8 transporter. The cell nucleus contains the transcription factors, Tiff1, Foxe1, Pax8, and Hhex that regulate the transcription of the genes encoding NIS, TPO, Tg, and TSH receptors (TSH-R) and other genes within the thyroid. To form the follicular structures, thyroid epithelia cells are connected to each other by thin junctions (TJ) proteins and adhesion molecules (AM).

DIFFERENTIATED THYROID FOLLICULAR CELLS The thyroid epithelial cells form follicular structures surrounding a lumen. These follicular structures are the functional units of the thyroid gland and are responsible for the biosynthesis, storage, and secretion of the thyroid hormones T3 and T4 (Chaper 4) (Figs. 2-5 and 2-6). Since thyroid hormones are iodinated, iodide is the rate limiting substrate and is actively transported into follicular thyroid cells by the NIS located at the basolateral membrane (106) (see Chapter 3). Iodide enters the lumen, at least in part, by the anion exchanger pendrin (PDS). It has recently been demonstrated that iodide may enter the lumen through the direct participation of other transporters— including anoctamin 1 (ANO1), cystic fibrosis transmembrane conductance regulator (CFTR) and sodium multivitamin transporter (SMVT); however, the level of participation of each in iodide transport in thyrocytes is still unclear (75). In the apical membrane facing the lumen, iodide is oxidized by TPO in a reaction that requires hydrogen peroxide (H2O2), which is generated by the dual oxidase. In the lumen, TG, synthesized by the follicular cells, serves as a matrix for the formation of mono- and diiodotyrosine (MIT and DIT), and the coupling of the two iodotyrosyls generates either T3 or T4. The iodinated TG is stored in the colloid and is internalized in follicular cells by micropinocytosis or endocytosis. It then undergoes proteolysis in lysosomes to release free T3 and T4 and other iodocompounds (MIT, DIT) (108). Iodide from MIT and DIT is recycled by the iodotyrosine deiodinase (IYD1 or DEHAL1) (109). Finally, thyroid hormones are transported to the bloodstream mainly by monocarboxylate transporter 8 (MCT8) (110). TSH

finely regulates this complex process after binding to its receptor (TSHR), a seven-transmembrane receptor coupling to G-proteins (111). Stimulation of the TSHR leads to the dissociation of trimeric G proteins into Gα and Gβγ subunits that, in turn, trigger complex signaling cascades. Most of the activities of TSHR are mediated through Gs, a Gα protein that activates the adenylyl cyclase/cAMP/PKA cascade. Nevertheless, it was recently shown that the Gβγ dimers play a role in thyroid cell differentiation in response to TSH acting via PI3K (112). In addition, mounting evidence supports a role for other kinases, such as MAPK, which are activated in response to TSH and downstream of cAMP (113). Consequently, we consider differentiated thyroid cells as those cells that express all of the main differentiation markers: NIS, PDS, DUOX1/2, TPO, TG, DEHAL1, and MCT8 (Fig. 2-5).

FIGURE 2-6. Electron micrograph of normal thyroid follicular cells with long microvilli (V) that extend into the luminal colloid (C). Pseudopods from the apical plasma membrane surround a portion of the colloid to form an intracellular colloid droplet (CD). Numerous lysosomes (L) are present in the apical cytoplasm in proximity to the colloid droplets. An intrafollicular capillary is visible at the lower left.

FIGURE 2-7. Microscopic section of the thyroid gland of a normal rat showing colloid-filled (C) follicles of varying size (arrows) lined by cuboidal follicular cells. An extensive network of capillaries is present between the thyroid follicles (periodic acid-Schiff stained section).

HISTOLOGY AND STRUCTURE The structure of the thyroid is unique among the endocrine glands as it is the only gland in which the hormonal products are stored in an extracellular location. The thyroid consists of individual structural and functional units, named the thyroid follicles, made up of an epithelial monolayer that surrounds a lumen containing TG-rich colloid (Fig. 2-7). The differentiated epithelial phenotype is characterized by structural and functional polarization of the cell surface into apical and basolateral domains, and the formation of a tight junction complex that mediates strong and firm intercellular adhesion. The presence of junction proteins, for example, in apical and adherens junctions, in thyroid epithelial cells is thought to be essential for the integrity and maintenance of the follicular structure and in preventing the leakage of colloid from the lumen (reviewed in [95,103]). The tight junction is a multiprotein complex that includes the

transmembrane proteins occludin and claudin, and a number of cytoplasmic zonula occludens proteins (ZO-1, ZO-2, ZO-3, and others) that link the junctions to microfilaments and regulate their assembly (114). Adherens junctions have a similar structural organization in which E-cadherin forms contacts between neighboring cells and the catenins connect with the cytoskeleton. Follicular structure is maintained by the integrity of the cytoskeleton, including microtubules and microfilaments (115), through which TSH and intercellular contact may regulate adhesion of follicular cells to each other and to the extracellular matrix, also influencing thyroid cell behavior. The extracellular matrix plays a role in the adhesion, proliferation, differentiation, and migration of thyroid follicular cells. Molecules involved in these processes include type I and type IV collagens, fibronectin, laminin (116), and cadherins (117). Type IV collagen and laminin (major components of the basement membrane surrounding each follicle) play important positive roles in the proliferation and differentiation of follicular cells, and E-cadherin is crucial for the maintenance of the thyroid epithelial cell phenotype during organogenesis (94,118). E-cadherin is a member of the large superfamily of cadherin adhesion molecules and has a critical role in the establishment of cell polarity and firm contacts. It is one of the central molecules involved in cell–cell adhesion and polarization, and beyond its function in development, appears to play an important role in human thyroid malignancies where its expression is frequently impaired (119). Thyroid follicular cells have long profiles of rough endoplasmic reticulum and a large Golgi apparatus in their cytoplasm for the synthesis and packaging of TG, which is then transported into the follicular lumen (see Chapter 4), and of other glycosylated proteins such as NIS (Chapter 3), which are transported to the basal membrane. The cytoplasm also contains numerous electron-dense lysosomal bodies that are important in the proteolysis of iodinated Tg for thyroid hormone secretion (Fig. 2-6). The surface characteristics of the apical (luminal) and the basolateral sides of the cells are different, according to the role that the particular surface has in thyroid hormonogenesis. The secretory polarity of the cells is directed toward the lumen of the follicles—this polarity is important for iodine uptake. The apical surfaces of the follicular cells have numerous microvilli that protrude into the follicular lumen, greatly increasing the surface area in contact with the colloid (Fig. 2-6). The follicular structure

seems to be required for normal thyroid hormone synthesis and secretion (120). An extensive network of inter-and intrafollicular capillaries formed during development provides the follicular cells with an abundant blood supply. There is also a network of lymphatics in the gland. The stroma also contains nerve fibers; some of these are parasympathetic but most are sympathetic. These nerves terminate around the blood vessels or in apposition to the follicular cells. Growth factors and vasoactive factors are also produced in the thyroid. They include Fgf and Vegf, which are potent angiogenic proteins and which in cooperation with TSH may regulate the growth and function of follicular cells later in the adult (120). The role of these growth factors in thyroid development during embryonic life has not been explored in detail, but recently, as explained above, their participation in different process of thyroid development such as specification, growth, and folliculogenesis has been described (review in [74]).

THYROID TRANSCRIPTION FACTORS Fully differentiated follicular cells simultaneously express four genes encoding different transcription factors: NKX2-1 (formerly known as thyroid transcription factor 1, TTF1), FOXE1 (formerly known as thyroid transcription factor 2, TTF2), PAX8, and HHEX1 (hematopoieticallyexpressed homeobox protein). These factors are collectively known as the thyroid transcription factors (TTFs) (74,93,121). Each of the TTFs is expressed in several other tissues in embryos and adults, but they are expressed collectively only in epithelial thyroid follicular cells. The expression of the TTFs in the thyroid anlage of mice at E8.5 suggests that they may play a crucial role in thyroid organogenesis. Accordingly, in the primitive pharynx, a group of cells specified to a thyroid epithelial fate expresses these TFFs simultaneously. After the formation of the thyroid diverticulum, and at the start of its migration, these TFFs are expressed only in the thyroid primordium and not in the thyroglossal duct; they also remain expressed in adult thyroid cells and define the thyroid-differentiated phenotype. Nkx2-1, Foxe1, and Pax8 transcription factors were initially identified in

rat thyroid cells by their binding to the promoter regions of the Tg, Tpo, Tshr, and NIS genes (reviewed in [122–124]) (Fig. 2-8). Less information is available about the binding capacity of HHEX to thyroid gene promoters (125). Although NKX2-1, FOXE1, PAX8 and HHEX are intimately involved in thyroid differentiation, they belong to different families of transcription factors. NKX2-1 and HHEX are both members of the homeodomain family, FOXE1 is a forkhead domain protein and PAX8 is a paired domain family member. These four transcription factors present a high degree of conservation across different species.

NKX2-1 This transcription factor was identified and initially named TTF1 (126); it has been cloned simultaneously by two independent groups, who termed the gene Titf1 (127) or T/ebp (128). As it belongs to the NKX2 family of transcription factors, it is currently named NKX2-1 (129). Genomic regions and cDNAs for NKX2-1 have been cloned from various species and the protein is encoded by a single gene in humans and mice, mapping to chromosome 14q13 and 12C1-C3, respectively. The Nkx2-1 gene contains three exons and two introns of 150 and 850 bp, respectively, and its structure is well conserved among mammals, encoding a 42-kDa protein that binds to DNA through its homeodomain sequence of 61 amino acids comprising three helical regions (I, II, and III) folded in a globular structure.

NKX2-1 Expression During Development The expression of Nkx2-1 is well characterized in mice. It is expressed in the endodermal cells of the primitive pharynx in the thyroid anlage at E8.5 (26,105), and it remains expressed throughout thyroid development and in adult life. The establishment of its expression coincides with proliferating cells that give rise to the primitive thyroid bud, before the migration of the precursor follicular cells begins. It is also expressed in the fourth branchial pouch and in the ultimobranchial body between days E8.5 and E10 (reviewed in [74,92,121]). In addition, Nkx2-1 is expressed in thyroid C cells and parathyroid cells (130), in the trachea and lung, and in specific areas of the forebrain including the developing posterior pituitary, basal ganglia neurons,

cortical interneurons and the hypothalamus (26,105). The expression of Nkx2-1 in nonthyroid tissues is maintained during adult life except in the brain, where it is restricted to the periventricular regions and some hypothalamic nuclei (reviewed in [92]). A more detailed understanding of the functional role of Nkx2-1 in development has been derived from the generation of knockout mice. The phenotype of Nkx2-1−/− mice is complex, reflecting the widespread distribution of this transcription factor. Null mice die at birth because of impaired lung morphogenesis. In addition, they have no thyroid or pituitary glands and there are severe alterations in the ventral region of the forebrain (26).

FIGURE 2-8. Schematic diagram showing interactions of multiple transcription factors with the promoter regions of the genes for Tg (thyroglobulin), TPO (thyroid peroxidase), TSH-R (TSH receptors), and NIS (sodium/iodide symporter). The structures of the promoter regions of the Tg and TPO genes are similar. NUE denotes the NIS Upstream Enhancer element that is the main regulatory promoter element of the NIS gene.

A more thorough study of the thyroid phenotype of Nkx2-1 null mice revealed that the thyroid bud begins to be formed, but is later eliminated by apoptosis (26,131). Therefore, Nkx2-1 is necessary for survival and proliferation of thyroid precursor cells, but not for initial specification of endodermal cells to give rise to the primitive thyroid. The phenotype of

Nkx2-1 null mice demonstrates that human athyreosis can be caused by a single hereditary genetic defect, likely because of the inability of the thyroid precursor cells to survive and not because of their lack of specification. The data obtained from Nkx2-1 null mice show that this factor plays different roles during development by controlling survival at the beginning of organogenesis and differentiation in adult life. Clearly, this last event cannot be studied in Nkx2-1 null mice, as thyroid cells are lost before terminal differentiation. However, a conditional knockout mouse in which Nkx2-1 disruption occurs late in organogenesis revealed an altered follicular organization of the thyroid gland, leading to the conclusion that Nkx2-1 is required for maintenance of the thyroid architecture (132). Furthermore, a mouse line expressing Nkx2-1 mutated in the phosphorylation sites also presents a thyroid gland with deranged follicular organization (133). The phenotype of this latter model suggests that the pleiotropic functions of Nkx2-1 are not all due to the protein as a whole unit, since some of them can be assigned to separate domains or to specific posttranslational modifications. In addition, this model reinforced the concept that Nkx2-1 could be one of the factors involved in folliculogenesis, as was later confirmed by Antonica et al. (98). Nkx2-1 is detectable only in the ventral wall of the anterior foregut and, consequently, there is no septation between trachea and esophagus in Nkx2-1 null mice (134). In the lung, Nkx2-1 expression is first detected at E9.5, and is restricted to the ventral migrating edge of the lung bud where the tracheal diverticulum forms. Later, Nkx2-1 is expressed in all pulmonary epithelial cells. As stated above, Nkx2-1 null mice have severely hypoplastic lungs, with dilated sac-like structures and only rudimentary bronchial branching (26). These findings suggest that Nkx2-1 is not required for initial commitment of endodermal cells to originate the lung, but it is required for the differentiation of lung cells (135). The mechanisms responsible for the initiation of Nkx2-1 expression are still not completely known and, given its critical role at the initiation of thyroid organogenesis, it will be important to define them to better understand the initial events that control thyroid specification. As discussed below, in vitro models of mESc and iPSc demonstrated an important control of Nkx2-1 expression by Fgfs and BMPs. However, very little is known about the transcription factors that regulate Nkx2-1 expression in the embryo.

In vitro studies showed that the zinc finger protein Gata6 regulates Nkx2-1 transcription, but analysis of Gata6−/− mice demonstrated that Nkx2-1 is expressed normally (136). Finally, Hoxa genes regulate Nkx2-1 gene transcription in vitro (137), and because these genes play a role in thyroid development (see later), they are potential candidates to regulate Nkx2-1 expression. The identification of upstream Nkx2-1 gene regulators should give important clues about the players responsible for patterning the foregut. It will be equally important to identify the genes downstream of and directly regulated by Nkx2-1, as well as other interacting factors that modify Nkx2-1 transcriptional activity. Regarding downstream factors, new technologies such as expression microarrays and massive parallel sequencing (RNA and chromatin immunoprecipitation) are beginning to provide more information. Microarray analysis of thyroid cells has revealed new putative Nkx2-1 target genes, including the genes that encode the other TTFs—Foxe1, Pax8 and Hhex—as well as other thyroid-related genes including Duox1, Duoxa1 and also other genes such as Cdh1 (reviewed in [121]). Moreover, Nkx2-1 regulates its own expression via an autoregulatory loop that is driven by the presence of Nkx2-1 binding sequences in its promoter (138,139). Interestingly, a microarray analysis of gene expression in the foregut has provided new gene expression profiles that reveal common and diverse mechanisms in thyroid and lung development. One of the most interesting regulated pathways involves the antiapoptotic gene Bcl2, which controls cell survival in early thyroid development (140). In conclusion, NKX2-1 plays different roles during development, controlling survival at the beginning of organogenesis, subsequent folliculogenesis, and differentiation in adult life.

FOXE1 The Foxe1 transcription factor was identified in rat cells in the same study that identified Nkx2-1, and was originally termed Ttf2 (126). The mouse Ttf2 gene (141) was cloned before the human gene, which was initially termed FKHL1 (142). The genes are located on chromosome 9q22 in mouse and chromosome 4 in humans. FOXE1 is a single-exon gene encoding a 38–42kDa protein that contains a forkhead DNA-binding domain, two putative nuclear localization signals flanking the DNA-binding domain, and a

polyalanine stretch that ranges from 11 to 19 residues in length. FOXE1 is a pioneer factor, as it has an intrinsic capacity to bind to and open chromatin structures via its winged-helix DNA-binding domain, which functions to facilitate binding of transcription factors to DNA (143,144). Maintenance of the differentiated thyroid is conditional on expression of Foxe1, which mediates TSH-driven expression of the Tg and Tpo genes (145–147). The promoter regions of both genes contain Foxe1-binding sites, with this transcription factor generally functioning as a transcriptional activator, although it can also act as a transcriptional repressor (148).

Foxe1 Expression During Development The expression of Foxe1 is detected in mice at E8.5 in the endoderm lining the foregut. Its expression at the initiation of development in the anterior pharynx is restricted to a group of cells in the midline that give rise to the thyroid anlage, whereas its expression is more widespread in the posterior region of the primitive pharynx. Thus, Foxe1 expression is more evident in the region of the posterior pharynx than in the thyroid precursor cells, where its expression is maintained during the entire process of development and in adult life. In addition to the thyroid anlage, Foxe1 is expressed in the epithelium facing the anterior pharynx and the pharyngeal arches, but is absent in the pouches. It is detected throughout the entire foregut including the future esophagus from which it disappears in adults. At later stages of development Foxe1 is expressed in tongue, epiglottis, and palate. Foxe1 is also expressed in ectodermal-derived structures at the beginning of development, such as the posterior stomateum, the bucopharyngeal membrane and the roof of the oral cavity that gives rise to Rathke pouch, which will form the various components of the anterior pituitary. At later stages, its expression in the pituitary is downregulated, but can be observed in the secondary palate, in the choanae and in the whiskers and hair follicles (149). In humans, FOXE1 mRNA is also detected in adult testis, brain, heart, pancreas, lung, liver, skeletal muscle, kidney, colon and small intestine (reviewed in [121]). The role of Foxe1 during development has been elucidated by the generation of knockout mice. Foxe1−/− mice are born at the expected Mendelian ratio but die within 2 days. Thyroid budding occurs with a normal

anlagen, but at E9.5 the thyroid precursor cells are unable to migrate downward and remain contiguous to the pharyngeal endoderm (93) (Fig. 210). The nonmigrating thyroid cells are able to complete the differentiation program, as Tg expression is detected, indicating that terminal differentiation of thyroid follicular cells is independent of the position of the cells. Later in development, Foxe1−/− mice have either a small thyroid remnant, still attached to the floor of the pharynx (thyroid ectopia), or no thyroid gland (thyroid agenesis). The cause of these two phenotypes has been attributed to a stochastic phenomenon during morphogenesis and to the different genetic backgrounds of the knockout mice. The definitive role of Foxe1 in thyroid development comes from the observation that reintroduction of a normal Foxe1 gene exclusively in these cells in a mouse missing the normal, endogenous gene, restores proper gland positioning. The expression pattern of Foxe1 suggests that this factor has a marginal role in the specification of thyroid follicular cell precursors. Therefore, its main function in thyroid development seems to be to control the migration of the thyroid to its position alongside the trachea. In addition, Foxe1−/− mice have a severe cleft palate that could be responsible for their early death after birth. The shelves of the palate do not fuse, and this anomaly correlates with the expression of Foxe1 in the posterior region of the primitive pharynx. As Foxe1 null mice die very soon after birth, it will be necessary to generate new animal models with a thyroid-specific conditional knockdown of Foxe1 to better understand the role of this factor in the adult gland. The mechanism responsible for Foxe1 expression at the beginning of thyroid development is unknown. The data provided by Antonica et al. (98) would suggest that Foxe1 is regulated by Nkx2-1 and Pax8. In addition, Foxe1 interacts with the ubiquitous transcription factor NF1/CTF to form a complex that can regulate the expression of the Tpo gene (150). Regarding Foxe1 downstream actions, genomic analysis has identified Duox2 and NIS as novel and direct Foxe1–NF1/CFT targets. Other genes including Cdh1 and Nr4a2 were also identified as transcriptional targets of Foxe1 (151); however, the potential involvement of these genes in thyroid differentiation remains to be determined. In conclusion, FOXE1 controls thyroid cell migration from the primitive origin to its normal position at both sites of the trachea. Furthermore, the fact that 50% of Foxe1−/− mice have a missing thyroid suggests that Foxe1 could

also be involved in the control of thyroid cell survival.

PAX8 The PAX8 (paired box gene 8) transcription factor is a member of the PAX family of paired domain (Prd) transcription factors well conserved through evolution (152). There are nine known members of this family, grouped into four classes based on the similarity of the DNA-binding Prd domain, belonging PAX8 to group 2. In addition to the Prd domain, the proteins of these three subfamilies have a truncated homeodomain, a conserved octapeptide sequence, and sequence homology in the transcriptional activation domain located at the carboxy terminal end of the protein. Human PAX8 is located on chromosome 2q12–q14, whereas the mouse ortholog is located on chromosome 2, and both encode a protein of approximately 46 kDa. The molecular structure of many PAX genes is known (153,154): the paired (Prd) domain is 128 amino acids in length and consists of two different subdomains—each containing a helix-turn-helix motif, joined by a linker region. The amino-terminal subdomain is named PAI and the carboxy-terminal subdomain is called RED. Because of this independent subdomain structure, each PAX protein binds to different DNA sequences. The binding sites for Pax8 and Nkx2-1 in the promoter regions of the Tg and Tpo genes partially overlap (Fig. 2-8), and the two factors therefore compete for binding. In the case of the Tg promoter, both subdomains of Pax8 are required for efficient binding. In addition, Pax8 interacts with Nkx2-1 for full expression of the Tg gene (154). Pax8 binds, in close proximity to Nkx2-1, efficiently to NIS upstream enhancer (termed NUE) region (124) (Fig. 2-8). In addition to the thyroid gland, human PAX8 is expressed in the renal excretory system, epithelial cells of the endocervix, endometrium, ovary, Fallopian tube and seminal vesicle, as well as in the epididymis, pancreatic islet cells and in lymphoid cells (155,156).

PAX8 Expression During Development Pax genes display dynamic patterns of expression during ontogenesis and are involved in pattern formation during organogenesis. In mouse endoderm, Pax8 is expressed only in the thyroid anlage coincident with Nkx2-1

expression, at E8.5 at the time of specification (Fig. 2-9). Its expression is maintained during all stages of development and continues throughout adulthood. Pax8 is also expressed transiently during development in the myelencephalon and along the entire length of the neural tube, but no expression is detected in brain at later stages or in adults. In the kidney, Pax8 is expressed together with Pax2 in the nephrogenic cords and the mesonephric tubules at E10.5. Subsequently, both are expressed in mesenchymal condensations at E13, in the cortex of the metanephros at E16, and in the adult kidney.

FIGURE 2-9. Schematic representation of the stages of thyroid development, indicating the morphologic features, the expression of relevant genes and the onset of thyroid hormone production, both in human and mouse. The names of the different steps of thyroid organogenesis and differentiation are shown in the upper boxes. The middle boxes show representative diagrams of these steps. The arrows in the lower boxes show the time of expression of the thyroid transcription factors and their target genes. (Modified from Fernandez LP, Lopez-Marquez A, Santisteban P. Thyroid transcription factors in development, differentiation and disease. Nat Rev Endocrinol 2015;11(1):29–42.)

