Molecular, Genetic, and Nutritional Aspects of Major and Trace Minerals

Molecular, Genetic, and Nutritional Aspects of Major and Trace Minerals

Editor

James F. Collins

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Series Preface

Part I. Calcium

Chapter 1. Calcium-Sensing Receptor Polymorphisms and Human Disease

Introduction

Calcium-Sensing Receptor Gene Polymorphisms and Calcium–Phosphate Homeostasis

Calcium-Sensing Receptor Gene Polymorphisms and Disorders of the Parathyroid Glands

Calcium-Sensing Receptor Gene Polymorphisms and Disorders of the Kidney

Calcium-Sensing Receptor Gene Polymorphisms and Bone Mineral Density

Conclusions

Summary Points

Key Facts

Mini Dictionary of Terms

Chapter 2. Molecular Aspects of the Calcium-Sensing Receptor and Calcium Homeostasis

Introduction

Structural Components of the Calcium-Sensing Receptor

Calcium-Sensing Receptor Function in the Parathyroid Gland

Calcium-Sensing Receptor Function in Bone

Calcium-Sensing Receptor Function in the Kidney

Pathophysiological Disorders of the Kidney and Calcium-Sensing Receptor Modulation

Calcium-Sensing Receptor Function in the Gastrointestinal System

Calcium-Sensing Receptor Function in the Skin

Conclusion

Chapter 3. New Developments in Our Understanding of the Regulation of Calcium Homeostasis by Vitamin D

Introduction

Bioactivation of Vitamin D

Regulation of Vitamin D Metabolism

New Insights Into Mechanisms Involved in the Effects of 1,25(OH)2D3 in Intestine, Kidney, and Bone

Genomic Mechanism of 1,25-DihydroxyVitamin D3 Action

Summary Points and Key Facts

Mini Dictionary of Terms

Chapter 4. Calcium in Obesity and Related Diseases: The Calcium-Sensing Receptor as a Novel Mediator

Introduction

Obesity: A Serious Public Health Problem Worldwide

Cellular Mechanisms for Fat Dysfunction: Role of Calcium

Conclusion

Chapter 5. Calcium: Basic Nutritional Aspects

Introduction

Summary Points

Key Facts

Chapter 6. Molecular Aspects of Calcium and Bone Mineralization

Introduction

Metabolism and Regulation

Calcium, Genetics, and Genetic Disorders

Other Genetic Factors Affecting Calcium Homeostasis

Conclusions

Part II. Copper

Chapter 7. Copper: Basic Physiological and Nutritional Aspects

Copper: Historical Facts

Copper Biochemistry

Biochemical and Physiologic Functions of Copper

Catalytic Functions of Copper (Copper-Dependent Proteins)

Physiological Functions of Copper

Copper Bioavailability and Nutrient Interactions

Copper Metabolism

Copper Deficiency in Humans

Copper: Dietary Considerations and Requirements

Evaluation of Copper Status

Copper Toxicity and Upper Limits

Chapter 8. Copper and Molecular Aspects of Cell Signaling

Introduction

The Copper Metalloproteome

Copper Modulation of Cell Signaling

Copper as a Regulator of Epigenetics

Cell–Cell Signaling Regulated by Copper

How Other Minerals Are Affected or Behave

Perspectives

Summary Points

Mini Dictionary of Terms

Chapter 9. Copper and Hypoxia-Inducible Transcription Factor Regulation of Gene Expression

Introduction to Hypoxia-Inducible Factors

Relationship between Copper and Hypoxia-Inducible Factor-1

Mechanisms by Which Cu Regulates Hypoxia-Inducible Factor-1 Activity

Clinical Implications

Open Questions

Chapter 10. Copper in Wilson’s and Alzheimer’s Diseases, Copper-Lowering Therapy in Cancer and Other Diseases, and Copper Deficiency

Introduction

Wilson’s Disease

Alzheimer’s Disease

The Promise of Copper-Lowering Therapy in Cancer and Other Diseases

Clinical Copper Deficiency

Part III. Iodine

Chapter 11. Iodine: Basic Nutritional Aspects

Role of Iodine

Iodine Metabolism

Sources of Iodine Nutrition

Recommended Dietary Intake of Iodine

Factors Influencing Iodine Bioavailability

Assessment of Iodine Nutrition

Iodine Deficiency

Treatment and Prevention of Iodine Deficiency

Toxic Effects of Excess