The role of Pax8 in thyroid development has been elucidated by examining the phenotype of Pax8 knockout mice. These mice do not have any apparent defect in the spinal cord or kidney, probably due to the redundant activity of

other Pax family proteins such as Pax2 and Pax5 in these tissues (157,158). However, they are growth retarded, likely due to thyroid deficiency, as they are hypothyroid with very low levels of T4 (99). Pax8−/− mice are infertile and die at an early age (2–3 weeks) unless given thyroid hormone, which increases their length of survival but does affect their fertility, due to a primary impairment in the development of the reproductive system. Accordingly, Pax8−/− mice have a severely affected thyroid gland and neither thyroid cell precursors nor follicles are detected. Thus, most of the cells in this rudimentary gland are calcitonin-producing C cells, which express Nkx21, but do not contain Tg or Tpo. In Pax8−/− mice, the thyroid anlage that derives from the endodermal cells of the primitive pharynx is present at E10.5 to E11.0, but fails to expand laterally at E11.5, as occurs in wild-type mice, and the lobular structure is completely missing. The absence of Pax8 is compatible with the initial events of thyroid development, but not with later stages such as formation of thyroid follicles and functional differentiation of thyroid cells. These phenotypes demonstrate that Pax8 is essential for the survival of thyroid precursor cells at the later stages of thyroid development. Similar to Nkx2-1, Pax8 might contribute to the maintenance of survival of thyroid precursor cells by inhibition of apoptosis, as demonstrated by the low levels of Bcl2 in the thyroid bud of Pax8 null mice (140). Interestingly, the expression of Foxe1 and Hhex is severely downregulated in the thyroid rudiment of Pax8 null mice, suggesting a crosstalk between transcription factors at the early stages of development. Additionally, Pax8 is a critical gene for maintaining the differentiated thyroid phenotype in adults (159). Little is known regarding the transcriptional regulation of Pax8 at the early stages of development, although recent studies with mESc and iPSc models are contributing to identify new players such as Fgfs and BMPs. The upstream transcription factors targeting Pax8 are still not identified, although Hhex could play a role in this mechanism. Pax8 has been demonstrated to regulate the expression of thyroid genes such as Hhex (160) and Foxe1 (161), Dio1 and Duox2 (162). Finally, similar to Nkx2-1, Pax8 expression is also autoregulated due to the presence of Pax8 binding sites in the regulatory regions of the Pax8 promoter. Whole-genome sequencing has expanded our knowledge of Pax8 function in thyroid biology with the identification of several target genes involved in

cell-cycle processes (Cdkn2B, Ccnb1 and Ccnb2) (162). Moreover, Pax8 binds preferentially to nonpromoter CpG-rich genomic regions and regulates genes involved in cell proliferation and differentiation (Cited2, Taz, Runx2, Trib1), signal transduction (Wnt4), apoptosis, cell polarity and transport (Myo5b, Rab17, Kcnj16), cell motility and adhesion (Rab11a, Rab8a, Ncam and Cdh16), and a plethora of DNA–protein-related processes has also been described (162). Other Pax8 binding partners have been described, including its cooperation with Taz (163) to regulate expression of Tg, with histone acetyltransferase p300 to mediate Tg and Tpo transcription (164,165), and with CTCF, SP1 and β-catenin at the NIS enhancer (162,166). Of particular interest is the observation that Nkx2-1 and Pax8 bind the transcriptional co-regulator Taz, a protein of the Hippo pathway that has been described to govern thyroid size in zebrafish (167), and is involved in the Brain–Lung–Thyroid syndrome (31). In conclusion, similar to Nkx2-1, Pax8 is required for the survival of thyroid precursor cells and not for their specification. It also plays a role in the genetic regulatory cascade that controls thyroid development and functional differentiation. Finally, based on the function of other Pax factors, it is accepted that Pax8 has a relevant role in initiating and maintaining the tissue-specific gene expression program, and is therefore considered as a master gene in thyroid differentiation.

HHEX The transcription factor HHEX is encoded by a gene located on chromosome 10q23.32 in humans and on chromosome 19 in mice (168). The gene comprises four exons and codes for a protein of 30 kDa. HHEX contains an evolutionary conserved 60-amino acid DNA-binding homeodomain with a proline-rich region at the N-terminus, important for its transcriptional control.

Hhex Expression During Development Hhex is expressed in the anterior visceral and rostral endoderm in early mouse embryogenesis. At E8.5, Hhex marks the primordium of several organs derived from the foregut such as thyroid, liver, thymus, pancreas, and

lung, and is expressed at high levels in both developing and adult thyroid and liver (169,170). Hhex null mice die during mid gestation and the embryonic phenotype varies from severe to mild according to the degree of malformation of organs such as the liver and brain. Severely affected embryos do not express Foxe1 and Nkx2-1 at E8.5 and these mice have thyroid agenesis in later developmental stages. Although the phenotype might suggest that Hhex plays a critical role in thyroid specification, the general defects in the anterior endoderm of these mice indicate that this developmental impairment is not thyroid-specific (171). Conversely, embryos with moderate to mild malformations show some degree of thyroid specification and develop a hypoplastic thyroid that remains connected to the pharynx (172). At E9, mildly-affected mice express normal levels of Pax8, Foxe1 and Nkx2-1; however, by E10, Pax8 and Foxe1 expression is dramatically downregulated, which might account for the small and ectopic thyroid gland phenotype seen at E15.5 (172). These data demonstrate that Hhex is not required for specification or budding of the primordial thyroid, but it does regulate the expression of Pax8 and Foxe1, which control bud formation and survival of thyroid precursor cells in the later stages of thyroid development. The relationship between these factors could be even more complex because Hhex is not expressed in Nkx2-1 and Pax8 null mice. The role of Hhex in thyroid organogenesis might be to regulate cell movement in the anterior foregut endoderm via a non–cell-autonomous mechanism, as has been described in liver. Thus, this factor is essential for thyroid morphogenesis and is an early marker of thyroid cells (Figs. 2-9 and 2-10).

FIGURE 2-10. Network of interactions between transcription factors during thyroid morphogenesis. A hypothetic model shown the relationship between Hhex, Titf1, Pax8, and Foxe1 (circles) and the possible functions regulated (squares) thought early and late organogenesis and in postnatal life (indicated at left). Most of the data for integrating this scheme come from studies in different animal models. A determined transcription factor can regulate the other factor (dashed arrows) or control critical steps of thyroid organogenesis (solid arrows). (Modified from De Felice M, Di Lauro R. Minireview: intrinsic and extrinsic factors in thyroid gland development: an update. Endocrinology 2011;152[8]:2948–2956.)

In the adult, Hhex may act as a transcriptional repressor, as reported in vivo and in cell culture experiments (173). Consequently, overexpression of Hhex in thyroid cell lines partially inhibits Tg transcription and Hhex is downregulated by TSH. Furthermore, Hhex expression decreases in thyroid cell lines transformed with oncogenes as well as in human thyroid cancer cells (174), whereas Nkx2-1 increases Hhex promoter activity. Moreover,

Hhex and Nkx2-1 interact to increase Hhex gene expression, demonstrating the existence of direct crossregulation between thyroid transcription factors (Fig. 2-10). In conclusion, Hhex is decisive for thyroid morphogenesis and acts by maintaining the expression of the other three TFFs. However, more detailed studies are needed to clarify the role of this factor in thyroid development.

Thyroid Transcription Factor Networks All four TTFs are expressed in thyroid precursor cells; however, whether all of the factors are necessary to commit an undifferentiated cell to a thyroidcell fate is not clear. Experiments in mESc showed that the transient expression of only Nkx2-1 and Pax8 was sufficient to drive differentiation of an epithelial thyroid lineage, and that overexpression of Nkx2-1 was sufficient to induce the expression of Foxe1 (98). Moreover, studies in developing mice showed that at the initiation of development, during placode formation (E8.5–9), Nkx2-1, Pax8 and Hhex are co-expressed in thyroid progenitors in an independent manner, as the absence of any one of these genes does not affect the expression of the others. With the exception of Foxe1, which requires Pax8 for its expression, the transcription factors do not crossregulate each other, at least based on current knowledge. After the thyroid bud is formed (E10), each of the three transcription factors influences the expression of the genes that encode the other TTFs, and together these proteins control the expression of Foxe1, which is downstream in the thyroid regulatory network (172). Autoregulation of Nkx2-1 and Pax8 expression has been demonstrated in thyroid cells, suggesting that a similar feed-forward transcriptional loop might also be operating in development, contributing to the propagation of thyroid lineage. These data support a previous concept that a hierarchy exists in the functional network of interactions between the TTFs (172), and crosstalk between all of these transcription factors is decisive for the control of the first stages of thyroid development. The functions that these factors control have been described in detail and are schematized in Figure 210, adapted from different articles of the group of Di Lauro ([172] and reviewed in [74,92,121]). Since the thyroid primordium is formed in all TTF-mutant mice, none of these transcription factors is individually necessary for the development of

this structure, but they are required for the emergence and survival of the thyroid bud (Fig. 2-10). The transcription factors upstream of Nkx2-1, Pax8, Foxe1 and Hhex will need to be identified to fully understand the initial events that control thyroid specification in thyroid progenitor cells (175). In this case, mESc and/or iPSc will be important tools for identifying these upstream factors and other contributors to thyroid development. In conclusion, all of the above genes temporally and spatially regulate different aspects of thyroid development, including budding, migration, survival and proliferation of thyroid follicular precursor cells, differentiation, and follicle formation. Finally, they are the genes responsible for instructing the thyroid differentiation program and controlling the expression of the thyroid genes Tshr, Tg, Tpo, and Nis with the subsequent onset of thyroid hormone production. From the above information, the temporal expression of the thyroid-specific transcription factors during thyroid development occurs before the expression of thyroid-specific functional genes necessary for thyroid hormone formation. A schematic representation of the initial mechanisms that govern thyroid gland formation is shown in Figure 2-10. In the future, the complete regulatory networks controlling the expression of thyroid transcription factors, and the signals and factors that govern thyroid cell specification, will need to be disentangled.

OTHER TRANSCRIPTION FACTORS INVOLVED IN THYROID DEVELOPMENT The identification of other transcription factors controlling thyroid development has been elucidated from the detailed study of mice deficient in different genes.

Hox Genes The analysis of Hox null mutant mice has revealed that these genes are involved in the morphogenesis of several structures. Some genes of the Hox family, expressed in the foregut during embryonic life, are involved in the development of the thyroid. In particular, Hoxa genes are expressed in the anterior neuroectoderm and in the branchial arches and their derivatives,

including the thyroid bud. Hoxa3 is expressed in the floor of the pharynx, in the developing thyroid, and in the mesenchymal, endodermal, and neural crest-derived cells of the fourth pharyngeal pouch. The generation of Hoxa3−/− mice has confirmed the role of this gene in thyroid organogenesis, as these mice present an abnormal development of the thymus and parathyroid glands, together with a poorly developed thyroid gland (176). Indeed, in the thyroid, the isthmus is either absent or is displaced cranially with a reduced number of follicles, and with one lobe absent or hypoplastic. In addition, Hoxa3 null mice have severe alterations in the development and migration of the ultimobranchial bodies, which do not fuse with the thyroid primordium that has very few C cells. The phenotype of mice carrying various mutant combinations of Hoxa3 and its paralogs Hoxb3 and Hoxd3 has also revealed alterations in the thyroid and in the ultimobranchial bodies, suggesting that the defects of thyroid follicular cells could be secondary to and the consequence of alterations in the migration of the ultimobranchial bodies. In conclusion, these Hox genes carry positional information contributing to the initiation of the expression of the Nkx2-1 gene (137). The Hoxa5 gene also has a role in thyroid organogenesis, as Hoxa5 null mice have manifestations of hypothyroidism, including transient growth retardation and delayed eye opening and ear elevation. Thyroid gland morphogenesis begins normally, but formation of thyroid follicles and processing of Tg are impaired (177).

Eya1 Gene The Eya1 (Eyes absent 1) gene regulates organogenesis in both vertebrates and invertebrates, and it has an important role in the morphogenesis of organs derived from the pharyngeal region, including the thymus and the parathyroid and thyroid glands (178). Eya1 is expressed in the third and fourth pharyngeal regions and in the ultimobranchial bodies. Eya1 null mice have a thyroid phenotype similar to that of Hoxa2 null mice. Thus, they have persistent ultimobranchial bodies, hypoplasia of the thyroid lobes, no isthmus, and a severe reduction in the number of follicular cells and C cells. Since Eya1 is not expressed in the thyroid diverticulum, it is possible that the defects in follicular cells are due to the lack of fusion of the ultimobranchial

bodies to the thyroid lobes. This hypothesis is also supported by the fact that the early stages of thyroid development are normal in Eya1 null mice (178). Altogether, the above data on Hoxa and Eya1 suggest their critical functions as early inductive events involved in the morphogenesis of the thyroid gland and other organs derived from the pharyngeal region.

Foxa2 Gene The forkhead transcription factor Foxa2, formerly called hepatocyte nuclear factor-3β, is expressed early in the primitive endoderm and is decisive for organogenesis of the liver and control of liver gene expression (179). It is also expressed in the invaginating foregut endoderm and in endodermderived structures including the developing thyroid. The expression of Foxa2 is downregulated during thyroid development before thyroid follicular cells reach their terminal differentiation. Its expression is maintained during adult life. Its relevance for thyroid morphogenesis is not yet clear because Foxa1 disruption causes a lethal phenotype at a stage before the thyroid bud emerges. In cultured thyroid cells, Foxa2 binds to the Tpo promoter at the same site at which Foxe1 binds (180).

Genes of the Nkx2 Family In addition to Titf1/Nkx2-1, members of the same family such as Nkx2-6, Nkx2-3, and Nkx2-5 are expressed in the endoderm layer of the developing pharynx, including the thyroid anlage and other tissues. However, the phenotype of Nkx2-6 or Nkx2-3 null mice does not show any apparent thyroid defect. By contrast, Nkx2-5 may play a role in thyroid organogenesis. This gene is expressed in the ventral side of the pharynx and in the thyroid anlage at early stages of development, although its expression decreases later (reviewed in [92]). Nkx2-5 null mice die early in development, but at E9.5 a small thyroid bud is present and Nkx2-1, Foxe1, and Pax8 are expressed. The expression of members of the Nkx2 family could be redundant and the absence of one factor may be compensated by the others.

Sox9

The SOX transcription factors play a fundamental role in the development of numerous organs, but usually with very divergent functions. A common feature of their structure is a highly conserved DNA-binding domain with a High Mobility Group (HMG), which was initially described in the transcription factor SRY. The SOX family comprises 20 factors, among which Sox9 has been recently described to play a role in thyroid morphogenesis in mice (85). Specifically, Sox9 controls a branching morphogenesis program that contributes to the growth of the thyroid in the late phases of embryonic development, specifically after thyroid bilobation. This process is essential in the development of other endoderm-derived organs such as the pancreas. In the case of the embryonic thyroid, Sox9 is differentially expressed in the distal progenitors where it shapes the branched growth pattern. Likewise, this process is also controlled by Fgf10, which is produced by the surrounding mesenchyme. Given the phenotype of the Fgf10−/− mouse, which presents a hypoplastic thyroid with a normal shape (85), it is postulated that Fgf10 is involved in the migration and branching of the progenitors, the increase in size and in folliculogenesis, but not in the proliferation itself. Furthermore, Fgf10 does not control the expression of Sox9 in the case of the thyroid, in contrast to what occurs in other endodermic organs such as pancreas. Also, from a phylogenetic perspective, a more recent report stipulates an interesting hypothesis about an exocrine origin of the thyroid (85), which may open a new field of study on the role of Sox9 in the transcriptional network that controls thyroid specification and differentiation.

IN VITRO GENERATION OF THYROID CELLS FROM PLURIPOTENT STEM CELLS ESCs emerged as a promising model to dissect and recapitulate the molecular events and gene networks that regulate cell-fate determination, cell differentiation and organogenesis. Tissue-specific genes and signaling pathways previously found to regulate the early steps of organogenesis have been applied in step-wise differentiation protocols starting from ES cells to recapitulate developmental steps of several organs, generating highly

promising in vitro models. In the thyroid field, several approaches have been used in the last years in order to generate in vitro ESC-derived thyroid tissue due to its potential applicability to the study of early thyroid embryogenesis and becoming a source of thyrocytes for genetic manipulation and cell transplantation. The first indications that ES cells could be used to generate thyroid tissue were provided in the early 2000s through the use of mES-derived embryonic bodies (EBs; 3D cell aggregates which express specific embryonic markers for the three germ layers) (181). When EBs were treated with TSH, thyrocyte-like cells expressing thyroid-specific genes such as Pax8, Nis, Tg, Tpo and Tshr could be detected, and these cells started forming some follicular-like structures (182,183). However, this approach had variable success and the thyrocyte-like phenotype was transient. Later, Jiang et al. improved the culture conditions by using TSH, insulin and potassium iodide to differentiate thyroid follicular cells from the E14 mouse ES cell line (184). These studies resulted in improved expression of Tg in the generated thyrocytes, as well as expression of other thyrocyte-restricted genes such as Tpo, Tshr and Nis. Moreover, Nkx2-1 and Pax-8 could also be detected (184). Despite the improvements obtained through these modifications, the low efficiency to obtain thyroid cells in vitro, and the difficulties in generating tridimensional follicular structures remained a major challenge. Thyroid, lung, liver and pancreatic cells are known to be derived from definitive endoderm, and studies have demonstrated that Activin A induces embryonic Nodal/Activin signaling efficiently deriving definitive endoderm progenitors from mouse and human stem cells (185–187). In order to improve the efficiency of thyroid differentiation from ES cells, Ma et al. tested the ability of Activin A to induce endodermal transformation (188). The addition of Activin A to cultures of EB cells markedly decreased the stem cell markers, and increased the expression of endodermal markers. Despite the promising developmental recapitulation of the thyroid endodermal progenitor cells, the culture of these Activin-induced EBs in a chemical conditioned differentiation medium supplemented with TSH and IGF-I resulted in a small population of cells expressing Pax8, Tshr and Nis genes. Moreover, the absence of Tg and Tpo expression, associated with a later downregulation of Nis, demonstrated that the induction of endoderm progenitors is insufficient to lead to efficient thyroid differentiation (188).

Subsequent studies demonstrated that even though the Activin-induced definitive endoderm is largely multipotent, thymus, thyroid, and lung epithelia, which form the most anterior foregut endodermal lineages, could not be efficiently derived from those progenitors (189). In contrast to specified foregut progenitors, such as the pancreatic lineage where specific markers or “knock-in reporter cell lines” have been employed to facilitate isolation (190), no tools to allow the isolation of the most primordial murine lung and thyroid progenitors were available. As briefly described previously, prior to differentiation, thyroid epithelia must first progress through a primordial progenitor stage defined by the coexpression of the transcription factors Nkx2-1, Pax8, Foxe1, and Hhex. The initially used protocols summarized above demonstrated the failure to access the multipotent primordial thyroid progenitors at their moment of specification, thus resulting in a lack of information about the mechanisms controlling their differentiation. This hampered a rational approach to developmentally derive thyroid tissue from ESCs in vitro. However, in 2012 a major breakthrough in the field of thyroid research was achieved by demonstrating the efficient generation of functional thyroid tissue by using thyroid-specific transcription factors overexpressed in mESCs (98). Moreover, subsequent studies have also obtained thyroid tissue in vitro by using distinct strategies such as forced expression of transcription factors, and knock-in reporter ES cell lines treated with chemical stimulation of specific pathways (81,191–194). The impact of these models on the knowledge concerning thyroid development, physiology and disorders, as well as the advantages and disadvantages of each strategy are summarized in the following section.

MODELING THYROID BY TRANSCRIPTION FACTORS OVEREXPRESSION Induction of organ-specific transcription factor expression in pluripotent stem cells has been used to derive cell lineages, and to improve the understanding of mechanisms involved in cell specification (195–198). In the thyroid field, functional thyroid tissue was efficiently derived from mESCs after a transient inducible co-expression of the thyroid transcription factors Nkx2-1 and Pax8

(98). The applied differentiation protocol involved generation of a recombinant mESC line in which the expression of these transcription factors can be temporally induced under incubation with doxycycline (Dox) (199). EBs derived from mES-modified cells were embedded in 3D Matrigel and then incubated with Dox (3 days) and subsequently with human recombinant TSH (2 weeks). The findings showed that the co-expression of these two transcription factors is required to induce high expression of functional TFC markers such as Tshr, Nis, Tg and Tpo, as well as to obtain follicular structures. Moreover, this approach was shown to have a great efficiency on thyroid differentiation, with around 60% of total cells presenting thyroid markers and follicular organization. A three-dimensional follicular structure of TFCs is a prerequisite for TH biosynthesis. Indeed, the follicles generated by this protocol are able to organify iodine. Of note, when Nkx2-1 or Pax8 where induced alone, mature thyroid follicles were not detected, in agreement with previous studies that have demonstrated the requirement of synergism between Nkx2-1 and Pax8 to induce thyroid maturation markers such as Tg, Tpo, and Nis (121,124,154,200,201). Finally, when ESC-derived mature thyroid cells were grafted in the kidney capsule of athyreotic mice, they were able to vascularize the transplanted thyroid tissue, restored thyroid hormone and TSH levels, resulting in the recovery from hypothyroidism 4 weeks after transplantation. This highly efficient 3D mESC-derived thyroid tissue generation protocol provides an abundant source of thyroid follicles and an invaluable tool to better dissect the role of candidate genes in early stages of thyroid development. The requirement of the co-expression of both Pax8 and Nkx2-1 transcription factors to generate thyroid tissue derived from mESC in vitro was subsequently confirmed by Ma et al. (192). In addition, they showed that after exposition to Activin A inducing the endodermal phenotype, the levels of Tg expression were higher in the Pax8/Nkx2-1 positive cells, and that these cells are capable to form thyroid follicles de novo (192). Later, the same group also demonstrated that this strategy for the generation of thyroid tissue generation is not restricted to murine embryonic stem cell, but that thyroid epithelial cells can also be differentiated from iPS cells, derived from murine fibroblasts, which exhibit very similar properties compared to thyroid cells derived from mESCs (202).

MODELING THYROID BY CHEMICAL PATHWAYS MANIPULATIONS In 2012, Longmire et al. published an in vitro step-wise protocol to derive, purify, and expand primordial lung and thyroid endodermal progenitors from pluripotent stem cells, basically by time-specific manipulations of signaling pathways (191). An Nkx2-1-GFP knock-in reporter ESC line was created aiming to track thyroid and lung progenitors and thus investigate mechanisms involved in the development of both tissues given that Nkx2-1 is the first protein induced during the specification to lung or thyroid fates (191). Based on the role of Activin and BMP4 in the endodermal cell patterning and specification (186,203), and the findings demonstrating that a precise modulation of TGFβ and BMP signaling can optimize early germ layer patterning in vivo and in vitro (189,204,205), these pathways were modulated and their effect on thyroid and lung specification was evaluated. By exposing mESC cells to Noggin (a BMP inhibitor) and SB431542 (a TGFβ inhibitor), the anterior endodermal state was maintained. Next, to induce Nkx2-1 and specification, the competent cells were exposed to a so-called WFKBE medium (containing Wnt3a, Fgf10, KGF, BMP4, EGF), previously reported to potentiate endodermal ventralization in hESC-endoderm (189), combined with high doses of Fgf2 and heparin (WFKBE+F2/H). This pathway manipulation resulted in efficient derivation of Nkx2-1GFP-positive cells from mESCs. By sorting GFP-positive cells, a gene expression signature suggesting initial lineage specification toward Nkx2-1-positive lung and thyroid progenitors was detected. Those progenitors were then submitted to a differentiation step by withdrawing Wnt3a, KGF, BMP4, and EGF from the media, maintaining Fgf2 and Fgf10 supplementation. This protocol resulted in upregulation of Tg and Tshr expression, however the resulting thyroid-like cells did not display expression of maturation makers such as Nis and Tpo (191). More recently, Kurmann et al. performed additional studies to better understand the role of BMP, Wnt, EGF, and Fgf signaling on thyroid development in order to enhance the efficiency of thyroid tissue generation in vitro (81). By sequential withdrawal of each factor from the WFKBE+2 medium, it became apparent that BMP4 and Fgf2 are mandatory for efficient

Nkx2-1-positive endodermal induction. The combination of BMP4, Fgf2 and Wnt3a in the induction media resulted in maximum efficiency of Nkx2-1positive cell specification, and sorted Nkx2-1GFP-positive cells (with or without Wnt3a) were subsequently expressing thyroid differentiation markers such as Tg and Tshr, and maturation markers including Nis and Tpo. Interestingly, Fgf2 alone induced predominantly neuroectodermal Nkx2-1positive cells without signals of lung or thyroid specification. Despite the discovery of the importance of those factors in thyroid specification, the percentage of Pax8-positive cells among Nkx2-1/GFPR-positive cells was around 5%. Later, Nkx2-1/GFP-positive cells were cultured in Matrigel conditions, and among those cells, around 50% were shown to express Pax8 and Tg. Moreover, higher levels of thyroid gene expression and an increased formation of follicular-like clusters were observed at the end of the differentiation protocol. In addition, after incubation in medium containing iodide and TSH, the thyroid organoids displayed functional capacity in terms of iodide organification and synthesis of small amounts of T4. Using a similar approach for in vivo studies, Antonica et al. showed that thyroid NKX2-1-positive cells grafted into hypothyroid mice are able to form functional thyroid tissue that is able to normalize T4, T3, and TSH levels (98). More recently, the essential minimal signaling pathways regulating distinct lung and thyroid-lineage specification from foregut endoderm have been characterized (193). Using the protocol established by Kurmann et al. to derive anterior foregut endoderm (81), Serra et al. tested the capacity of the combinations of BMP4+Fgf2, Wnt3a+BMP4, BMP4 alone, or all three factors in combination, to induce Nkx2-1-positive cells. Then Nkx2-1positive cells produced under each condition were sorted and cultured in either lung or thyroid maturation medium. Nkx2-1-positive cells exposed to Fgf2+BMP4 were capable to express markers of thyroid lineage differentiation without upregulation of lung markers. In contrast, in the sorted Nkx2-1-positive cell population specified with Wnt3a+BMP4, markers of airway and alveolar lung epithelial differentiation were significantly increased. Moreover, RNAseq analysis demonstrated that the transcriptional profiles obtained from in vitro-derived lung and thyroid progenitors resemble those of in vivo Nkx2-1-positive lung and thyroid progenitors (193).

Combination of Transcription Factor Overexpression and Chemical Pathway Manipulation Dame et al. showed that a transient overexpression of NKX2-1 combined with chemical pathway manipulation might be a powerful tool to obtain a robust and efficient derivation of thyroid cells from mESC (194). Moreover, the protocol allows identifying the precise developmental stage where NKX2-1 acts as a signal for thyroid lineage specification (194). By using an mESC line with a Dox-inducible Nkx2-1 transgene in combination with a knock-in reporter for Foxa2, it has been observed that the induction of Nkx21 resulted in a consistent increase of the Nkx2-1-positive cells only at the anterior foregut endoderm stage. This then resulted in mature differentiated thyrocytes expressing thyroid markers at a level comparable to adult mouse thyroid tissue, indicating a singular and narrow window where Nkx2-1 acts as a thyroid lineage specification signal. Moreover, when applying the differentiation protocol by Kurmann et al. (81) to Dox-induced Nkx2-1positive cells at the anterior foregut endoderm stage, organoid formation and production of T4 could be observed after exposure to iodide (194). Interestingly, subpopulations of anterior foregut endoderm cells seem to harbor thyroid competency. By sorting cells at the anterior foregut endoderm stage based on Foxa2 expression, it was shown that the overexpression of Nkx2-1 in the Foxa2-negative cells compared to a population with high expression of Foxa2, increased the number of Nkx2-1 and Pax8-positive cells, as well as the expression of thyroid markers. In addition, there was no thyroid specification in mesodermal cells with low FOXA2 expression treated with Dox and maintained in thyroid specification media, indicating that the FOXA2-negative population possibly acquires this stage-specific thyroid competence due to a sequential progression from FOXA2-positive definitive endoderm to FOXA2-negative anterior foregut endoderm. Remarkably, based on the fact that an immediate activation of the endogenous Nkx2-1 was only observed at the anterior foregut endoderm stage in response to Dox stimulation of the transgene, it has been suggested that there is a time-dependent bistable switch that underlies the effect of Kkx2-1 overexpression on cell-fate decisions during thyroid differentiation in mESCs (194). Taken together, these thyroid models derived from mESCs have

demonstrated to be a powerful tool to study early stages of thyroid formation and the mechanisms involved in thyroid specification. Moreover, these systems are a unique source for thyroid follicles that can easily be manipulated in vitro (98,194), thus providing a consistent alternative for studies addressing mechanisms of thyroid physiology, hormonogenesis, and toxicity. As already demonstrated by using knock-in mESC lines to track developmental steps (81), these systems can be manipulated efficiently permitting to test candidate genes associated with thyroid development and disorders, for example, candidate genes associated with congenital hypothyroidism.

Human Stem Cell Models: Challenges and Potentials Based on the experience obtained from mESC-derived thyroid models, Ma et al. have used human embryonic stem (hESC) cells with the goal to recapitulate the mouse protocols and produce functional human thyroid cell lines (206). Lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line, followed by the thyroid differentiation protocol that combined Activin A and TSH treatment. The results revealed enhanced thyroid-specific gene expression in NKX21/PAX8-expressing hESC cells at the end of the differentiation protocol. Moreover, around 50% of those differentiated cells were able to form some follicle-like structures, which were also capable of cAMP generation and radioiodine uptake, suggesting they are formed by functional thyroid epithelial cells (206). Despite these data on mature thyroid cells generated from hESC, it is important to consider that this does not reflect cell reprogramming because NKX2-1 and PAX8 are constantly induced in these hESC. Moreover, several in vitro and in vivo studies have demonstrated that several thyroid differentiation markers such as Tg, Tshr, Nis and Tpo are regulated by Nkx21 and Pax8, which bind to specific target sequences in the promoter regions of these genes (see above) (121,126,128,154,200,201,207–212). Interestingly, a recent study has demonstrated that hESCs and iPSCs are intrinsically programmed to form cystic structures containing lumina when dissociated and plated at low density in 2D or 3D cultures (213). Kurmann et al. also used their mESC protocol to differentiate hESCs and

iPSCs into thyroid progenitors (81). Based on the observation that in mESCs Nkx2-1 haploinsufficiency does not affect specification, they used iPSC from hypothyroid children previously diagnosed with the Brain–Lung–Thyroid syndrome harboring three different NKX2-1 coding sequence mutations that were predicted to cause NKX2-1 haploinsufficiency. Initially, definitive endoderm was induced in each iPSC line, as well as in control hESCs (RUES2) and control iPSCs. Next, the co-treatment with BMP4 and Fgf2 resulted in induction of clusters of NKX2-1 and PAX8 co-expressing cells in all iPS and hES cell lines. After further culture in the same conditions used for mESCs thyroid differentiation, upregulation of NKX2-1, PAX8, and TG was observed and maintained for at least 42 days. However, due to the absence of a human NKX2-1 reporter in those hPSC lines, thyroid progenitors could not be sorted from these heterogeneous endodermal cultures for further analysis and maturation steps (81). Additional studies where then performed by Serra et al. using hESC lines engineered to carry a GFP reporter targeted to the NKX2-1 locus (193). By treating human PSCderived foregut-staged cells with maturation medium containing Fgf2+BMP4 they also observed induction of PAX8 and TSHR expression (193). These findings indicate that BMP and Fgf signaling can trigger thyroid specification from human developing endoderm. The transcriptional co-activator with PDZ binding motif (TAZ) has been shown to function as a co-activator that regulates PAX8 and NKX2-1 (163). Based on the functional role of TAZ, Kawano et al. explored the potential of its induction on the promotion of hESC differentiation to thyroid cells (214). Ethacridine, a TAZ activator, was used in Activin-derived endodermal cells, followed by a differentiation protocol using ethacridine and TSH. Endodermal cells treated with ethacridine demonstrated TAZ induction followed by an increase in PAX8 and NKX2-1 levels. After differentiation with ethacridine and TSH, thyroid markers such as TG, TPO, TSHR, and NIS were augmented in the differentiated hES cells. When these cells where cultured in Matrigel, follicle-like structures were observed, which contained abundant TG protein (214). In addition, hES cell–derived thyroid follicle-like structures showed significant radioiodine uptake and organification (215). Despite the advances obtained using hESC to model thyroid tissue in vitro, these systems are less advanced than the mESC-based models, possibly because of several significant differences between these systems. It is clear

that the efficiency of thyroid-fate induction and reprogramming in hESCs is very low compared to mESCs. To date, it has been a big challenge to translate the knowledge obtained from mESC-derived thyroid models to generate a sufficient amount of human thyroid progenitors for the use as source to explore the mechanisms involved in human thyroid maturation.

CONCLUSIONS Development is a dynamic process and a thorough elucidation of the underlying mechanisms is key for the understanding of physiologic and pathologic processes, including congenital diseases and cancer. The knowledge on the development of hypothalamus–pituitary–thyroid, between the previous edition of this book and the present, has increased significantly. For example, mutations in several genes involved in thyroid organogenesis have been described in humans with congenital hypothyroidism (see Chapter 40). Many of the genes involved in different processes that occur at the beginning of organogenesis, as well as the signals that control them, have now been identified. In the future, new cell and animal models will accelerate efforts to discover new functions of these genes. In addition, next generation sequencing will help to identify new genes and epigenetic studies will contribute to generate new concepts. The new insights allowing to generate thyroid cells able to secrete thyroid hormones from pluripotent stem cells open a novel field of investigation and, perhaps, therapy in the future. Stem cell technology constitutes a powerful tool to dissect mechanisms involved in tissue development, function and defects. The in vitro models derived from mESC are a unique source for follicular structures that permit investigating basic processes. The generation of a functional thyroid model from hESC remains a big challenge, but will be required for modeling specific thyroid disorders and the development of novel therapeutic modalities, for example for congenital hypothyroidism.

ACKNOWLEDGMENTS Pilar Santisteban acknowledges the support of Grants: SAF2016-75531-R

(Spanish Ministry of Science, Innovation and Universities); S2017/BDM3724 TIRONET2-CM (Community of Madrid, Spain); European Regional Development Fund (FEDER); GCB14142311CRES (Foundation of Spanish Association Against Cancer (FAECC).

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THYROID HORMONE PART B SYNTHESIS AND SECRETION

3 Thyroid hormone synthesis: thyroid iodide transport Nancy Carrasco

Iodine is an essential micronutrient because it is an indispensable constituent of the thyroid hormones T3 and T4 [triiodothyronine and thyroxine (or tetraiodothyronine), respectively), the only iodine-containing hormones in vertebrates. Therefore, thyroid function ultimately depends on an adequate supply of iodide (I−), which can only be obtained through the diet, to the gland. A remarkably efficient and specialized system has evolved in the thyroid that ensures that ingested dietary I− is accumulated in the gland and thus made available for T3 and T4 biosynthesis. The significance of this becomes more apparent when one considers that I− is rather scarce in the environment. Endemic goiter and cretinism caused primarily by an insufficient dietary I− supply remain major health problems in many parts of the world, affecting millions of people (1). This situation dramatically underscores the health value of I− as a nutrient and the consequences to society of its environmental scarcity. Iodine was discovered in 1811 by Courtois, who isolated it from treating seaweed ash with sulfuric acid (2,3). The ability of the thyroid to concentrate I− was first reported as early as the 19th century (4,5). The thyroid gland concentrates I− by a factor of >40 with respect to its concentration in the plasma under physiologic conditions. Hence the existence of a thyroid I− transporter was inferred, and some of its properties were elucidated over the years (see (6–8) for reviews). Briefly, I− accumulation in the thyroid has long been known to be an active transport process that occurs against the I− electrochemical gradient, stimulated by thyrotropin (TSH), and inhibited by

the anions thiocyanate (SCN−) and perchlorate (ClO4−). Since the 1940s, the ability of the thyroid to accumulate I− has provided the basis for diagnostic scintigraphic imaging of the gland with radioiodide (RAI), and has served as an effective means for administered RAI to target and destroy hyperfunctioning thyroid tissue, such as in Graves disease, or I− transporting thyroid cancer cells. Indeed, RAI treatment of thyroid cancer administered after thyroidectomy is the most effective targeted internal radiation cancer therapy. Nevertheless, no molecular information about the I− transporter was available until 1996, when, after a decades-long search by numerous investigators, the cDNA encoding the elusive transporter was finally isolated by Dai et al. by expression cloning in Xenopus laevis oocytes (3). The protein was named the Na+/I− symporter (NIS) because it couples the inward “downhill” translocation of Na+ to the inward “uphill” translocation of I−. The driving force for this process is the inwardly directed Na+ gradient generated by the Na+/K+ ATPase (Fig. 3-1). One of the most remarkable properties of NIS is precisely its ability to transport I−, both because it is difficult to bind a halide ion such as I− with high affinity (9) and because NIS discriminates in an exquisite way between I − and Cl−: unlike Cl− transporters and channels, which transport both anions (10–12), NIS does not transport Cl−, even though its concentration in the extracellular fluid (∼100 mM) is >105 times that of I− (sub-µM). NIS mediates the first step in thyroid hormone biosynthesis, which is crucial: the active transport of I− against its electrochemical gradient across the basolateral plasma membrane into the cytoplasm of the follicular cells (I− uptake) (Fig. 3-1). After reaching the cytosol, I− is passively translocated across the apical membrane into the colloid, located in the follicular lumen (I − efflux). I− efflux to the follicular lumen has been suggested to be mediated by transporters and/or channels located on the apical side of the follicular cells, facing the colloid (see “I− efflux” below) (Fig. 3-1). NIS research at the molecular level is an exciting field in thyroidology. This chapter, after giving some historical perspective, focuses on the most recent advances and their impact on our understanding of thyroid pathophysiology and on a wide variety of basic and clinical fields, including structure and function of and mechanistic information on membrane transporters, metabolism, cancer, gene transfer studies, and public health.

IDENTIFICATION BY FUNCTIONAL EXPRESSION OF THE CDNA ENCODING RAT NIS A promising development in the search for the I− transporter was the expression of ClO4−-sensitive Na+/I− symport activity in Xenopus laevis oocytes by Vilijn et al. (13), by microinjection of poly-A+ RNA isolated from FRTL-5 cells, a highly functional line of rat thyroid–derived cells. Thus, in the absence of oligonucleotides based on protein sequence data or anti-NIS antibodies, the oocyte system was shown to be of value for the possible expression cloning of the cDNA that encodes NIS. After a few years, this cloning strategy was successful. Dai et al. (3) generated several cDNA libraries from poly-A+ RNA of FRTL-5 cells, and subjected them to expression screening in Xenopus laevis oocytes. The expression cloning of NIS was carried out by measuring ClO4−-sensitive Na+/I− symport activity in oocytes microinjected with cRNAs made in vitro from pools containing decreasing numbers of cDNA clones, until the single clone was identified. The cloning of NIS marked the beginning of its molecular characterization, as discussed below.

THE NIS MOLECULE The complete nucleotide sequence of the cloned rNIS cDNA and the deduced 618 amino acid sequence are presented in reference (3). The hydropathic profile and initial secondary structure predictions of the protein led Dai et al. (3,14) to propose that NIS was an intrinsic membrane protein with 12 transmembrane segments (TMSs). However, this model has subsequently been revised upon extensive experimental testing, as detailed below. The NH2 terminus was originally placed on the cytoplasmic side, given the absence of a signal sequence. The COOH terminus, which was also predicted to be on the cytoplasmic side, is a hydrophilic region of ∼70 amino acids. Levy et al. (15) confirmed that the COOH terminus faces intracellularly, as an anti-COOH terminus antibody binds to its epitope only after cell permeabilization. NIS has three potential N-glycosylation sites, at positions 225, 485, and 497. Levy et al. (16) demonstrated that, contrary to previous

suggestions (17), neither partial nor total lack of N-linked glycosylation impairs activity, stability, or targeting of NIS. Their studies also showed that N225, which was originally predicted to face intracellularly, is indeed glycosylated, indicating that the hydrophilic loop that contains N225 faces the extracellular milieu rather than the cytosol. The authors thus proposed and experimentally showed that NIS traverses the plasma membrane 13 times and that the NH2 terminus faces extracellularly (16) (Fig. 3-2).

FIGURE 3-1. NIS mediates active I− transport in thyroid follicular cells, the first step in thyroid hormone biosynthesis. The basolateral surface of the cell is shown on the left side of the figure, and the apical surface on the right. Red circle: NIS translocating 2 Na+ per I−; black and gray rectangles: KCNQ1-KCNE2 K+ channel subunits; green diamond: Na+/K+ ATPase, which pumps 3 Na+ out per 2 K+ pumped in per ATP hydrolyzed; light blue: proteins and Cl− channels mediating the efflux of I−into the colloid; magenta: TPO (thyroid peroxidase); dark blue: DUOX2 (dual oxidase); green coil: Tg (thyroglobulin).

NIS, the product of the SLC5A5 gene, is a member of solute carrier family 5 (SLC5), which also includes—among many other proteins that mediate key physiologic transport processes—Na+/glucose cotransporter 1 (SGLT1) (18). The current 13-TMS model for NIS has been confirmed with the determination, by Faham et al. (19), of the crystal structure of vSGLT, the

Vibrio parahaemolyticus Na+/galactose transporter, with which NIS shares significant identity (27%) and homology (58%). vSGLT is the bacterial homolog of the human SGLT1.

ISOLATION OF THE CDNA ENCODING HUMAN NIS (hNIS) AND ELUCIDATION OF THE GENOMIC ORGANIZATION OF hNIS The hNIS cDNA was identified on the expectation that hNIS would be highly homologous to rNIS, given that transporters are highly conserved across different species. Using primers to the cDNA rNIS sequence, Smanik et al. (20,21) amplified a cDNA fragment of hNIS from human papillary thyroid carcinoma tissue by PCR, utilized this cDNA fragment to screen a human thyroid cDNA library, and isolated a single cDNA clone encoding hNIS. The nucleotide sequence of hNIS has an open reading frame of 1,929 nucleotides, which encodes a protein of 643 amino acids. hNIS exhibits 84% identity with and 93% similarity to rNIS. hNIS differs from rNIS only on account of a five–amino acid insertion between the last two TMSs (amino acids 485 to 488 and 499) and a 20–amino acid insertion in the COOH terminus (amino acids 618 to 637). Subsequently, Smanik et al. (21) examined the expression, exon–intron organization, and chromosome mapping of hNIS. Fifteen exons encoding hNIS were found to be interrupted by 14 introns (Fig. 3-3), and the hNIS gene was mapped to chromosome 19p. The human NIS promoter has been sequenced by three different groups (22–24).

FIGURE 3-2. Structural models of NIS. A: Experimentally tested secondary structure model. Cylinders: TMS (transmembrane segments); Trees: Nlinked glycomoieties. Iodide transport defect–causing mutations are shown in the rounded rectangles, which contain the wild-type amino acid letter, the amino acid’s position, and the letter for the amino acid causing the mutation. Cylinders: TMS (transmembrane segments); trees: N-linked glycomoieties. B: NIS homology model based on the crystal structure of vSGLT. TMSs represented in different colors, match those used in A.

NIS IN EXTRATHYROIDAL TISSUES The field of I− transport systems outside the thyroid has changed considerably since the extensive review published on the topic in 1961 by Brown-Grant (25). The main vertebrate nonthyroid tissues reported to actively accumulate I− are salivary glands, stomach, and lactating mammary gland (Fig. 3-4). Many of these transport systems exhibit functional similarities with their thyroid counterpart, notably a susceptibility to inhibition by SCN− and ClO4−. However, they also display important differences: (a) Nonthyroid I− transporting tissues do not have the ability to organify accumulated I−; therefore, they behave like MMI-treated thyroid tissue; (b) TSH exerts no regulatory influence on nonthyroid I− accumulation; (c) at least salivary glands and gastric mucosa concentrate SCN−, unlike the thyroid, where SCN− is metabolized after uptake and therefore not concentrated. NIS is clearly regulated and processed differently in each tissue. The cloning of human NIS cDNAs has been reported from gastric mucosa, parotid and lactating glands, all of which exhibited full identity to thyroid hNIS cDNA. Whereas hNIS gene expression has been detected in many other tissues by RT–PCR, it must be pointed out that the RT–PCR technique yields a large number of false positives due to its high sensitivity (26,27). Therefore, the detection of the NIS amplified product by RT–PCR in a given tissue cannot be regarded as sufficient evidence that NIS is functionally expressed in that tissue. A thorough characterization of NIS protein expression is necessary to properly evaluate the significance of results obtained by RT– PCR and Northern analysis. Still, even with the use of a wide variety of techniques (Northern analysis, RT–PCR, Western analysis, and immunohistochemistry), different groups have often obtained inconsistent

and sometimes conflicting results on whether or not NIS is expressed in a particular tissue. Hence, once NIS protein expression has been demonstrated, a correlation with Na+-dependent, ClO4−-sensitive, active I− accumulation in that tissue must be established. By these criteria, and taking into consideration the above results, NIS is expressed and active in extrathyroidal tissues previously known to actively accumulate I−, such as salivary glands, stomach, lactating breast, and placenta (Fig. 3-4). Physiologically, I− transport in the mammary gland occurs during late pregnancy and lactation, resulting in the transfer of I− to the milk. An adequate supply of I− for sufficient thyroid hormone production is essential for proper development of the newborn’s nervous system, skeletal muscle, and lungs. In contrast, no I− transport is observed in normal breast tissue in the absence of pregnancy and lactation (28). In the placenta, NIS is expressed at the apical membrane of cytotrophoblasts, where it transports I− from the maternal to the fetal circulation, a process critical for adequate fetal thyroid function (29,30).

FIGURE 3-3. Correlation between the structural organization of the hNIS gene and that of the hNIS protein. Exons in the hNIS gene are represented by gray rectangles. The 13 TMS making up the protein are represented by cylinders. Exons are connected to the corresponding amino acid regions of the protein by dotted lines. The localization of the iodide transport defect mutations is indicated under the schematic representation of the hNIS protein.

FIGURE 3-4. Immunohistochemical analysis of NIS protein expression in tissues that display active I− transport. (1) Thyroid; (2) Salivary gland; (3) Stomach; (4) Lactating breast; (5) Small intestine; (6) Kidney. Note that NIS is expressed basolaterally in 1 through 4 but apically in 5 and 6.

In the small intestine, where dietary I− is absorbed, Nicola et al. (31) showed that the apparent affinity of intestinal NIS for I− (Km ∼ 10 to 20 μM) is indistinguishable from that of thyroid NIS. Interestingly, NIS is expressed apically in the small intestine (Fig. 3-4), unlike in other tissues, where it is expressed basolaterally. In addition, high I− downregulates NIS protein expression and I− uptake in the small intestine in a time-dependent manner in vivo. Therefore, it seems that NIS downregulation after high I− exposure may be a general autoregulatory mechanism to protect the thyroid or other I−transporting tissues from the possibly deleterious consequences of I− overload. It seems plausible that NIS-mediated secretion of I− into the saliva and gastric juice and NIS-mediated absorption of I− in the small intestine are part of a systemic mechanism that tends to conserve and recycle I−, considering that most of the I− secreted in the upper gastrointestinal tract is not lost but rather reabsorbed in the small intestine alongside newly ingested dietary I−.

Beyond the tissues already mentioned, more recent evidence points to the kidney as yet another tissue where functional NIS is expressed (Fig. 3-4). The excretion and reabsorption of I− by the kidney are crucial for adequate TH secretion and for maintaining euthyroid conditions. To determine the role of NIS in the renal proximal tubule, Reyna-Neyra et al. (32) generated a renal proximal tubule (i.e., tissue-specific) NIS KO mouse model by crossing the Slc5a5flox/flox mice with cGT-Cre mice, in which CRE recombinase expression is driven by the gamma-glutamyl transpeptidase promoter (cGTCRE). When the resulting Slc5a5flox/flox cGT-Cre mice were fed a diet supplying them with the minimum amount of I− that mice require daily (0.15 µg I−/g food), they excreted 30% more I− in their urine than control mice. These results suggest that NIS mediates the reabsorption of I− in the kidney and may thus be critically important for I− homeostasis.

NIS MECHANISM AND STOICHIOMETRY Eskandari et al. (33) investigated the mechanism, stoichiometry, and specificity of NIS using electrophysiologic, tracer uptake, and electron microscopy methods in Xenopus laevis oocytes expressing NIS. Using the two-microelectrode voltage clamp technique, they showed that an inward steady-state current (i.e., a net influx of positive charge) is generated in NISexpressing oocytes upon addition of I− to the bathing medium, leading to depolarization of the membrane and indicating that NIS-mediated I− transport is electrogenic. Simultaneous measurements of tracer fluxes and currents revealed that two Na+ ions are transported with one anion, demonstrating unequivocally a 2:1 Na+/I− stoichiometry. Therefore, the observed inward steady-state current is due to a net influx of Na+ ions. Eskandari et al. (33) determined that the turnover rate of NIS is ∼36 s−1, and reported that expression of NIS in oocytes led to a ∼2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles, as ascertained by freeze-fracture electron microscopy. This was the first direct electron microscopy visualization of ostensible NIS molecules. A long-standing question in the field was how NIS transports I− even though its plasma concentration is in the submicromolar range. The answer lies in the conformational changes that occur when NIS binds Na+. Nicola et

al. (34) obtained extensive kinetic data, which were analyzed using a statistical thermodynamic formalism. They determined that the intrinsic affinity of NIS for I− (Kd = 223 µM) is 10 times higher (Kd = 22.4 µM) when Na+ is already bound to the transporter. Even so, this Kd is still one or two orders of magnitude greater than the concentrations of I− available under typical physiologic conditions (35–37). Ultimately, transport is possible because, at physiologic Na+ concentrations, ∼79% of NIS molecules are occupied by two Na+ ions, and hence ready to bind and transport I− even when the I− concentration is well below its Kd. This may explain at the kinetic level why, under conditions of extreme I− scarcity, the only way to increase the transport rate and the resulting level of I− accumulation is to increase the number of NIS molecules. Because of this, the long-term physiologic response to an inadequate dietary supply of I− is an enlargement of the thyroid gland (goiter) (38).

SUBSTRATE SPECIFICITY OF NIS As mentioned above (see Introduction), NIS discriminates exquisitely between Cl− and I−: it transports I− but not Cl−, even though the concentration of Cl− in the extracellular fluid is >105 times that of I−. However, NIS does transport several other anions. In electrophysiology experiments using NISexpressing Xenopus laevis oocytes, steady-state inward currents were generated not only by I− but also by ClO3−, SCN−, SeCN−, NO3−, Br−, BF4−, IO4−, and BrO3− when they were added to the medium, indicating that these anions are all transported by NIS electrogenically (33). However, ClO4−, the most widely used inhibitor of thyroidal I− uptake, was surprisingly found not to generate a current, strongly suggesting that it was not transported. Yoshida et al. (39,40) reported, similarly, that ClO4− did not induce an inward current in mammalian cells expressing NIS. The most likely interpretation of these observations seemed at the time to be that ClO4− was not transported by NIS, although the unlikely possibility that ClO4− was translocated by NIS with an electroneutral 1 Na+:1 ClO4− stoichiometry could not be ruled out—and the unavailability of 36ClO4− made flux experiments impossible.

NIS TRANSPORTS THE ENVIRONMENTAL POLLUTANT ClO4− WITH A 1 Na+:1 ClO4− STOICHIOMETRY Using a bioassay consisting of a monolayer of polarized epithelial cells expressing NIS, Dohán et al. (41) conclusively—and surprisingly— demonstrated that ClO4− is in fact actively translocated by NIS after all. These authors extended their in vitro observations by building upon their discovery that NIS is expressed in the lactating breast (28). When 131I− or 131I − plus ClO − was injected into lactating dams but not into their pups, 4 thyroidal 131I− uptake in both dams and pups was markedly lower in the ClO4−-treated animals than in the controls. In conclusion, it is clear that ClO4− is not a nontransported blocker but rather a substrate that is actively transported by NIS. It is now evident that ClO4− elicited no currents in electrophysiology experiments because Na+-dependent ClO4− transport is electroneutral and, therefore, its kinetic analysis could only be carried out by direct substrate flux measurements (which, as indicated above, are not feasible). Thus, Dohán et al. (41) instead analyzed the kinetic parameters of NIS-mediated transport of a structurally similar anion, perrhenate (186ReO4−). The initial rates of NIS-mediated, Na+-dependent ReO4− transport yielded a hyperbolic curve indicating an electroneutral stoichiometry, strongly suggesting that the stoichiometry of NIS-mediated Na+/ClO4− transport is also electroneutral. This stoichiometry stands in stark contrast to the electrogenic 2 Na+:1 anion stoichiometry observed with most other NIS substrate anions, demonstrating that NIS translocates different substrates with different stoichiometries, a remarkable property that had not previously been discovered in any transporter. These authors developed a simple mathematical model that accurately predicts the degree of competition between ClO4− and I− and that the affinity of NIS for ClO4− should be 10 times higher than for I− (34). The results of Dohán et al. have been confirmed by Tran et al. (42), who showed, using a sensitive chromatography– electrospray ionization–tandem mass spectrometry method, that ClO4− is actively transported in FRTL-5 cells. Significant insights have been gained into the ability of NIS to translocate

different substrates with different stoichiometries by characterizing a NIS mutant, G93R (Fig. 3-2), found in a patient (43). Although G93R NIS is inactive, Paroder-Belenitski et al. have reported that a single amino acid substitution at position 93, for example, G93T or G93N, changes the stoichiometry of NIS-mediated ClO4− or 186ReO4− transport from electroneutral to electrogenic (see “Iodide Transport Defect” below).

IODIDE TRANSPORT DEFECT, A CONGENITAL CONDITION CAUSED BY MUTATIONS IN THE NIS (SLC5A5) GENE Mutations in the SLC5A5 gene, which encodes NIS, lead to congenital hypothyroidism due to an I− transport defect (ITD). ITD is a rare autosomal recessive condition clinically characterized, when untreated, by hypothyroidism and goiter of varying degrees, stunt growth, and even mental retardation. ITDs are diagnosed by reduced I− uptake on scintigraphy, and a low I− saliva-to-plasma ratio (normal >30). To date, 16 ITD-causing NIS mutations have been reported: V59E, G93R, R124H, Q267E, V270E, C272X, Δ287–288, T354P, Y358D, G395R, frame-shift 515X, Y531X, G543E, G561E, Δ143–323, and Δ439–443 (Fig. 3-2). They are either nonsense, alternative splicing, frame-shift, deletion, or missense mutations of the NIS gene. In addition, Nicola et al. have also reported one mutation in the 5′ untranslated region of NIS, a C-to-T transition (44). Invaluable mechanistic and structural information on NIS has been obtained by characterizing the amino acid positions bearing mutations in ITD patients and elucidating the molecular requirements of NIS at those positions. Nine of the aforementioned NIS mutants (V59E (45), G93R (43), R124H (46), Q267E (47), V270E (48), T354P (49), Y358D (50), G395R (51), and G543E (52)) have been analyzed in detail, yielding a wealth of mechanistic information. For example, the first ITD-causing mutation found, T354P, led De la Vieja et al. (53) to identify the Na2 binding site, and Ravera et al. (54) to gain insight into Na+ cooperativity —a central part of the mechanism of NIS function. The analysis of the T354P substitution by Levy et al., which revealed that this position requires an OH group at the β-carbon (49), led these authors to study the importance of other

β-OH–containing residues in TMS 9 (the region of NIS where such residues are most abundant). De la Vieja et al. (53) showed that these residues are involved in Na+ binding/translocation, and proposed a structural homology between LeuT and NIS, even though there is no primary sequence homology between the two. LeuT is the leucine transporter from Aquifex aeolicus, the structure of which was determined at very high resolution (1.6 Å) by Yamashita et al. (55). The crystal structure of vSGLT, the Vibrio parahaemolyticus Na+/galactose transporter, which belongs to the same family as NIS, was determined by Faham et al. (19), who showed that, as predicted by De la Vieja et al. (53), vSGLT does indeed have the same fold as LeuT (55). This fold consists of two inverted repeats (TMS 1–5 and 6–10) that are related by a pseudo–2-fold axis of symmetry parallel to the membrane, and two unwound helices that provide the right environment for substrate coordination. Below is a summary of the main findings obtained from the detailed study of the NIS positions bearing amino acid substitutions in ITD patients, in the order in which they were characterized (for more details, see reference (56)). Dohán et al. (51) showed that the presence of an uncharged amino acid residue with a small side chain at position 395 is required for NIS function, and that the presence of a charge or a long side chain at position 395 decreases the turnover rate of the transporter, without affecting its apparent affinity for I−. Based on flow cytometry data, it was initially suggested that the Q267E mutation results in impaired trafficking of the mutated NIS protein to the plasma membrane (57). However, De la Vieja et al. (47), have reported that the Q267E mutant NIS is modestly active and properly targeted to the plasma membrane. The mutant protein has a lower Vmax for I− than the wild-type protein, which could result in the lower transport rate of Q267E NIS. In contrast to T354P, G395R, and Q267E, De la Vieja et al. (52) reported that G543E NIS matures only partially and was the first mutant identified that is not targeted properly to the cell surface, apparently because of faulty folding. Therefore, the G543 residue plays significant roles in NIS maturation and trafficking. Remarkably, NIS activity was rescued by small neutral amino acid substitutions (volume A [R2223H]) resulting in fetal goitrous hypothyroidism. J Clin Endocrinol Metab 2003;88(8):3546–3553. Targovnik HM, Vono J, Billerbeck AE, et al. A 138-nucleotide deletion in the thyroglobulin ribonucleic acid messenger in a congenital goiter with defective thyroglobulin synthesis. J Clin Endocrinol Metab 1995;80(11):3356–3360. Medeiros-Neto G, Kim PS, Yoo SE, et al. Congenital hypothyroid goiter with deficient thyroglobulin. Identification of an endoplasmic reticulum storage disease with induction of molecular chaperones. J Clin Invest 1996;98(12):2838–2844. Kim PS, Ding M, Menon S, et al. A missense mutation G2320R in the thyroglobulin gene causes non-goitrous congenital primary hypothyroidism in the WIC-rdw rat. Mol Endocrinol 2000;14(12):1944–1953. Godlewska M, Banga PJ. Thyroid peroxidase as a dual active site enzyme: focus on biosynthesis, hormonogenesis and thyroid disorders of autoimmunity and cancer. Biochimie 2019;160:34–45. Taurog A. Hormone synthesis: thyroid iodine metabolism. In: Braverman L, Utiger R, eds. Werner and Ingbar’s the Thyroid: a Fundamental and Clinical Text. 7th ed. Philadelphia, PA: LippincottRaven; 1996:47–81. Taurog A. Hormone synthesis: thyroid iodine metabolism. In: Braverman L, Utiger R, eds. Werner and Ingbar’s the Thyroid: A Fundamental and Clinical Text. 8th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2000:61–85. Kimura S, Kotani T, McBride OW, et al. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs. Proc Natl Acad Sci U S A 1987;84(16):5555–5559. de Vijlder JJ, Dinsart C, Libert F, et al. Regional localization of the gene for thyroid peroxidase to human chromosome 2pter-p12. Cytogenet Cell Genet 1988;47(3):170–172. Kimura S, Hong YS, Kotani T, et al. Structure of the human thyroid peroxidase gene: comparison and relationship to the human myeloperoxidase gene. Biochemistry 1989;28(10):4481–4489. Magnusson RP, Gestautas J, Taurog A, et al. Molecular cloning of the structural gene for porcine thyroid peroxidase. J Biol Chem 1987;262(29):13885–13888.

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PERIPHERAL THYROID HORMONE PART C BINDING AND METABOLISM

5 Thyroid hormone transport proteins David Halsall and Carla Moran

Triiodothyronine (T3) and its precursor thyroxine (T4) are classic endocrine hormones in that they are secreted at a distant site from that of their actions. As these molecules are hydrophobic, carrier molecules are required to facilitate transport via the bloodstream. Three major proteins have been identified that fulfil this function, which together bind 99.97% and 99.7% of circulating T4 and T3 (thyroid hormones—THs), respectively. In order of the amount of T4 bound in human plasma and in reverse order of the plasma concentration these are T4-binding globulin (TBG), transthyretin (TTR), and albumin (ALB). The plasma concentration and fraction of bound T4 in humans for TBG, TTR, and ALB are 0.015 g/L, 75%; 0.25 g/L, 15%; and 42 g/L, 10%, respectively (1). TBG binds thyroid hormone (TH) specifically. TTR also binds retinol-binding protein and is the major TH carrier protein in the central nervous system (CNS) as well as other extracirculatory sites. ALB also binds a vast array of other ligands in addition to THs (Table 5-1) (2). Other thyroid carrier proteins exist but these bind proportionately less TH and are less well studied. A possible thyroid-binding motif has been identified (Tyr, Leu/Ile/Met, X, X, Val/Leu/Ile), which is conserved in human TBG, TTR, ALB, and apolipoproteins (3); this has been used to speculate on the existence of other potential TH-binding proteins (THBPs). TABLE 5-1. Main Characteristics of the Major Protein Transporters of Thyroid Hormones in Humans TBG Protein

TTR

Albumin

Molecular mass (kDa)

54

55 (tetramer)

66.5

Residues (n)a

395

127

585

Carbohydrate (%)

20

0

0

Electrophoretic mobility

α-globulinb

Prealbumin

Albumin

Serum concentration (mg/L)

12–30

150–400

∼35,000–50,000

Serum concentration (μM)

∼0.22–0.56 μM

∼2.8–7.4 μM

∼540–770 μM

Half-life (days)

5

2

15

Number

1

2

4/5c

Ka for T4 (M−1)d Primary site

1.0 × 1010

2.0 × 108

1.5 × 106

Ka for T3 (M−1)d Primary site

1.0 × 109

1.0 × 106

2 × 105

% Bound plasma T4d

75

20

5

% Bound plasma T3d

75

1 mM). PTU probably inhibits D1 by competing with the endogenous thiol-containing cofactor for a putative selenenyl iodide (E-Se-I) intermediate (42). Supporting this interpretation is the fact that PTU inhibition is uncompetitive with the first D1 substrate (iodothyronine), but competitive with the second (e.g., dithiothreitol) (42). Because of the PTU insensitivity, D2- or D3-catalyzed deiodination proceeds by removal of an iodonium (I+) ion by the endogenous cofactor, resulting in an enzymethyronine intermediate [D2-T3 (or reverse T3)] complex and a cofactor-Se-I complex (43). In this regard, it is notable that the sequence Thr-Sec-ProPro/Ser-Phe is identical in D1, D2, and D3, but that in all D1 sequences, excepting the PTU-insensitive D1 of the blue tilapia (Oreochromis aureus), the uncharged polar side chain of the Ser residue substitutes for the nonpolar side chain of Pro at position 128 (44). The existence of this natural variation prompted the creation of the Ser128Pro D1, Pro135Ser D2, and Pro146Ser D3 proteins (Fig. 6-3). Remarkably, replacement of Pro135 with Ser in D2 results in a two orders of magnitude increase in Km (T4) to approximately 250 nM, approximately 10-fold lower than that of D1 for T4 and the enzyme operates with ping-pong kinetics. Furthermore, the Pro135Ser D2-catalyzed deiodination is two orders of magnitude more sensitive to PTU (Ki 4.0 μM), although PTU inhibition is noncompetitive with dithiothreitol. Thus, the substitution of Ser for Pro135 in D2 results in changes in the enzyme that

make its kinetics more similar to those of D1, indicating a critical influence of the amino acid occupying this position on enzyme function. Similar observations were made with the Pro146Ser D3 protein, which has a fivefold higher Km (T3) and is highly sensitive to inhibition by PTU (Ki 1.0 μM). As mentioned above, Ser128Pro D1 catalyzed deiodination is resistant to PTU (Ki >1 mM), suggesting that there is no longer an accessible E-Se-I intermediate, again illustrating the pivotal role of this position in the active center of the deiodinase molecule. The presence of Pro in the 128/135/146 positions of D1, D2, and D3 may result in tighter binding of the substrate in the D2 (and D3) binding pockets, perhaps explaining the approximately 1,000-fold higher affinity of D2 and D3 for T4 as compared with D1. This could reflect an interaction between the phenolic hydroxyl group of T4 and the Pro residue at these positions, and explain why sulfate conjugation of the phenolic hydroxyl group dramatically increases the maximum velocity (Vmax)/Km and changes the T4 deiodination site from an outer to an inner ring iodine (45). Three conserved amino acids with charged polar side chains mark the transition between the β2-strand and the a-helix in the IDUA-like insertion, Glu/Asp155, Glu156, and His158 (D1 residues). When the invariant Glu156 (D1), Glu163 (D2), and Glu174 (D3) are replaced with Ala, the resulting enzymes have no deiodinase activity. Replacement of Glu156 with Asp in D1 supported deiodination, but with an approximately 4.5-fold higher Km (rT3), whereas a similar substitution at position 163 in D2 did not alter the Km (T4). Thus, the acidic amino acids in this region of the deiodinase pocket are important for substrate binding or enzyme function, although the length of the side chain can vary. This is further supported by mutational studies of His at position 158 in D1: mutation to Asn, Gln, or Phe resulted in complete loss of deiodinase activity (46). Replacement of the corresponding His 165 in D2 with Asn also resulted in loss of deiodinase activity. According to the model, residues in this acidic pocket could interact with either the amino or the carboxyl group in the alanine side chain of the iodothyronines, a hypothesis supported by previous studies indicating that the positively charged T4 analogue (3,5,3′,5′-tetraiodothyroethylamine), which lacks a carboxyl group, is not a substrate for D1 (47). In addition, the compounds with the highest

affinity for D1 (lowest apparent Km values) are those that lack positively charged functional groups (NH3+), such as tetraiodothyroacetic acid. Furthermore, the Km (T4) values for D- and L-T4 are similar (47). These results argue that the carboxyl group in the iodothyronines interacts with the NH3 group of His in the 158 position of D1 and that the other acidic residues in this pocket act to reduce the ionization of the His residue. The critical role played by the IDUA-like insertion is further strengthened by the complete loss of deiodinase activity when the conserved Trp163 in D1 and the corresponding Trp 170 in D2 are replaced with Ala.

SPECIFIC PROPERTIES OF THE DEIODINASES TYPE 1 DEIODINASE PTU-inhibitable, D1-catalyzed conversion of T4 to T3 supplies approximately 20% of the T3 in the serum in normal humans, but 50% or more in patients with thyrotoxicosis. D1 is the only selenodeiodinase that can function as either an outer (5′)-or inner (5)-ring iodothyronine deiodinase, D2 and D3 being exclusively outer and inner ring deiodinases, respectively (48). The molecular basis for these differences is not known. The gene (DIO1) for human D1 is on chromosome 1 p32-p33, in a region syntenic with mouse chromosome 4, the location of mouse Dio1 (49). The complete cDNA sequences have been determined for rat, human, mouse, dog, chicken, and tilapia D1 (9,44,50–53). The size of the mRNAs for these D1s is about 2 to 2.1 kb, and all contain a UGA codon in the region encoding the active center, which is highly conserved among species. By Northern analysis, Dio1 is expressed in many tissues of most vertebrates (54–56). In rats, these include the liver, kidneys, CNS, anterior pituitary gland, thyroid gland, intestine, and placenta. In humans, D1 activity is notably absent from the CNS, but is present in the liver, kidneys, thyroid and pituitary (57,58). At the same time, clues obtained from the Dio1 knockout mouse hint that D1 might also affect thyroid economy by playing a scavenger role in iodine metabolism (59). Conjugated iodothyronines are the preferred substrates for

D1 and have increased water solubility and are thus preferentially eliminated in the urine and bile. However, loss of iodine through these pathways is minimized because conjugated iodothyronines are excellent substrates for D1, which is highly expressed in liver and kidneys (59). Regulation of D1 Synthesis Changes in D1 activity have been investigated in developing rats; in general, D1 activity is low in all tissues of fetal rats. It appears soon after birth in the intestine, liver, kidneys, cerebrum, cerebellum, and gonads (60). The agerelated changes in D1 mRNA content are similar, indicating that the changes in activity arise at a pretranslational level. The mechanism for the age-related changes in Dio1 expression is unknown. The physiologic benefit of the low D1 activity in the fetus is presumably low serum T3 concentrations, thus permitting changes in intracellular T3 to be determined by the developmentally programmed changes in D2 and D3 activity (61).

Thyroid Hormone Thyroid hormone–induced increases in D1 activity and mRNA content are well documented in rats, mice, and humans (9,62). The increases are due to increased transcription, which in the human DIO1 gene can be attributed to the presence of two thyroid hormone-response elements (TREs) in the 5′flanking region of the gene (63–65). Studies in thyroid receptor (TR) knockout mice indicate that the T3-induced D1 stimulation is largely mediated by the β subtype of the receptor (66). Given this finding, the levels of the human DIO1 gene to T3 production would be expected to be greatest in patients with thyrotoxicosis. In fact, the D1 mRNA content of peripheral blood mononuclear cells is increased in proportion to the degree of thyrotoxicosis (58). This increase can explain the acute decrease in serum T3 concentrations that occurs in response to PTU in patients with thyrotoxicosis (67).

Cytokines Interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), and other

cytokines are potential mediators of the alterations in thyroid function that occur in patients with severe nonthyroidal illness (see section on nonthyroidal illness in Chapter 14) (68–70). TNF-α, IL-1β, and interferon-γ decrease D1 activity and mRNA in rodent thyroid (FRTL5) cells (71). The effects of TNFα have been examined in hepatocytes and HepG2 cells with contradictory results. TNF-α decreased the stimulatory effect of T3 on D1 mRNA production in HEPG2 cells, an action that is blocked by nuclear factor kappa B (72). In dispersed rat hepatocytes, IL-1β and IL-6 blocks T3 stimulation of D1 mRNA and activity; however, TNF-α has no effect (73). The effect of IL1β is blocked by coexpression of the nuclear steroid receptor coactivator-1 (SRC-1), but not by CREB-binding protein (CBP) or CBP-associated factor (pCAF). Because IL-1 does not alter the amount of SRC-1 in hepatocytes, the effect is attributed to competition between IL-1 and T3-stimulated transcriptional events for limiting quantities of SRC-1. More recent studies describing decreases in D1 activity in liver and possibly D2 activity in skeletal muscle and increases in D3 activity in liver and skeletal muscle in patients dying of multiple organ failure and related conditions are discussed in the section on nonthyroidal illness in Chapter 14.

Nutritional Influences on D1 Expression A decrease in serum T3 concentrations relative to those of T4 and an increase in serum reverse T3 concentrations during fasting in humans was one of the earliest indications that the peripheral metabolism of thyroid hormones in humans is modulated by physiologic or pathophysiologic events (74). Similar changes occur in virtually all acutely ill and many chronically ill patients (75,76). Because thyroidal secretion accounts for only about 20% of daily T3 production in humans, the illness-associated decrease in serum T3 concentrations must be caused, largely if not completely, by decreased T4 to T3 conversion by D1 or D2 or by increased T3 clearance by D3 (77,78). Early studies of liver D1 activity in rats suggested that the decrease in T4 conversion to T3 that occur during fasting might be caused by a decrease in the thiol cofactor that serves as the cosubstrate for D1-catalyzed T4 to T3 conversion (79,80). However, this cofactor has not been identified. Although rats have been studied extensively as a model for the effects of fasting (and illness) on T4 to T3 conversion in humans, they are a poor model for the

effects of fasting in humans because of their low body fat content and the fact that, unlike humans, their serum TSH and T4 concentrations decrease rapidly when they are starved (81). Also, despite reduced hepatic D1 activity, total body conversion of T4 to T3 is not reduced during starvation in rats (82,83). The marked fasting-induced reduction in serum TSH, T4 and T3 concentrations (i.e., central hypothyroidism) in rats is probably due, at least in part, to leptin deficiency (84). Prefeeding rats with a high-fat diet to induce obesity results in less urinary nitrogen loss and a lesser decline in serum T4 and T3 concentrations during starvation, and serum T3 concentrations actually increase if the period of starvation is prolonged (81). In contrast, in humans, serum T3 concentrations decrease rapidly to about 50% of baseline during fasting, and they remain low for up to 3 weeks of fasting, but serum T4 and TSH concentrations change little (85).

Selenium Availability A decrease in hepatic D1 activity of Se-deficient rats and the demonstration that D1 could be labeled with 75Se were the first clues that this trace element is critical to the function of D1 (86–89). However, the effects of Se deficiency on the synthesis of intracellular selenoproteins, such as the selenodeiodinases, depend on the tissue being examined. For example, in Sedeficient rats, thyroidal D1 activity is preserved, while that in the liver declines precipitously (90), serum T3 concentrations increase, and serum T4 concentrations do not change (91). Se deficiency also decreases D1 activity in the kidneys; this is accompanied by a decrease in D1 mRNA, which does not occur in the liver (92). Se deficiency can occur in patients receiving diets that are restricted in protein content, such as those given for phenylketonuria, and has also been found in elderly patients (93–96). In Se-deficient humans, serum T4 concentrations and the serum ratio of T4 to T3 are slightly increased, but serum TSH concentrations are normal. In one endemic goiter region in Africa, there is concomitant Se deficiency (97,98). When Se was supplied to these iodine-deficient subjects, their thyroid function deteriorated, as evidenced by an increase in serum TSH concentrations and a decrease in serum T3 concentrations, suggesting that the reduction in D1 activity during Se deficiency can protect against iodine deficiency, presumably by reducing inner ring deiodination of T4 or T3 (99,100).

TYPE 2 DEIODINASE Type 2 deiodinase is an obligate outer ring selenodeiodinase that catalyzes the conversion of T4 to T3. The Km of D2 for T4 is in the nanomolar range under in vitro conditions in the presence of 20 mM dithiothreitol. The presence of D2 activity in many tissues, including bone, skin and skeletal muscle provides a plausible source for a substantial fraction of the extrathyroidally generated T3 in human serum (41). T4 causes a posttranslational decrease in D2 activity because catalysis induces ubiquitin conjugation to D2, inhibiting the enzyme (101).

Gene Structure and Chromosomal Localization The DIO2 gene is present as a single copy located on the long arm of chromosome 14 (14q24.3) in humans (102,103). It is about 15 kb in size, and the coding region is divided into two exons by an intron of approximately 7.4 kb. The exon/intron junction is located in codon 75, and is at the same position in the human and mouse DIO2 genes (102,104,105). For the human gene, there are three transcriptional start sites, 707, 31, and 24 nucleotides 5′ to the initiator codon ATG. The longest 5′-untranslated region of human D2 mRNA contains an intron of approximately 300 bp that can be alternatively spliced (105). Other splicing variants involving the coding region have also been identified (106). The human, mouse, and rat DIO2 5′-flanking regions have been isolated and functionally characterized. All contain a functional cyclic adenosine monophosphate (cyclic AMP) response element, but only human DIO2 has binding sites for thyroid transcription factor-1 (TTF-1) (105,107,108).

D2 mRNA and Protein Human, mouse, and chicken D2 cDNAs containing intact 3′-untranslated regions (5-to 7.5-kb) have been identified using GenBank searches and library screening. These D2 cDNAs encode functional D2 proteins, as determined by expression in Xenopus laevis oocytes (104,109,110). Rat and human D2 mRNAs are approximately 7.5 kb, and chicken cDNA is

approximately 6 kb (41,109–111). A detailed analysis involving nuclease mapping, primer extension, and Northern blots indicated that human D2 mRNA exists as four different transcripts in thyroid, brain, and possibly other tissues (105). The longest transcript is approximately 7.5 kb, starts 708 nucleotides upstream from the initiator ATG codon, and is the only transcript found in placenta. A shorter (approximately 7.2-kb)-minor D2 species uses the same transcriptional start site, but the approximately 300-bp intron is spliced out. Two shorter transcripts of approximately 6.8 kb, differing by only seven nucleotides, use 3′ transcriptional start sites close to the translation initiation site. It is not known whether the rat and mouse genes use the same two major transcriptional start sites, but this is likely to be so for D2 in rat brain (41,105,111). The deduced amino acid sequences of the chicken, mouse, rat, and human D2 enzymes contain two selenocysteine residues. The first is in the active center of the enzyme, whereas the second is located close to the carboxy-terminus. In fish and frog D2, there is only one selenocysteine codon, which is located in the active center of the enzyme (41,104,110–113). Truncating the C-terminal amino acids, including the C-terminal selenocysteine, in human D2 has no effect on D2 enzyme kinetics or activity (114).

Tissue Distribution In rodents, D2 activity is predominantly expressed in the pituitary, brain, and brown adipose tissue (BAT) (36,115–119). D2 activity is also present in the bone, gonads, pineal, thymus, and uterus of rats, mammary gland of mice (60,120–125). High levels of D2 mRNA and activity are found in the mouse liver at the first postnatal day (126) and mouse cochlea at the eighth postnatal day (127), suggesting a role for D2 in generating T3 for postnatal development. In the cerebral cortex of neonatal rats, D2 mRNA is present in astrocytes (128) and tanycytes; the latter are specialized ependymal cells lining the third ventricle that have multiple cellular processes that express D2 mRNA and that extend to the median eminence (128–131). A monosynaptic pathway has also been identified between the arcuate nucleus, which contains D2, and the paraventricular nucleus, which contains thyrotropin-releasing hormone (TRH) (132). In humans, D2 mRNA or activity is expressed in vascular smooth muscle

cells, thyroid, heart, brain, spinal cord, skeletal muscle, and placenta, and small amounts of D2 mRNA have been detected in the kidneys and pancreas (41,105,111,133–135). Thyroid tissue contains relatively more D2 mRNA than D2 activity, with the exception of thyroid tissue in patients with thyrotoxicosis caused by Graves disease and follicular adenomas, in which both D2 mRNA and activity are present in large amounts (133). The discrepancy between D2 mRNA and activity is probably due to substrateinduced D2 ubiquitination. D2 mRNA sequences are also present in libraries from prostate, breast, and uterus, but none of these tissues have D2 activity (109). D2 mRNA or activity is present in human pituitary glands and brain tumors (133,136–138), and D2 activity has been found in human keratinocytes (139) and mesothelioma cells (29).

Regulation of D2 Synthesis The DIO2 gene is regulated by a cyclic AMP-mediated pathway. Cold exposure increases D2 mRNA and activity in BAT in rodents, and α1- or βadrenergic antagonist agents block this effect (119). In isolated brown adipocytes, the increase of D2 activity during catecholamine treatment is actinomycin D sensitive (140–142). In addition, D2 activity in BAT is induced by norepinephrine, isoproterenol, insulin, and glucagon, and it is inhibited by growth hormone (143,144). Cyclic AMP increases D2 activity in mesothelioma cells (29) and in rat astroglial cells (116,145), as does both nicotine and cyclic guanosine monophosphate (cyclic GMP) (146,147). As noted above, D2 mRNA and activity are increased in thyroid tissue from patients with Graves thyrotoxicosis, and forskolin increases D2 mRNA in dispersed human thyroid cells (105,133). It is therefore not surprising that human, rat, and mouse Dio2 contains a cyclic AMP response element approximately 90 nucleotides upstream of the transcriptional start site (105,107,108,148). The promoter activity of human DIO2 increases 10-fold when cells are cotransfected with DIO2 constructs and the α-catalytic subunit of protein kinase A. Mutation of the latter element abolishes the effect and decreases basal expression of DIO2 by approximately 90% (105). Acting via a plasma membrane receptor known as TGR5, bile acids can also stimulate the cAMP pathway and activate D2 expression in BAT and

skeletal muscle (149). This has been shown to activate thermogenesis and protect against diet-induced obesity. In addition, certain flavonols such as kaempferol also cause cAMP accumulation and D2 induction in BAT and skeletal myocytes (150), opening the interesting possibility that pharmacologic intervention of this pathway might have clinical benefit in the treatment of obesity. Although there is a high level of D2 mRNA in human thyroid tissue, no D2 mRNA or activity is present in FRTL-5 rat thyroid cells, and in adult rat thyroid tissue D2 mRNA levels are very low and D2 activity is undetectable (108,133,151). Expression of the DIO2 gene in human thyroid tissue is under the control of TTF-1 but is not affected by PAX-8 (108). The human DIO2 gene has two TTF-1 binding sites, which are not present in the rat Dio2 gene, despite an overall 73% cross-species homology. The lack of these sites may explain the very low expression of D2 mRNA and activity in rat thyroid tissue. Cells respond rapidly to endoplasmic reticulum stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and endoplasmic reticulum–associated degradation pathways. This process affects D2 synthesis. Endoplasmic reticulum stress causes rapid loss of D2 activity, to as low as of 30% of basal levels, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells engineered to express D2. The rapid loss of D2 results from slower rate of D2 synthesis and reduction intracellular D2-mediated T3 production. Remarkably, endoplasmic reticulum stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity (152).

Regulation of Degradation of D2 D2 is the critical T3-generating deiodinase due to its substantial responsiveness to physiologic signals. For example, Dio2 responsiveness to cyclic AMP constitutes the basis for the adrenergic stimulation of D2 activity in BAT, human skeletal muscle and thyroid tissue. This links D2 expression with the sympathetic nervous system and widens the spectrum of environmental and endogenous stimuli that can potentially influence adaptive

T3 production (see Ref. 2 for review). Several transcriptional and posttranslational mechanisms have evolved to ensure tight control of tissue levels of D2, which is inherent to its homeostatic function. The D2 mRNA in higher vertebrates is more than 6 kb in length, containing long 5′ and 3′ untranslated regions. The D2 5′untranslated regions are greater than 600 nucleotides in length, and they contain three to five short open reading frames, which reduce D2 expression by as much as fivefold (153). Alternative splicing is another mechanism that regulates the level of D2 synthesis, because mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs, but not encoding an active enzyme, are present in both human and chicken tissues (153). The ratios of D2 activity to D2 mRNA level in tissues vary, indicating substantial posttranslational regulation of D2 expression (154). In fact, the decisive property of D2 that characterizes its homeostatic behavior is a halflife of approximately 40 minutes that can be further reduced to approximately 25 minutes by exposure to physiologic concentrations of its substrate, T4, or extended to approximately 300 minutes when cells are grown in medium lacking T4 (155–162). This constitutes a rapid, potent regulatory feedback loop that efficiently controls T3 production and intracellular T3 concentrations based on how much T4 is available. The potency of T4 in inducing loss of D2 activity mirrors the enzyme’s affinity for the substrate, indicating that enzyme–substrate interaction must occur in order to induce loss of D2 activity. At the molecular level, D2 activity is regulated by conjugation to ubiquitin, a protein of approximately 8 kd. The ubiquitinated D2 is subsequently recognized and degraded by proteasomes (163,164) (Fig. 6-5). The first evidence for this process was obtained in GH4C1 cells, in which the half-life of endogenous D2 was noted to be stabilized by MG132, a proteasome inhibitor (165). Substrate-induced loss of D2 activity was also inhibited by MG132 in these cells, indicating that both pathways affecting loss of D2 activity were mediated by the proteasomes. This implies that the loss of D2 activity is at least partially due to proteolysis of D2, a premise that was confirmed when the levels of immunoprecipitable D2 were found to parallel D2 activity both under basal conditions and after exposure to T4 (166). In subsequent studies it became clear that D2 is ubiquitinated (101), and the various enzymes involved in this process were identified. In studies in which

human D2 was expressed in yeast, Ubc6p and Ubc7p were identified as the ubiquitin conjugases involved in ubiquitination of D2 (167), and it is now clear that these conjugases play a role in ubiquitination of human D2 (168,169).

FIGURE 6-5. Posttranslational regulation of type 2 deiodinase (D2). ER stress activates PERK/elF2a, inhibiting translation of Dio2 mRNA and D2 synthesis; Ub, ubiquitin; E2, Ub conjugase; Ubc7, E2 involved in D2 ubiquitination; Cue1, endoplasmic reticulum–docking protein for Ubc7; WSB-1 and TEB4 are E3 ligases that interact with D2 and direct D2 ubiquitination; USP33/22, deubiquitinases that reactivate Ub-D2; Ub-D2 can be removed from ER via p97/Atx3 complex and delivered for degradation in the proteasomes.

WSB-1 is a SOCS-box–containing WD-40 protein that is induced by Hedgehog signaling in embryonic structures during chicken development. Using a yeast two-hybrid screening system WSB-1 was found as part of an E3 ubiquitin ligase for D2 (170). The WD-40 propeller of WSB-1 recognizes an 18–amino acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modeled as Elongin BC-Cul5-Rbx1 (ECS (WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS (WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation and possibly playing a role in skeletogenesis (170). D2 was also found to be a substrate of TEB4, a ubiquitin ligase involved in degradation of misfolded protein in the endoplasmic reticulum (171). In a setting of transient coexpression in HEK-

293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by approximately 50%. TEB4 knockdown decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 µM of the proteasomal inhibitor MG132 for 24 hours nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and BAT, where TEB4 expression is minimal. D2 ubiquitination occurs via K48-linked ubiquitin chains and exposure to its natural substrate, T4, accelerates UbD2 formation and retrotranslocation to the cytoplasm via interaction with the p97-ATPase complex. D2 retrotranslocation includes deubiquitination by the p97-associated DUB Ataxin-3 (Atx3). Inhibiting Atx3 with eeyarestatin-I did not affect D2:p97 binding but decreased UbD2 retrotranslocation and caused endoplasmic reticulum accumulation of high–molecular-weight UbD2 bands possibly by interfering with the D2-ubiquitin–specific peptidases binding. Once in the cytosol, D2 is delivered to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit S5a and increased colocalization with the 20S proteasome (172). Fusion of the 8–amino acid FLAG sequence to the carboxyl-, but not the amino-, terminus of D2 prolongs its activity and increases the size of the ubiquitin-D2 pool by 20- to 30-fold (101), suggesting that D2 ubiquitination is reversible, because not all Ub-D2 undergoes proteolysis. Enzymatic deubiquitination of ubiquitin-D2 occurs in vitro (173) and could explain recycling in vivo. D2 was identified as a substrate for the deubiquitinating enzymes USP33 and USP20 (174). Confocal studies indicate that both USPs colocalize with D2, itself an integral endoplasmic reticulum membrane protein. The physical colocalization of these two USPs with D2 provides the opportunity for deubiquitination of D2. USP33-catalyzed D2 deubiquitination is an important part of the adaptive mechanism that regulates thyroid hormone action. In stimulated brown fat tissue, D2 increases intracellular T3 production, resulting in isolated tissue

thyrotoxicosis (175–177). Increased USP33-catalyzed deubiquitination of ubiquitin-D2, and therefore rescue of D2 from proteasomal degradation, could amplify this mechanism. In BAT, USP33 mRNA levels are markedly upregulated by cold exposure or norepinephrine, which amplifies the transcriptional increase in D2 activity, and hence T3 production increases by approximately 2.5-fold. The availability of a reversible ubiquitination-dependent mechanism to control the activity of D2 constitutes a biochemical and physiologic advantage that allows for rapid control of thyroid hormone activation. The finding that USP33 and USP20 are coexpressed with D2 in many human tissues, including brain, heart, and skeletal muscle (2,178), suggests that the importance of this mechanism may extend well beyond thermal homeostasis to include brain development, cardiac performance, glucose utilization, and energy expenditure. Using an in silico analysis of published array data, a significant positive correlation was found between the relative mRNA expression levels of WSB1 and USP-33 in a set of 56 mouse tissues (179). Subsequently, in situ hybridization combined with immunocytochemistry in rat brain showed that in addition to neurons, WSB-1 and USP-33 are differentially expressed in astrocytes and tanycytes, the two main D2 expressing cell types in the brain. Tanycytes, which are thought to participate in the feedback regulation of TRH neurons, express both WSB-1 and USP-33, indicating the potential for D2 ubiquitination and deubiquitination in these cells. Notably, only WSB-1 is expressed in astrocytes throughout the brain, a distribution that might affect how T4-signaling is transduced in different brain areas (179). Indeed, in adult thyroidectomized rats chronically implanted with subcutaneous L-T4 pellets, there was reduction in whole-body D2-dependent T4 conversion to T3, but in the hypothalamus D2 activity was only minimally affected. In vivo studies in mice harboring an astrocyte-specific WSB-1 deletion as well as in vitro analysis of D2 ubiquitination driven by different tissue extracts indicate that in the hypothalamus D2 ubiquitination is relatively lower, or D2 deubiquitination is relatively higher. As a result, in contrast to other D2expressing tissues, the hypothalamus is uniquely equipped to have increased sensitivity to T4 (180).

TYPE 3 DEIODINASE D3, acting by inner ring deiodination, is the major T3- and T4-inactivating enzyme, although D1 also has some activity as an inner ring deiodinase (181). D3, which has almost exclusively inner ring deiodination activity, catalyzes the conversion of T4 to reverse T3 and the conversion of T3 to 3,3′diiodothyronine (T2), both of which are biologically inactive (Fig. 6-1). That reverse T3 and T2 do not support thyroid hormone–dependent gene expression is illustrated by the severe consumptive hypothyroidism that occurs in patients with hemangiomas, in whom tumor overexpression of D3 results in very high serum reverse T3 concentrations, and the blockade of metamorphosis that occurs in tadpoles overexpressing D3 (38,182). D3 contributes to thyroid hormone homeostasis by protecting tissues from an excess of thyroid hormone. It was identified in monkey hepatocarcinoma cells (NCLP6E), and the first physiologic studies were performed in the CNS of rats (183–186). In humans, D3 is present in not only the CNS, but also in skin and placenta; it is also present in fetal liver and in the uterus of pregnant rats (187). The highest activity found to date is in hemangioma-type tumors in humans (38). In amphibians, D3 plays a critical role in development (188); it is present in tadpoles from premetamorphosis to the onset of the metamorphic climax, after which it declines to barely detectable levels. In mammals, D3 is critical for thyroid hormone homeostasis, because it protects the fetus from premature exposure to excessive amounts of thyroid hormone, which can result in malformations, altered growth, mental retardation, and even death. In fetal and neonatal animals, D3 expression is highly regulated in tissue-specific patterns that are likely to be critical to the coordinated regulation of thyroid hormone effects on development.

Gene Structure and Chromosomal Localization The DIO3 gene is located on human chromosome 14q32 and mouse chromosome 12F1 (189). A unique feature of the human and mouse DIO3 gene is that it has no introns (190), which is a rarity among eukaryotic genes (189,191). The gene is preferentially expressed from the paternal allele in mice whereas the maternal allele is silenced through imprinting (192). The

DIO3 gene likely belongs to the same cluster of imprinted genes in mouse chromosome 12 and human chromosome 14, and as such it might play a role in the phenotypic abnormalities associated with uniparental disomy of those chromosomes, a condition in which gene expression is altered due to abnormal genomic imprinting (193). Human D3 mRNA contains 2,066 nucleotides. There are 220 bp in the 5′untranslated region, an 834-bp open reading frame, and a 3′-untranslated region of 1,012 bp (194). All D3 cDNAs identified to date include a selenocysteine-encoding TGA codon, as well as a SECIS element in the 3′untranslated region. There is a high degree of identity between the DIO3 gene in human and other species, particularly in the putative active center where the selenocysteine is located. The conservation of this enzyme from tadpoles to humans implies that its role in regulating thyroid hormone inactivation during embryologic development is essential. The most common form of D3 mRNA in most tissues is 2.3 kb, but there are at least four differently sized mRNAs in the CNS of rats, and thyroid hormone causes increases in the relative intensity of these mRNAs (195).

Tissue Distribution D3 has been identified in various tissues in several animal species, among which rats have been studied most extensively. In adult rats, D3 is found predominantly in the CNS and skin. In neonatal rats, it is found not only in these tissues, but also in skeletal muscle, liver, and intestine (60,184,196–199). In particular, using in situ hybridization analysis, D3 mRNA has been identified throughout the brain in adult rats, especially in hippocampal pyramidal neurons, granule cells of the dentate nucleus, and layers II to VI of the cerebral cortex (195). It is noteworthy that these regions also contain the highest concentrations of thyroid receptors in the brain, and they have critical roles in learning, memory, and higher cognitive functions (200–202). Furthermore, the distribution of D3 mRNA in the CNS changes during the early stages of development. At postnatal day 0, D3 is selectively expressed in the bed nucleus of the stria terminalis, the preoptic area, and other areas related anatomically and functionally to the bed nucleus, such as the central amygdala; all of these areas are involved in the sexual differentiation of the brain (203). D3 expression in these areas is transient and

is no longer detected at postnatal day 10. The overall pattern of distribution of D3 in the brain of rats strongly suggests that it is primarily expressed in neurons, but it is also present in astrocytes (204–206). Very high levels of D3 activity and mRNA have been identified in hemangiomas in infants. In subjects with very large tumors, the result is hypothyroidism, caused by very rapid deiodination of T4 and T3 (38). This syndrome, termed consumptive hypothyroidism, has also been described in an adult with a large hepatic hemangiopericytoma (207). D3 activity has also been detected in the retina in rat fetuses and, in lesser amounts, in adults (208). In Xenopus laevis, the localized expression of D3 in the cells of the marginal zone of the retina accounts for the asymmetric growth of the retina (209). As noted above, D3 is highly expressed in the skin of adult rats (60,210), and in them skin contains more reverse T3 than any other tissue, suggesting that the high levels of D3 activity in homogenates of skin accurately reflect the activity of this enzyme in vivo (210). In this regard, T4 applied to normal human skin is largely converted to reverse T3 (211). Large amounts of D3 are present in the placenta of rats, guinea pigs, and humans, and it is by far the predominant deiodinase present in this tissue (197,212–215). High levels of D3 are also present in the uterus of pregnant rats, initially in decidual cells and later in the single-cell layer of the epithelium (216). The levels of D3 are highest at the implantation site, nearly double the highest values obtained for placental tissue. In humans, the highest levels of D3 are in the endometrium, and it is also found in the amnion and chorion (214), fetal skin, tracheal and bronchiolar epithelium, mesothelium, and intestinal epithelium (39). Regulation of D3 Synthesis Thyroid Hormone

Parallelism between D3 activity and thyroid status has been demonstrated in several species, although the underlying molecular mechanisms remain obscure. In Xenopus laevis tadpoles, administration of T3 before the climax of metamorphosis results in a rapid and marked increase in D3 activity (56). In rats, D3 activity in the CNS is increased by thyroid hormone

administration and decreased by hypothyroidism (184). In in situ hybridization histochemical studies, D3 gene expression within the CNS increased 4- to 50-fold in rats made thyrotoxic, with the greatest increase in the cerebellum. Conversely, D3 mRNA is not detectable in Northern blots of brain tissue of hypothyroid rats (195). Whether the dramatic increase in D3 mRNA in rats given T3 is due to increases in gene transcription, mRNA stabilization, or a combination of these factors is not known. In Xenopus laevis, this T3 effect is not blocked by inhibition of protein synthesis. The promoter regions of the DIO3 gene are stimulated by T3, but the magnitude of this stimulation is modest as compared with the effect of T3 on D3 activity. Regulation of D3 activity by thyroid hormone has also been demonstrated in cultured astroglial cells. In these cells, the addition of 10 nM T3 (or T4) to the culture medium causes a slow increase in D3 activity, which reaches a plateau in 48 hours (217).

Sex Steroids D3 is expressed in the uterus, and the content increases during pregnancy. In rats, uterine D3 activity increases immediately after implantation, or artificial decidualization of the uterus in pseudopregnant rats, whereas the increases in activity are minimal in the nondecidualized uterine horn in the latter rats. In spontaneously cycling female rats, D3 activity is three to eight times higher during estrus, as compared with diestrus. Furthermore, the uterine levels of D3 activity are synergistically increased in ovariectomized rats given estradiol and progesterone in various combinations. Thus, estradiol and progesterone regulate thyroid hormone metabolism in the uterus, and the implantation process is a potent stimulus for the induction of D3 activity in this organ.

Nonthyroidal Illness Most critically ill patients have low serum T3 and high serum reverse T3 concentrations (see section on nonthyroidal illness in Chapter 14). A study of 16 critically ill patients revealed that the drop in circulating T3 levels is the result of a major decrease, down to approximately 27% of healthy individuals, in T3 production. In contrast, the elevation of the rT3 levels stems from a slower clearance rate from the circulation, which is down to

42% of healthy individuals (218). The finding of ectopic expression of D3 in tissues of critically ill patients recently deceased suggest that rT3 production rate could also be accelerated in some settings of nonthyroidal illness (78). In patients with multiple organ system failure and other serious illnesses who died, hepatic D1 activity is low, and hepatic and skeletal muscle D3 activity is high (78). These findings suggest that T4 and T3 metabolism is altered in tissue-specific ways in illness, particularly with respect to reducing T4 formation or increasing T3 degradation. Similarly, D3 activity is increased in cardiac muscle of rats with cardiac hypertrophy and failure (219); among these rats, right ventricular D3 activity is significantly higher in those animals in which hypertrophy progresses to heart failure, as compared with the animals in which it did not. Evidence of increased D3 expression and rT3 production has also been documented in the myocardium of patients with left ventricular hypertrophy caused by aortic stenosis (220). A similar D3 reactivation was documented in critically ill patients with compromised organ perfusion (78). The induction of D3 in cardiac muscle would be expected to result in reduced intracellular concentrations of T3, which might reduce cardiac work and therefore help maintain cardiac compensation (37,221). Hypoxia seems to be a key signal inducing D3 expression in disease states. Indeed, hypoxia induces D3 expression via a hypoxia-inducible factordependent (HIF-dependent) pathway; hypoxia mimetics that increase HIF-1 also induce D3 activity and mRNA levels. HIF-1α was found to interact specifically with the DIO3 promoter, indicating that DIO3 may be a direct transcriptional target of HIF-1 (37). In this system, D3 activity decreased T3dependent oxygen consumption, suggesting that hypoxia-induced D3 slows down the rate of metabolism. Using a rat model of cardiac failure due to right ventricle (RV) hypertrophy, HIF-1α and D3 proteins were induced specifically in the hypertrophic myocardium of the RV, creating an anatomically specific reduction in local T3 content and action (37). In addition, hypoxia leads to nuclear import of D3 in neurons, without which thyroid hormone signaling and metabolism cannot be reduced. After unilateral hypoxia in the rat brain, the D3 protein level is increased predominantly in the nucleus of the neurons in the pyramidal and granular ipsilateral layers, as well as in the hilus of the dentate gyrus of the hippocampal formation (222). In hippocampal neurons in culture, as well as in a human neuroblastoma cell line, a 24-hour hypoxia period redirects active

D3 from the endoplasmic reticulum to the nucleus via the cochaperone Hsp40 pathway. Preventing nuclear D3 import by Hsp40 knockdown increases the effects of thyroid hormone on cellular metabolism and transcription of the thyroid hormone target gene ENPP2. In contrast, increasing nuclear D3 import minimizes thyroid hormone effects in cell metabolism (222).

Extracellular Receptor Kinase–Activated Pathways In cultured rat astroglial cells, factors that alter cellular processes through signaling cascades originating at the plasma membrane increase D3 activity. For example, D3 activity increases markedly and rapidly after exposure of the cells to 12-O-tetradecanoyl phorbol-13-acetate (TPA), fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and cyclic AMP analogues (223). The stimulatory effects of TPA and fibroblast growth factor on D3 mRNA and activity appear to be mediated at least partially by activation of the MEK/ERK signaling cascade (224).

NONDEIODINATIVE METABOLISM OF IODOTHYRONINES Iodothyronines are mostly metabolized by deiodination. However, there are other pathways of metabolism, such as ether bond cleavage (mainly of T4 in leukocytes (225)), deamination and decarboxylation of the alanine side chain (226,227), sulfoconjugation mediated by cytosolic phenol sulfotransferases in several tissues (228), and glucuronidation and O-methylation, which renders the products more hydrophilic and thereby facilitates their excretion by bile, feces, and urine (229). Sulfoconjugation is the most important alternative iodothyronine metabolic pathway. The sulfotransferases of liver are normally involved in inactivation and detoxification reactions, with a preference for lipophilic substrates (230,231). Sulfation of iodothyronines facilitates rapid inner ring deiodination by D1 but not by D3. All iodothyronines except reverse T3, the preferred substrate of D1, are sulfated to some extent (228). Iodothyronines with two iodine atoms at the phenolic ring are preferentially conjugated with glucuronic acid, whereas iodothyronines that

contain only one iodine atom in the phenolic ring are sulfated with the following preference: 3′-T1 = 3,3′-T2 > T3 > rT3 > T4 (186,228,230,232,233). T3 can be sulfated by human cytosolic liver phenol sulfotransferase (EC 2.8.2.1), with Km values in the 100 mM range, considerably greater than that for D1 (234). Biliary and urinary excretion of iodothyronine sulfates is a minor route for thyroid hormone elimination in humans. Considerable amounts of iodothyronine sulfates are detectable in plasma and bile after inhibition of D1 by PTU in rats (228,235,236). Moreover, at a high substrate concentration (>1 mM) in vitro, metabolism proceeds mainly by sulfation, whereas at lower concentrations (isoleucine) in the extracellular domain of the thyrotropin receptor. J Clin Invest 1997;100(6):1634–1639. 45. Rodien P, Bremont C, Sanson ML, et al. Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensitive to human chorionic gonadotropin. N Engl J Med 1998;339(25):1823–1826. 46. Coulon AL, Savagner F, Briet C, et al. Prolonged and severe gestational thyrotoxicosis due to enhanced hCG sensitivity of a mutant thyrotropin receptor. J Clin Endocrinol Metab 2016;101(1):10–11. 47. Kleinau G, Worth CL, Kreuchwig A, et al. Structural-functional features of the thyrotropin receptor: a class A G-protein-coupled receptor at work. Front Endocrinol (Lausanne) 2017;8:86. 48. Kobe B, Kajava AV. The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 2001;11(6):725–732. 49. Miller-Gallacher J, Sanders P, Young S, et al. Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain. J Mol Endocrinol 2019;62(3):117–128. 50. Sanders J, Chirgadze DY, Sanders P, et al. Crystal structure of the tsh receptor in complex with a thyroid-stimulating autoantibody. Thyroid 2007;17(5):395–410. 51. Sanders P, Young S, Sanders J, et al. Crystal structure of the TSH receptor (TSHR) bound to a blocking type TSHR autoantibody. J Mol Endocrinol 2011;46(2):81–99. 52. Caltabiano G, Campillo M, De Leener A, et al. The specificity of binding of glycoprotein hormones to their receptors. Cell Mol Life Sci 2008;65(16):2484–2492. 53. Costagliola S, Panneels V, Bonomi M, et al. Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors. EMBO J 2002;21(4):504–513. 54. Westmuckett AD, Hoffhines AJ, Borghei A, et al. Early postnatal pulmonary failure and primary hypothyroidism in mice with combined TPST-1 and TPST-2 deficiency. Gen Comp Endocrinol 2008;156(1):145–153. 55. Sasaki N, Hosoda Y, Nagata A, et al. A mutation in Tpst2 encoding tyrosylprotein sulfotransferase causes dwarfism associated with hypothyroidism. Mol Endocrinol 2007;21(7):1713–1721. 56. Palczewski K, Kumasaka T, Hori T, et al. Crystal structure of rhodopsin: A G protein- coupled receptor. Science 2000;289(5480):739–745. 57. Ridge KD, Abdulaev NG, Sousa M, et al. Phototransduction: crystal clear. Trends Biochem Sci

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11 Aging and the thyroid Anne R. Cappola

The importance of adequate thyroid function to maintain metabolic processes persists into advanced age. However, age-related changes in thyroid function tests occur in individuals who have no evidence of underlying thyroid dysfunction or alterations in thyroid structure. Discerning between normal aging and thyroid dysfunction is an important distinction to avoid unnecessary treatment. In addition, the prevalence of benign thyroid nodules increases with age (1), as does the incidence of aggressive papillary thyroid carcinoma (1). The additional challenges in diagnosis and management of thyroid dysfunction and thyroid nodular disease in older patients require an appreciation of features not typical in younger patients.

CHANGES IN THYROID ANATOMY WITH AGE In the absence of thyroid disease, the size of the thyroid is smaller at advanced ages (2). Thyroidal volume is more highly correlated with body weight than with age (3). On pathologic examination, lymphocyte infiltration and fibrosis increase, and sizes of colloid and follicles diminish (4). In addition, the location of the thyroid becomes more caudal with increasing age (5). A more caudal position is associated with lower cervical spine height, a shorter hyoid bone to hard palate distance, and a tracheal angle that is more horizontally oriented (5).

PHYSIOLOGIC CHANGES IN THYROID FUNCTION WITH

AGE Both cross-sectional and longitudinal studies have demonstrated a gradual increase in TSH concentrations without concomitant changes in free thyroxine concentrations with age, even in individuals without thyroid disease or antithyroid antibodies (6–8). The shape of the TSH distribution suggests a population shift rather than increased incidence of hypothyroidism at older ages (6). The etiology of this physiologic change is unknown. Possibilities include a decrease in the bioactivity of TSH or a diminished thyroidal response to TSH. In turn, this leads to a higher prevalence of TSH above the reference range, which is found in 14.5% of those aged 80 years and older, compared with 2.5% of 20 to 29 year olds (6). Individuals aged 70 years or older with mildly elevated TSH levels and normal free thyroxine levels (which has been called subclinical hypothyroidism) do not have an increased risk of cardiovascular disease, disability, or mortality (8–10) and may have better mobility and functional status compared with euthyroid peers (11,12). These data suggest that the increase in TSH level represents an age-associated adaptive response and not thyroid dysfunction. In addition, a randomized trial in patients with persistent subclinical hypothyroidism who were aged 65 years and older (mean age 74.4 years) did not show benefit of a low dose of levothyroxine (mean dose 50 mcg daily) for hypothyroid symptoms or physiologic outcomes compared with placebo (13). Of note, subclinical hypothyroidism is frequently transient in this population; 62% of participants whose TSH was initially elevated were ineligible for inclusion in this trial due to reversion of the TSH to the euthyroid range (13). In the community, both initiation of treatment with levothyroxine and overtreatment to low TSH levels is common in older individuals (14–16). The combination of lack of harm in the absence of treatment, lack of benefit from levothyroxine treatment, and potential harm from accidental overtreatment has led multiple groups to recommend annual surveillance rather than treatment in older patients with mild subclinical hypothyroidism with a TSH of up to 7 to 10 mU/L (Fig. 11-1) (17–19). Whether current data are sufficient to support a change in the upper limit of the TSH reference range to 7 mU/L in individuals who are 65 years of age or older is a topic of

debate. T3 concentrations are lower in healthy centenarians than in younger individuals, suggesting an age-related decline in deiodinase activity (20). This has also been shown in individuals aged 75 years and older (8). Of note, these studies were conducted in study participants who did not have acute illness, and therefore these findings are unlikely to represent acute effects from nonthyroidal illness. However, whether a persistent effect on T4 to T3 conversion occurs with chronic nonthyroidal illness in older individuals is not known.

FIGURE 11-1. Recommendations for levothyroxine treatment in individuals aged 65 years or older by serum TSH concentration. The TSH concentration should be persistently in the specified range, based on repeat testing performed at least 1 month after the initial test.

CHALLENGES IN THE DIAGNOSIS AND MANAGEMENT OF THYROID DYSFUNCTION WITH AGE In addition to age-related changes in thyroid function testing, the incidence of thyroid dysfunction also increases with increasing age. Hypothyroidism and hyperthyroidism both are more common in older individuals, as are comorbid conditions and medications that affect thyroid function (21,22). Recognition of thyroid dysfunction can be challenging; the classic symptoms of hypothyroidism and hyperthyroidism are reported less frequently in older patients with overt thyroid disease than in their younger counterparts with a similar degree of thyroid dysfunction (23–25). Furthermore, many symptoms, such as fatigue and constipation, are frequent in older individuals without thyroid disease, and clinicians may fail to identify these symptoms as related to thyroid dysfunction. In addition, in overt hyperthyroidism in older patients,

atypical symptoms, such as apathy and anorexia, are more common and hyperadrenergic symptoms are less common (24). Goiter and ophthalmopathy may be absent in Graves disease, and thyroid function testing and thyrotropin receptor antibodies are lower in older than in younger individuals with Graves disease (26). A toxic multinodular goiter is a common etiology of hyperthyroidism in an older person, and it may first present after an iodine load from iodinated contrast (27). The prevalence of antithyroid antibodies also increases with age, particularly in women, consistent with an age-related increase in autoimmune thyroid disease, though antibody levels are lower in the oldest old (6,20). Subclinical hyperthyroidism—low TSH levels with normal levels of free thyroxine—is associated with an increased risk of atrial fibrillation, hip fracture, and dementia if left untreated (28–30). Even patients with low, but not suppressed TSH levels (TSH 0.1 to 0.44 mU/L) are at increased risk of atrial fibrillation, coronary heart disease, and hip fracture in analyses stratified by degree of subclinical hyperthyroidism (28,30). Because older patients have a high baseline risk of these outcomes, subclinical hyperthyroidism is more likely to have clinically meaningful effects in these patients. The American Thyroid Association recommends treatment of individuals with subclinical hyperthyroidism who are aged 65 years and older and have a TSH 70 to 75 years (18). It also follows that the time between dose adjustments should be longer than the standard 6 weeks. A waiting period of six half-lives between dose adjustments, accounting for a 9-day half-life, would be closer to 8 weeks.

CHALLENGES IN THE DIAGNOSIS AND MANAGEMENT OF NODULAR DISEASE AND THYROID CANCER

The thyroid gland may be more difficult to palpate in older individuals, particularly those who have pulmonary disease or kyphosis, and therefore recognition of thyroid nodular disease may be more difficult on physical examination. However, thyroid nodules are more likely to be incidentally discovered in older individuals due to the high frequency of imaging studies that include the neck (38). Both benign and malignant thyroid nodules are more common in older individuals. The higher incidence of malignant thyroid nodules with increasing age is not thought to be due to detection bias, as papillary thyroid carcinoma is more likely to present at a more advanced stage in an older than in a younger person (39). The management of thyroid nodules and thyroid cancer does not differ between younger and older individuals. However, life expectancy and general health status should be taken into consideration when deciding the management plan. Micropapillary thyroid cancer, defined as a papillary thyroid cancer less than 1 cm in diameter and confined to the thyroid, is more indolent in patients ≥60 years of age than in those 160 mg/day), propranolol inhibits thyroxine monodeiodination leading to a decrease in serum T3 and an increased in FT4 (65). Nadolol has a similar effect (65). The fall in T3 has been shown to be the result of decreased deiodination of T4 and not an alteration in T3 clearance. Some in vitro

studies have suggested that the action of propranolol may relate to membrane effects decreasing the transport of T4 into the cells rather than a direct inhibition of enzymatic activity (65). In euthyroid individuals, the inhibition of monodeiodination may lead to an increase in T4, FT4, and even a reduction of TSH and the TSH response to TRH (66). This blockade of monodeiodination is not seen with other betaadrenergic blocking drugs like metoprolol or atenolol, or the mixed alpha and beta blocker, labetalol (67,68). There is no evidence that the symptomatic improvement in hyperthyroidism is different for propranolol compared to the other beta blockers that do not affect T4 monodeiodination.

DRUGS THAT ALTER THE TRANSPORT OR ACTION OF THYROID HORMONES (FIG. 12-1, PANEL 3) While many drugs inhibit the binding of T4 and T3 to their binding sites on serum transport proteins in vitro, these effects are often not seen in vivo, as they require high drug concentrations (69). The following paragraphs describe the effects of drugs that alter serum thyroid hormone levels through alterations in thyroid hormone binding in doses that are used clinically. When such alterations occur, serum total T4 and T3 concentrations are affected, but the actual free hormone concentrations are not altered. Drugs altering the levels of thyroxine-binding globulin (TBG) will result in parallel changes in the total thyroid hormone levels, but the free hormone levels will remain normal. In patients with normal thyroid function, these changes in TBG are not clinically significant. However, in hypothyroid patients on thyroid hormone supplementation, they may alter dose requirements with an increase in TBG requiring an increase in the thyroid hormone dose and a decrease in TBG requiring a decrease in the thyroid hormone dose.

NONSTEROIDAL ANTI-INFLAMMATORY DRUGS (SALICYLATES, SALSALATE, FENCLOFENAC) Salicylates inhibit T4 and T3 binding to both TBG and transthyretin (TTR),

resulting in reductions in serum total T4 and T3 concentrations (70,71). Other nonsteroidal anti-inflammatory drugs also displace T4 from its binding sites, particularly salsalate and fenclofenac (71,72).

FUROSEMIDE Furosemide inhibits protein binding of T4. The effect does not occur with oral doses of less than 100 mg but is found consistently with very large intravenous doses of furosemide, and may be enhanced in the setting of renal failure (69) (where use of very high doses is especially common).

HEPARIN AND FATTY ACIDS Heparin does not directly displace thyroid hormone from binding proteins, but a transient increase in free T4 has been noted in vivo, especially when free T3 or T4 was measured by equilibrium dialysis (73,74). This is the result of heparin-induced lipoprotein lipase activation leading to the release of nonesterified fatty acids (NEFA) from triglycerides; the NEFA then displace thyroid hormone from the binding proteins resulting in an increased measured level of free hormone (74). In vitro activation of lipoprotein lipase can be seen with even low doses of heparin, but can be minimized by rapid and careful handling of blood samples and by avoiding repeated freezing and thawing. These findings have also been observed with enoxaparin, and it has been recommended to delay free T4 measurements for 10 hours after a dose of enoxaparin (75). These effects are most relevant to interpretation of diagnostic tests, as patients treated with these agents do not appear to become hyperthyroid although reduced serum TSH level have been reported in some of these patients, but may just have been the effect of illness or other drugs (69). To effectively displace thyroid hormone from the binding proteins, serum FFA levels have to exceed 2.5 to 3 mEq/L. In vivo, such concentrations are uncommon. They are more likely to be seen in patients on hemodialysis and receiving intravenous lipid infusions especially in patients with low serum albumin levels.

ESTROGEN AND SELECTIVE ESTROGEN MODULATORS (SERM) (TAMOXIFEN, RALOXIFENE) Increased levels of estrogens, from either endogenous (from pregnancy, or estrogen-producing tumors) or exogenous sources, lead to an increase in serum concentrations of TBG and a decrease in TTR (76,77). Estrogen alters the composition of oligosaccharide side chains on TBG and this reduces its clearance rate (78). Estrogens are the most common cause of TBG elevations. The effect is dose related, so low-dose estrogen oral contraceptives (OCPs) have less of an effect than older OCPs that had much higher levels of estrogen. Increased levels of TBG can be seen with hormone replacement therapy in postmenopausal women, and also when used in treatment of gender transition. Transdermal estrogens have less of an effect than oral forms (79). Paradoxically, tamoxifen blocks the estrogen-induced increase in TBG, but when used alone in postmenopausal women it leads to increases in TBG levels (80). Raloxifene, another SERM used to treat osteoporosis has minimal effects on TBG levels and thyroid function tests (81). The increase in TBG levels results in higher serum levels of T4, T3, and rT3 and, to a lesser extent, other metabolites of T4 deiodination. The fractional turnover rate of T4 is lower as the result of a higher intravascular T4 pool. At steady state, the free T4 and T3 concentrations and the absolute amount of hormone degraded each day remain normal, but transient changes in these parameters may be seen during early increases in the TBG concentration. During ovarian hyperstimulation for ovulation induction, an increase in TSH and FT4 has been observed attributed to marked acute increases in estrogen (82).

ANDROGENS Androgens decrease the concentration of TBG in serum leading to decrease in T4 and T3 while TTR levels are higher (83). As with estrogens, the free hormone concentrations and the absolute amount of hormone degraded daily remain normal, but in contrast, the fractional turnover rate is higher as the result of a smaller intravascular T4 pool.

OTHER DRUGS ALTERING TBG LEVELS An increase in TBG has been reported with chronic methadone use with the expected changes in thyroid function tests (84). A marked, but transient, decrease in TBG has been reported during L-aspariginase therapy occurring after only 2 days of therapy (85).

DRUGS ACTING AT MULTIPLE SITES TO ALTER THYROID HORMONE HOMEOSTASIS IODINATED RADIOGRAPHIC CONTRAST AGENTS The number of CT scans performed annually has been steadily rising with around 3 million performed in 1980 and over 70 million annually in 2007 (86) and many of these utilized iodinated contrast. Whether low or high ionic strength, low or high osmolality, all of these agents contain large amount of iodine ranging from 320 to 370 mg/mL (87). As a consequence of the high iodine content, both hyperthyroidism and hypothyroidism may occur (87). In a prospective study, 2.6% of adults receiving iodine-containing contrast developed hyperthyroidism, although many of these cases were minimally symptomatic and transient (88). In a study of hospitalized elderly patients with hyperthyroidism, 23% of them had had a contrast CT performed in the preceding 6 months (89). Examining a data base of 4,500,000 patients, it was found that the likelihood of developing hyperthyroidism within 2 years of being euthyroid was doubled by having had a recent contrast CT (90). In a small study, pretreatment with thionamides was not clearly shown to have an effect (91). Since many episodes of hyperthyroidism after iodinated contrast are transient, mild, and asymptomatic, this approach may only be appropriate for patients who are at high risk, such as those with multinodular goiters and severe heart disease. Other options include avoidance of iodinated contrast and definitive treatment of any underlying thyroid disorder after the patient has recovered. In a study of newly diagnosed hypothyroidism in children, the risk was

increased nearly threefold by recent administration of iodinated contrast (92). In a study in adults, the risk of hypothyroidism was doubled (93).

PHENYTOIN Phenytoin is a commonly used antiepileptic drug. As with other antiseizure medications (discussed previously) it induces P450 CYP3 activity increasing hepatic metabolism of T4 and T3. The structure of phenytoins (two phenyls and a hydantoin moiety) is similar to the two phenyls linked by an ether bond in T4. Although the affinity of T4 for TBG is much greater than that of phenytoin, in therapeutic doses the serum concentrations of the drug are high enough to displace some T4 and thus reduce the serum concentrations (94). Phenytoin also appears to increase the conversion of T4 to T3, so that while T4 and rT3 levels are lower, T3 levels are not, suggesting that increased formation compensates for increased degradation. There may also be effects on cellular entry and hormone action (95). Patients who were previously euthyroid and then placed on phenytoin usually have normal TSH values despite the low free T4, but both elevated and decreased TSH levels and responses to TRH have been reported, suggesting possible alterations of thyroid hormone nuclear effects in the pituitary. In hypothyroid patients placed on phenytoin, TSH usually rises and levothyroxine doses need to be increased consistent with the increased catabolism in the liver (96).

AMIODARONE Amiodarone (2-n-butyl-3,4′-diethylaminoethoxy-3′,5′diiodobenzoylbenzofurane) is used to treat both atrial and ventricular arrhythmias. It alters normal thyroid hormone regulation and metabolism and can cause both hypothyroidism and hyperthyroidism (97). It is stored in fat, has a half-life measured in months. A standard 200-mg tablet contains 75 mg of iodine, and its structure resembles both phenytoin and levothyroxine. Like ipodate and iopanoic acid, amiodarone inhibits both DIO1 and DIO2 and this effect is dose dependent (97). This inhibition initially leads to a marked decrease in T3, an increase in T4, free T4, and rT3, as well as a mild

increase in TSH (97). In patients, given an IV loading dose of amiodarone for 2 days followed by 600 mg oral amiodarone daily, TSH was already elevated on day one, T3 was reduced by day two, but T4 and free T4 were not elevated until day four (98). In patients who remain euthyroid on therapy, T4, free T4, and TSH normalize within the first few months, while T3 remains lower and reverse T3 remains elevated. While there is some decreased clearance of serum T4 by the liver (99), it is the inhibition of T4 conversion to T3 that is the main cause of the persistently low T3 levels. Amiodarone may also interfere with the entry of thyroid hormone into cells, binding to the thyroid hormone receptor, and may antagonize the effects of thyroid hormone at the cellular level (97,100,101). Amiodarone and its major metabolite desethylamiodarone are able to displace T3 from its nuclear receptor (102). This coupled with observations of decreased beta-adrenergic receptor density in the myocardium and the occurrence of bradycardia in treated patients has led to the concept that some to the antiarrhythmic effects might be from selective “myocardial hypothyroidism” but this is not supported by clinical studies (97,100). Amiodarone-induced hypothyroidism is directly related to its high iodine content, with resulting suppression of thyroid hormone production and secretion and an inability to escape from the Wolf–Chaikoff effect (97,103). Similar to other causes of iodine-induced hypothyroidism, this occurs more commonly in women and in patients with pre-existing anti-TPO antibodies (104). It has also been observed that the development of hypothyroidism is much more common in areas where iodine is replete (105). In patients with hypothyroidism, TSH will be elevated. If amiodarone is needed, the development of hypothyroidism does not require the drug be discontinued (97,103). However, even if it is discontinued, given the prolonged half-life, it may take months for normal thyroid function to recover. In contrast to hypothyroidism, amiodarone-induced thyrotoxicosis (AIT) is more prevalent in areas of the world with relative iodine deficiency, where it may occur in 10% of treated patients (97,105). It may develop within months of starting therapy or, given the long residence time in the body, months after amiodarone was discontinued (97,106,107). Patients with underlying nodular thyroid disease or Graves disease are at increased risk, but many patients who develop this complication had a normal thyroid gland prior to treatment with

amiodarone. Patients with AIT can be divided into a type 1 form (AIT type 1) with underlying Graves disease or thyroid nodules, and a type 2 form (AIT type 2), resembling a toxic drug-induced thyroiditis, perhaps from a direct cytotoxic effect of the drug (97,103). In areas of iodine deficiency, it may be possible to demonstrate increased iodine uptake in some AIT type 1 patients, but given the high iodine content of amiodarone, this is not possible in iodine-replete areas (97). Color flow Doppler on thyroid ultrasound has also been proposed as a way to separate these types, with low or normal flow being consistent with AIT type 2, but this has not proven to be consistently reliable (97,108). The uptake of 99mTc-SestaMIBI has also been proposed as a useful test with very low uptake in AIT type 2, higher uptake in AIT type 1, and intermediate results in patients who were thought to have a mixed type based on their clinical course (109). AIT type 1 typically occurs 3 to 12 months after initiation of amiodarone therapy, while AIT type 2 usually occurs 1 to 3 years after treatment was started or even after the drug was stopped (107). Given the issues discussed above, it is recommended that thyroid function tests be checked prior to initiating amiodarone and then every 6 months, including for up to 1 year after it has been discontinued (97,103,110). Evaluation of patients who develop hyperthyroidism should include ultrasound with color flow Doppler and serologic testing for Graves disease. Patients should be managed in close consultation with their cardiologist. In both types, beta blockade should be considered for symptomatic relief. If type 1 disease is suspected, therapy with high-dose thionamides (e.g., 40 to 60 mg per day of methimazole or 400 to 800 mg per day of PTU) should be initiated. If available, the addition of perchlorate (111) and/or ipodate (112) can be considered for severely ill patients or those not responding to thionamides. If type 2 is suspected, treatment should be initiated with glucocorticoids (40 mg per day of prednisone or equivalent) with improvement expected within a few weeks and euthyroidism in 6 to 12 weeks (97,103,113). In severe cases, or where there is uncertainty as to the type of hyperthyroidism, therapy may be initiated with both thionamides and glucocorticoids, with subsequent management based on the biochemical clinical response. If there is rapid improvement in thyroid function within 7 days, AIT type 2 is far more likely to be present, since thionamides do not lead to such rapid improvement in thyroid hormone levels. Thyroidectomy

may need to be considered in patients not responding to therapy (114). Therapy may need to be maintained for an extended period of time, especially with AIT type 1. Given its long half-life, the value of stopping amiodarone is uncertain, but it may be more helpful for AIT type 1 (97,103). Radioactive iodine therapy may be considered after recovery from thyrotoxicosis to prevent recurrence. In patients with a previous episode of AIT, reintroduction of the drug may be possible. Concomitant prophylactic thionamide treatment has been recommended to prevent recurrences of AIT type 1 (115).

GLUCOCORTICOIDS Glucocorticoids have multiple effects on thyroid homeostasis at both physiologic and pharmacologic concentrations. Even physiologic levels of glucocorticoids suppress TSH secretion, as serum TSH levels may be elevated with adrenal insufficiency and normalize with physiologic replacement (116). High levels, as attained in Cushing syndrome, suppress basal TSH and the response to TRH in both euthyroid and hypothyroid patients (117). Pharmacologic glucocorticoid effects include a decrease in serum TBG, inhibition of outer ring (5′) deiodinase, suppression of TSH secretion, decreased hepatic uptake of T4, and increased renal clearance of iodine (118,119). In euthyroid patients treated with glucocorticoids, a reduction in TBG leads to a lower serum T4 level while the level of free T4 remains normal. As with other drugs, inhibition of 5′ monodeiodination, leads to a greater reduction in serum T3, and this effect is rapid, occurring within 24 hours. These multiple effects may make it difficult to diagnose hypothyroidism in an ill patient receiving glucocorticoids. In addition to the glucocorticoid effects on thyroid hormone secretion, transport, and metabolism, immunosuppressive effects may reduce levels of stimulating antibodies in Graves disease, and decrease inflammation and thyroid hormone release in various forms of thyroiditis, including AIT type 2 (120).

CYTOKINES (INTERFERON, INTERLEUKIN, AND TUMOR NECROSIS FACTOR) These cytokines are used in the treatment of infectious diseases such as hepatitis, as well as malignancies including melanoma and renal cell carcinoma. Acute administration has been used as a model of illness as the effects are similar; interferon-α leads to a decrease in T3, an increase in rT3, and a fall in TSH (121). In a group of euthyroid HIV-infected patients, however, short-term administration of interleukin-2 was observed to lead to an increase in TSH, T3, T4, and free T4 (122). In vitro, interferons and interleukins, and tumor necrosis factor-α are known to inhibit iodine organification and hormone release, as well as to modulate thyroglobulin production and thyrocyte growth (123). Interferon and interleukin have been associated with the development of both hypothyroidism and thyrotoxicosis. Cytokine-induced thyroid disease is often immune mediated with the development of antibodies without clinical disease, painless thyroiditis with positive antibodies, or Graves disease (123–125). The frequency of immune-mediated thyroiditis and Graves disease is much greater in females and in patients with pre-existing positive antithyroperoxidase antibodies prior to the initiation of therapy (123,125,126). In patients treated for hepatitis with interferon, the frequency of autoimmune thyroid disease was much higher in those with hepatitis C than those with hepatitis B; however, with the availability of potent antivirals, interferon now is only used for the treatment of some patients with chronic hepatitis B. Patients with hepatitis C have an increased risk for thyroid autoimmunity even before receiving antiviral treatment (127). During therapy, patients who were anti–TPO antibody positive may have a rise in titer, while antibody positivity may develop in previously negative patients (123–125). Patients who develop autoimmune hypothyroidism can continue interferon and be treated with levothyroxine; if interferon is discontinued, about 40% will become euthyroid (128). In patients who develop Graves(two-thirds of the patients presenting with hyperthyroidism) it is unclear that there is a benefit of stopping interferon and treatment is with the usual modalities, but some authors express concern about the use of antithyroid drugs in patients with significant liver disease (124,128).

Cytokine-induced thyroid disease may also present as a nonautoimmune destructive thyroiditis presenting as hyperthyroidism which is always transient and often followed by transient hypothyroidism without antithyroid antibodies (124,125). Permanent hypothyroidism occurs in less than 5% of these patients.

TYROSINE KINASE INHIBITORS Tyrosine kinase inhibitors (TKIs) are used in the treatment of many cancer types, including renal cell cancers, GIST tumors, and hematologic malignancies (129,130). They are also used in the treatment of advanced papillary, follicular, oncocytic, and medullary thyroid cancers (130,131). These drugs inhibit the activity of multiple kinases and can reduce cellular activation via the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways. These activities result in inhibition of tumor growth and invasion, but are also the source of many side effects. Details of their current use in thyroid cancer are discussed in Chapter 57. In patients being treated for other malignancies, the most prevalent effect on the thyroid is the development of hypothyroidism. In some cases this has been documented to be preceded by transient thyrotoxicosis with elevated T4 and FT4 levels and/or suppressed TSH levels, thought to represent a destructive thyroiditis (132,133). The frequency of this complication is variable among the different agents, and appears to be related to the degree of effect on VEGF which may lead to loss of fenestrated capillaries. In some cases there has been sonographic evidence supporting decreased thyroidal blood flow, and over time the thyroid gland decreases in size (133). Sunitinib maleate has been most studied and is associated with a high rate of this complication (in many series over 50%). The hypothyroidism is permanent in most of the affected patients, but is transient in some who may demonstrate elevated TSH values after each treatment cycle, followed by TSH normalization (130,133,134). The risk for permanent hypothyroidism increases over time and the number of cycles of treatment, and may occur as late as 2 years after the initiation of treatment. The majority of affected patients do not develop antithyroid antibodies. Development of

hypothyroidism is much less common with sorafenib with a rate one-third that of sunitinib (134,135). An additional effect of the TKIs is enhanced catabolism of thyroid hormones. This is most clearly evident in thyroidectomized thyroid cancer patients who had stable TSH levels prior to TKI use, and then developed an elevated TSH requiring a dose increase (131,136). Patients have also been noted to have decreased T3/T4 and T3/rT3 ratios consistent with increased DIO3 activity (130,137). It is unclear if there may also be decreased intestinal absorption or reduced enterohepatic reabsorption contributing to the increased dose requirement (130,131). Given the high frequency of thyroid disorders, it is recommended that thyroid function be assessed prior to the initiation of therapy, then monthly for several months, and then at 2- to 3month intervals (138).

DRUGS ALTERING THYROID FUNCTION THROUGH ALTERATIONS IN IMMUNITY A number of drugs discussed above, including interferon and lithium, affect thyroid function either in part or completely by inducing thyroid immunity. Treatment of immunodeficiency states may also lead to the development of autoimmune thyroid disease. In the past few years, a number of agents have been developed to treat cancer and multiple sclerosis (MS) by altering immune regulation. Unintended side effects of these drugs have been the development of hyperthyroidism from Graves disease or thyroiditis and hypothyroidism as a consequence of autoimmune hypophysitis or chronic thyroiditis.

IMMUNE RECONSTITUTION SYNDROME With the initiation of HAART and the recovery of CD4 T cells some patients with HIV infection develop the capacity to mount an inflammatory response against infections. This is termed the immune reconstitution inflammatory syndrome (IRIS), and occurs in about 10% of treated patients. It usually develops within a few weeks to months after starting HAART, and is related

to an increase in CD4-positive memory cells. More recently, it has been recognized that 1% to 2% of HIV patients treated with HAART will develop Graves disease, but in contrast to the response to infections, this usually occurs more than 1 year after initiating HAART and involves CD4-positive naïve cells (139). Graves disease in the setting of immune reconstitution has also been reported in children receiving stem cell transplantation for immunodeficiency states (140). Similar to what is seen with the HIV patients, Graves disease developed 16 to 28 months after transplantation. Several patients had the onset of hypothyroidism prior to the onset of Graves disease (140).

ANTI-CD52 ANTIBODY ALEMTUZUMAB This monoclonal antibody reacts against CD52, a glycoprotein that is expressed on B cells and CD4+ T cells. It is approved by the FDA for the treatment of MS and B-cell chronic lymphocytic leukemia. Autoimmune thyroid disease has been noted to occur in a third of treated patients with MS, but not in leukemia patients (141,142). It has also been seen in patients being treated after islet cell transplantation, but at a rate much lower than that seen in patients treated for MS (143). It is unclear if these different rates relate to the underlying disease or different dosing regimens. Some cases are seen within weeks of initiating treatment, and most cases seen within the first 3 years, but cases have been reported up to 7 years after starting treatment (142). The most frequently observed disorder has been Graves disease, which affects 20% to 30% of treated patients (141,142). Most affected patients are TRAb positive. Most patients have overt hyperthyroidism, and some patients have experienced significant ophthalmopathy. While as many as 35% of these patients have been reported to have spontaneous resolution (141), most have been treated medically and a more recent series reported a much lower rate of spontaneous resolution, with some patients requiring radioiodine therapy (142). Hypothyroidism develops in 5% to 7% of treated patients. Most of these patients develop anti-TPO antibodies and the deficits are usually permanent (141,142). Less than 5% of patients will develop typical painless thyroiditis with transient hyperthyroidism sometimes followed by

hypothyroidism.

CHECKPOINT INHIBITORS These agents target systems that are utilized by cancer cells to limit activation of the immune system and thereby block immune-mediated destruction of the cancer. Use of these agents allows the activated immune cells to kill tumor cells (144,145). The same mechanism of action, however, leads to activation of immune-mediated damage to other tissues including skin, liver, pituitary, and thyroid glands (144,145).

Antibody against CTLA-4 (Ipilimumab, Tremelimumab) HLA and B7 expressed on antigen-presenting cells (and tumor cells) interact respectively with T-cell receptors and CD 28 expressed on T cells leading to T-cell activation. Activated T cells then express CTLA-4 that binds to B7 and thus prevents the binding of B7 to CD28. This leads to a loss of T-cell activation and, in the case of tumors, suppresses immunity directed against the tumor. The monoclonal antibodies, ipilmumab and tremelimumab, specifically bind to CTLA-4, allowing B7 and CD28 to interact, and markedly enhance T-cell activation (146,147). This promotes immunemediated destruction of tumor cells, but also results in immune-mediated side effects. Ipilimumab was initially approved by the FDA for the treatment of melanoma in 2011, but use has expanded to the treatment of other tumors. Tremelimumab is used in Europe, but has never been approved by the FDA. The development of hypophysitis is the most common associated autoimmune disorder affecting the thyroid (145,147,148). Prior to the introduction of this agent, autoimmune hypophysitis was typically seen in women in late pregnancy or postpartum, while hypophysitis secondary to ipilimumab has been seen mostly in men. Onset has been within weeks of initiating therapy until almost 2 years later. Most patients present with systemic symptoms while some present with headaches or visual symptoms. MRI abnormalities are common and include pituitary enlargement and stalk thickening (148,149). Similar to postpartum hypophysitis, the pituitary– adrenal axis is most commonly affected. Most deficits are permanent.

Hypothyroidism occurs in 4% to 6% of treated patients from 2 months to 3 years after starting treatment (145,148). Fatigue is the most common presenting symptom. The hypothyroidism is usually permanent. Some 1% to 3% of treated patients will develop typical painless thyroiditis with initial transient hyperthyroidism. The onset is usually 2 to 4 months after starting treatment, and is followed by hypothyroidism that may be transient or permanent. The rate of occurrence of these complications has been reported as 6% to 8% for hypophysitis, 4% to 6% for hypothyroidism, and 2% to 3% for thyroiditis (145,148). New onset Graves has also been reported within a few weeks to 8 years after starting therapy (150,151). It is recommended that baseline thyroid function tests be obtained before starting anti-CTLA-4 therapy, and then at intervals after that (138).

Antibodies Against Programmed Death (PD-1) Receptor (Pembrolizumab, Nivolumab) and Programmed Death Receptor Ligand (PD-L1) (Atezolizumab, Avelumab, Durvalumab, Cemiplimab) Recognition of tumor cells via MHC/T-cell receptor interaction leads to Tcell activation and interferon production, which stimulates tumor cell production of the PD-1 ligand. This then binds with the PD-1 receptor on the T cell and inhibits activation. Dendritic cells also express this ligand to inhibit T-cell activation. Pembrolizumab and nivolumab are monoclonal antibodies directed against the PD-1 receptor that act to increase T-cell activity and thus promote immune-mediated destruction of tumor cells. These drugs were approved by the FDA in 2014 for the treatment of melanoma. Indications for the use of these agents continue to expand and they remain under investigation for treating other tumors (152). Therapy with both pembrolizumab and nivolumab may lead to the development of immune-mediated hypothyroidism, thyroiditis, and hypophysitis with hypothyroidism being the most common problem (145,153,154). Hypothyroidism may occur within weeks of starting therapy or may not occur until after a year, and it is usually permanent. In contrast, reported cases of hyperthyroidism from thyroiditis have occurred from 2

weeks to 5 months after initiating therapy, almost always resolve in 5 to 10 weeks, and are usually followed by hypothyroidism. Many of the cases of hyperthyroidism are asymptomatic, so given the brief duration they may be missed and only the subsequent hypothyroidism may be identified (145,153,154). When measured, iodine uptake is uniformly low and most patients have low thyroid blood flow on ultrasound. For both pembrolizumab and nivolumab, the rate of occurrence of these complications has been reported as 4% to 8% for hypothyroidism, 2% to 6% for thyroiditis, and 2.5 mIU/L, and there was a negative association between serum 4,4′-DDE concentrations and serum FT4 levels (121). In two Spanish

studies examining the effects of prenatal organochlorine pesticide exposure (assessed by placenta levels) on neonatal TSH, one found elevated neonatal TSH levels in infants with HCH exposures greater than the 90th percentile (122), while the other noted variable TSH effects of different organochlorine pesticides without a single clear pattern (123). A U.S. study of men showed that serum DDE concentrations were positively associated with both serum free T4 and total T3 concentrations, while they were inversely associated with serum TSH (124). Animal studies suggest that subchronic exposure of rats to HCB results in irreversible hypothyroidism that develops within 21 days (125). HCB exposure was shown to be inversely associated with serum T4 levels in a cross-sectional study of 5,000 Spanish individuals residing in a highly contaminated area (55). In a South African case-control study, individuals with high DDT exposure had lower serum free T4, free T3, and TSH concentrations than controls (126). Among 259 preschool children in Spain, serum HCH levels were inversely associated with T3, but no associations with thyroid function were noted for HCB or DDE exposures (115). Another Spanish study of 387 newborns showed that plasma HCH was positively associated with plasma TSH levels; positive associations were also noted between TSH levels and both plasma DDE and HCB, but these relationships did not achieve statistical significance (116). HCB exposure has also been inversely correlated with serum T4 levels among Akwesasne Mohawk adolescents (56). Finally, serum free T4 concentrations were inversely associated with HCH in a cohort of 303 men from a rural area of heavy HCH contamination outside of Rio de Janeiro (127).

DIOXINS AND FURANS The term “dioxins” is often used for the family of structurally related polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The most toxic of these chemicals is 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). In addition, some PCBs are described as dioxin-like compounds, and may have similar effects on UDPGT induction. Dioxins are byproducts of a wide range of manufacturing processes including

smelting, chlorine bleaching of paper pulp, and the manufacturing of some herbicides and pesticides. Ingestion is the major route of human exposure, especially in fat-containing animal products such as fish and other seafood. Dioxins are lipophilic and tend to concentrate in adipose tissues, serum, and breast milk. The elimination half-life of TCDD in humans is approximately 7 to 11 years (128). In rat studies, dioxin exposure increases biliary T4 clearance and decreases serum T4 levels (129). Among U.S. Vietnam War Veterans who had sprayed Agent Orange (which contains TCDD), high serum TCDD levels were associated with increased serum TSH levels (130). Agent Orange exposure has also been associated with an increased risk of thyroid cancer among U.S. Veterans (131). As a result of an industrial accident in 1976 in Seveso, Italy, local residents were highly exposed to TCDD. Subsequent studies during the early 1990s demonstrated that infants of TCDD-exposed pregnant women from the Seveso area were found to have elevated neonatal TSH heelstick levels, compared to infants of nonexposed mothers (132). In contrast, in a region of Slovakia heavily polluted with dioxins and furans in addition to DDEs, HCB, and PCBs, serum PCB levels were positively associated with thyroid volumes, serum anti-TPO antibodies, and free T4 and total T3 concentrations and with decreased serum TSH concentrations (133). The thyroidal effects of dioxin exposures require further study though, as a recent systematic review concluded that there were no consistent effects of dioxin exposure on thyroid function across the 23 included studies (134).

COMPOUNDS THAT INHIBIT DEIODINASE ACTIVITY In addition to their other effects, described above, an in vitro study suggests that halogenated phenolic contaminants such as PBDE and triclosan may inhibit hepatic deiodinase (135). HCH has also been discussed above regarding its inhibitory effects on the deiodinase (119).

OTHER EXPOSURES

STYRENES Styrene is used in the production of plastics, rubber, and resins. Exposure may occur by inhaling styrene vapors from building materials, cigarette smoke, car exhaust, and from contaminant ingestion of polystyrene food containers. No adverse thyroid effects have been demonstrated in animal models (136), but in a study of 38 men with high occupational styrene exposure compared against 123 unexposed controls, there was a positive correlation between the duration of styrene exposure and thyroid volume (137). Free T4 levels and the FT4/FT3 ratio were positively correlated with urinary concentrations of a styrene metabolite, suggesting that styrene exposure may inhibit T4 to T3 conversion (137). There is overall limited human understanding regarding the thyroid effects of environmental styrene exposure.

PERFLUORINATED AND GEN X COMPOUNDS The perfluoroalkyl acids, including perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), are synthetic highly persistent perfluorinated compounds which have been used for a wide variety of industrial applications since the 1940s; they have been found in food packaging, commercial household products (e.g., stain-repellant fabrics, nonstick cooking surfaces), and drinking water. Although certain perfluoroalkyl substances are no longer used in the United States, they are still produced internationally, and environmental exposures are persistent. Gen X compounds are newer fluoropolymers (the primary chemical being hexafluorpropylene oxide dimer acid and its ammonium salt) that are synthesized without the use of PFOAs. There are limited data regarding the toxic risks of these exposures in humans. A meta-analysis of 12 included studies reported the association between exposure of perfluoroalkyl substances and decreased serum total T4 concentrations (138), similar to findings among pregnant women and their infants (139). In contrast, other occupational studies have not found clear evidence of a relationship between high-level PFOA exposure and thyroid function (140). The U.S. EPA is undergoing assessments to better understand

the health risks of this class of toxicants with potential antithyroid actions (141).

ORGANOPHOSPHATE PESTICIDES Organophosphate pesticides are unlike organochlorine pesticides and do not persist in the environment, but are currently widely used worldwide in residential and agricultural applications. Occupational studies of 136 male Mexican floriculture workers have recently demonstrated that high exposure levels are associated with increased serum TSH and total T4 levels and with decreased serum total T3 levels; individuals with low hepatic paraoxonase-1 enzyme activity levels may be particularly sensitive to these effects (142,143). In a Chinese study of 637 pregnant women, urinary levels of organophosphate pesticides were positively correlated with serum FT4 levels and negatively correlated with serum TSH levels (144).

SUNSCREENS Evidence from rat studies suggests that ultraviolet filters, including 4methylbenzylidene-camphor (4-MBC) and octyl methoxycinnamate (OMC), decrease both hepatic 5′-deiodinase activity and serum thyroid hormone concentrations in rats (145). These substances are found in cosmetics, plastics, and other personal care products. Although they have been detected in human urine, serum, and breast milk (146), there are limited human data regarding the thyroidal effects of sunscreen exposure (147). In a single prospective study of 15 young men and 17 postmenopausal women, no effects on thyroid function were noted following 1 week of daily whole-body topical application of a sunscreen containing 4-MBC, OMC, and benzophenone-3 (BP-3) (148).

LEAD Among its many toxicities, lead exposure (through inhalation or ingestion)

may adversely affect the pituitary–thyroid axis via an unknown mechanism. U.S. legislation in the 1970s began to reduce lead content from gasoline and smokestack emissions, and a complete ban was initiated in the United States in 1996. However, in many developing countries, leaded gasoline is still in use, and there is limited regulation of lead exposure and monitoring for lead toxicity. In the United States and other developed countries, ingestion of leaded paint chips from older structures remains a concern in some children. Occupational lead exposures occur among workers in mining, smelting, refining, battery manufacturing, soldering, electrical wiring, and ceramic glazing. The human studies assessing the thyroidal effects of lead exposure are inconsistent. A meta-analysis of 16 included studies assessing occupation lead exposure in men did not show any effects on thyroid status (149). However, other occupational studies have demonstrated a pattern of low serum T4 levels with inappropriately low serum TSH concentrations in moderately to highly lead-exposed workers, suggesting possible secondary hypothyroidism (150,151). In contrast, a study of 58 male gas station attendants and auto mechanics reported higher serum TSH concentrations in exposed individuals, compared to unmatched unexposed controls, and T3 and T4 levels did not differ (152). A population-level study of over 5,000 adults in China showed that serum lead levels were positively associated with both higher serum TPO and TSH titers (153).

SUMMARY There has been a growing awareness over the past several decades that common environmental exposures may affect thyroid function in humans and other species. Individuals may be most vulnerable to these effects in utero and in infancy, when thyroid hormone is needed for normal neurodevelopment. Perchlorate, thiocyanate, and nitrate are competitive inhibitors of thyroidal iodine uptake at pharmacologic doses, but their effects on human thyroid function at environmental exposure levels remain unclear. Many compounds, including PCBs, PBDEs, BPA, and triclosan may have direct actions on the thyroid hormone receptor and other effects that remain incompletely understood. Soy isoflavones inhibit TPO activity and may cause

goiter and hypothyroidism if ingested at high levels, particularly in iodinedeficient individuals. PBDEs and hydroxylated PCBs may displace T4 from binding proteins. Organochlorine pesticides and dioxins may decrease serum T4 half-life by activating hepatic enzymes. Styrene exposure may inhibit T4 to T3 conversion, and components of some sunscreens may inhibit hepatic 5′deiodinase activity and lower serum T4 levels. The thyroid risks of low-level environmental and occupational chemical exposures remain incompletely understood at present. In addition, most potential thyroid disruptors have been studied only in isolation, and it is likely that combined toxicant exposures may have additive or synergistic effects. Further studies employing in vitro and animal models combined with robust human epidemiologic and controlled data are needed to examine the potentially adverse effects of thyroid-disrupting chemical exposures.

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14 Nonthyroidal illness syndrome Anita Boelen, Lies Langouche, Wilmar Wiersinga, and Greet Van den Berghe

INTRODUCTION Changes in the regulation of the hypothalamic–pituitary–thyroid axis and in thyroid hormone transport and metabolism are common in patients with nonthyroidal illness, collectively known as the nonthyroidal illness syndrome (NTIS). Illness in this context comprises virtually all nonthyroidal disorders, surgical and nonsurgical trauma, and inadequate caloric intake. Many patients with nonthyroidal illness also receive drugs that affect thyroid hormone regulation and metabolism, but for the sake of clarity, pharmacologic interference is not considered an intrinsic part of NTIS. The effects of illness are not confined to the thyroid axis; in fact, illness induces changes in many neuroendocrine systems (Fig. 14-1). The changes in thyroid function are therefore only one component of the neuroendocrine response to illness and can be viewed as part of the general adaptation to stress (1). Whereas the acute stress response is considered beneficial, this is not necessarily true for the response in prolonged critical illness. NTIS is sometimes referred to as the “sick euthyroid syndrome.” This name is less appropriate because it is unclear if patients are really euthyroid in all target tissues of thyroid hormone. NTIS occurs in humans and animals, and has been reviewed extensively (2,3). NTIS in animals is discussed here only to clarify aspects for which human data are lacking. Although NTIS is heterogeneous, as most evident from particular changes in patients with specific disease entities, the majority of illnesses induce a rather uniform pattern of changes.

DESCRIPTION OF NTIS CHANGES IN SERUM THYROID HORMONE AND THYROTROPIN CONCENTRATIONS NTIS in the Acute Phase of Illness Within hours of the onset of acute stress (such as sepsis, surgery, myocardial infarction, or trauma), serum concentrations of triiodothyronine (T3) decrease and that of reverse triiodothyronine (rT3) increase (4–6). The fall in T3 and rise of rT3 are the most common changes in NTIS, often referred to as the “low T3 syndrome.” Few studies describe in detail the very early stages of NTIS. In patients who underwent elective abdominal surgery, a small but significant increase of serum thyrotropin (TSH) occurs peaking at 0.5 hours after skin incision and lasting for 2 hours, followed by a transient 20% increase of serum total and free thyroxine (FT4); a sustained fall in total and free triiodothyronine (FT3) and rise of rT3 are observed after 2 and 6 hours, respectively (7). The data support the sequence of events depicted in Fig. 141.

FIGURE 14-1. Simplified concept of the pituitary-dependent changes during the course of critical illness. In the acute phase, anterior pituitary secretion is essentially maintained or amplified, whereas anabolic target hormones are inactivated. Cortisol secretion is elevated in concert with ACTH. In the chronic phase anterior pituitary secretion appears uniformly suppressed in

relation to reduced circulating levels of target organ hormones, with cortisol being a notable exception. In the recovery phase, anterior pituitary sensitivity to reduced feedback control is restored. (From Van den Berghe G, de Zegher F, Bouillon R. Acute and prolonged critical illness as different neuroendocrine paradigms. J Clin Endocrinol Metab 1998;83:1827–1834, with permission.)

NTIS in the Chronic Phase of Illness The low serum T3/high rT3 pattern is found in patients with most acute and chronic illnesses, including infectious diseases, infiltrative and metabolic disorders, cardiovascular diseases, pulmonary diseases, gastrointestinal diseases, cancer, burns, and trauma (2,8). In general, serum FT3 falls to a lesser extent than does total T3. Serum rT3 increases to supranormal values in patients with mild illness and does not increase much more in patients with more severe illness. In contrast, serum T3 decreases further as the severity of illness increases (Fig. 14-2). Serum T3 concentrations are low or even undetectable in patients with critical illness, and can remain persistently low in patients with chronic illnesses. The severity of the illness is in general reflected by the magnitude of the changes in serum T3 and rT3. For example, there is an inverse relationship between serum T3 and glycosylated hemoglobin values in diabetes mellitus, between serum T3 and serum creatinine in renal insufficiency, and between serum T3 and burn severity (3). Similarly, the size of myocardial infarction is inversely related to the fall in serum T3 and directly related to the rise in serum rT3 (5). Patients with mild to moderate NTIS usually have normal serum total and free T4 concentrations. Some severely ill patients have low serum T4 (Fig. 14-2), and serum T4 concentrations are inversely correlated with the mortality rate. There has been uncertainty about serum FT4 concentrations in NTIS; the results vary depending on the assay method (see Chapter 17). The issue is complicated by the decrease in serum T4–binding proteins that may occur in severe NTIS. Furthermore, the serum of some patients with NTIS contains substances that inhibit the binding of T4 to serum-binding proteins (9). On the basis of the most accurate assays, it appears that most patients with NTIS have serum FT4 within the normal reference range (10,11). However, blood samples from critically ill patients are often collected through heparinized catheters and heparin is a known indirect inhibitor of

thyroid hormone binding to TBG, which artifactually can increase the free hormone concentration in the sample (12).

FIGURE 14-2. Serum thyroid hormone concentrations (filled circle, triiodothyronine (T3); open circle, reverse T3 (rT3); open triangle, thyroxine (T4); upside down open triangle, free T4 index; closed triangle, free T4, measured by equilibrium dialysis) in 504 newly hospitalized patients, divided as follows: Group I, all values within the normal reference range; group II, serum rT3 values high but serum T3 and T4 values normal; group III, serum T3 values low but serum T4 values normal; group IV, serum T3 and T4 values low. The hospitalization days are given as median days for the survivors. (From Docter R, Krenning EP, de Jong M, et al. The sick euthyroid syndrome: changes in thyroid hormone serum parameters and hormone metabolism. Clin Endocrinol (Oxf) 1993;39:499–518, with permission.)

Serum TSH is usually normal in NTIS. However, some critically ill patients who have low serum T4 concentrations also have low serum TSH, indicative of a poor prognosis (13), although TSH is rarely as low as in patients with thyrotoxicosis. The serum TSH response to thyrotropin-

releasing hormone (TRH) is usually proportional to baseline TSH. Critically ill patients are sometimes treated with dopamine infusions or high doses of glucocorticoids, which can lower serum TSH (14,15). In addition, stress levels of glucocorticoids may have a suppressive effect on TSH secretion. SH (16). The prevalence of low serum TSH in patients with NTIS is rather low. In patients admitted to an intensive care unit (ICU) who underwent invasive mechanical ventilation, low TSH was observed in 7%, low FT4 in 24%, and low FT3 in 79% (17).

NTIS in the Recovery Phase of Illness During recovery from illness a rapid rise of total and free T4 can be observed, associated with a marked increase in serum TSH. TSH concentrations may rise transiently above the upper normal limit in some patients, but rarely exceed the value of 10 mU/L. The TSH rise consistently precedes the T4 rise, suggesting an essential role of TSH in the return of T4 to normal. Normalization of serum T3 also occurs after the TSH rise (18,19).

NTIS and Nutrition Fasting in healthy individuals induces a fall in serum T3 and rise in serum rT3 within 24 to 36 hours, and the values return rapidly to baseline upon refeeding (20). When fasting is prolonged, also T4 levels decline, without a compensatory rise in TSH (20,21). A reduced nutritional intake also contributes to NTIS in critically ill patients as early feeding prevents part of the fall in plasma T4, T3 and TSH and the rise in rT3 (22). (Fig. 14-3). Importantly, tolerating a nutritional deficit during the first week in ICU was demonstrated to be clinical superior to early feeding, which was partly explained by the acute fall in T3 and T3/rT3 ratio, suggesting that this acute response to illness is likely adaptive.

CHANGES IN THYROID HORMONE KINETICS Kinetic studies of thyroid hormone production and metabolism have been conducted in normal subjects during fasting and in patients with chronic renal

failure, cirrhosis, diabetes mellitus, mild illness, and critical illness. The results can be summarized as follows: in patients with low serum T3, the production rate of T3 is decreased, but its metabolic clearance rate is unchanged (8). The decrease in T3 production is due to decreased extrathyroidal deiodination of T4 to T3, which normally provides ±80% of the daily T3 production. The fractional rate of transport of T3 into tissues is unaltered. For rT3, the production rate is increased, especially in more severe illness, and the metabolic clearance rate is decreased, due to decreased extrathyroidal deiodination of rT3 to 3,3′-diiodothyronine (23–25). For T4, the metabolic clearance rate to T3 is usually decreased, whereas the metabolic clearance to rT3 is increased, especially in patients with severe illnesses. The most severely ill patients have low serum T4, due at least in part to decreased production of one or more serum thyroid hormone–binding proteins. These severely ill patients also tend to have low serum TSH, and may have low T4 production rates. The fractional rate of transport of T4 from serum to tissues is reduced to ±50% of the normal value.

FIGURE 14-3. Effects of early macronutrient supplementation versus tolerating macronutrient deficit during the acute critical illness: partial reversal of NTIS in human patients. The bars (mean − standard error) represent the changes from the admission values (D) to day 3 in the intensive care unit or last day for patients with a shorter ICU stay. Open bars values of patients randomized to receiving early parenteral nutrition (early PN), filled bars values from patients randomized to nutrient restriction (late PN). (From Langouche L, Vander Perre S, Marques M, et al. Impact of early nutrient restriction during critical illness on the nonthyroidal illness

syndrome and its relation with outcome: a randomized, controlled clinical study. J Clin Endocrinol Metab 2013;98:1006–1013, with permission.)

CHANGES IN THE HYPOTHALAMIC–PITUITARY– THYROID AXIS The failure of serum TSH to increase despite marked reductions in serum T3 and sometimes T4, suggests that the sensitivity of TSH secretion to low serum thyroid hormone concentrations is decreased in NTIS. This could be due to changes in the thyrotropes or due to decreased TRH secretion. Evidence for the latter is a decrease in the amplitude (but not frequency) of the nocturnal pulses of TSH secretion; such decreases in pulse amplitude have been documented during fasting, after surgery, and in a wide variety of nonthyroidal diseases, including chronic renal insufficiency, diabetes mellitus, acute respiratory failure, infections, cancer, and critical illness (26,27). For example, fasting for 60 hours results in disappearance of the nocturnal TSH surge and a 50% reduction in 24-hour mean serum TSH concentrations. Among patients with NTIS, the nocturnal TSH surge is decreased in at least half, and the decrease is not closely related to changes in serum T3 or free T4 (28). The pattern of 24-hour TSH secretion in these patients is similar to that in central hypothyroidism. If the abnormal pattern of TSH secretion in NTIS persists for a week or longer, then the T4 production rate should decrease, as it does in patients with central hypothyroidism. In a study in deceased patients with NTIS, the TRH messenger RNA (mRNA) content in the paraventricular nucleus (PVN) was directly correlated with serum TSH and T3 obtained just before death, but not with serum T4 or free T4 (Fig. 14-4). In another study, the T3 content of the hypothalamus and anterior pituitary were, respectively, 64% and 46% lower in patients who died as a result of illness as compared with patients who died suddenly (29). Furthermore, the TSH that is secreted in patients with NTIS is less glycosylated, and therefore less biologically active, than normal, a change that also occurs in patients with overt hypothalamic disease and TRH deficiency (30). Little is known about the function of the thyroid gland itself in NTIS. The increase of serum TSH induced by exogenous TRH or that occurs during

recovery from illness is followed by an increase in serum T3 and T4, indicating that the thyroid gland can respond normally to TSH. Thyroid weight is lower and thyroid follicular size is reduced in deceased chronically ill patients, as compared with subjects with sudden death (31), possibly related to decreased TSH secretion in severe NTIS.

FIGURE 14-4. Top: Macroscopic film autoradiograms of hypothalamic sections of two patients, showing the hybridization signal of thyrotropinreleasing hormone (TRH) messenger RNA (mRNA) in the paraventricular nucleus along the wall of the third ventricle. The left panel (A) shows a lowintensity signal of a patient with low serum triiodothyronine (T3), thyroxine (T4), and thyrotropin (TSH) concentrations measured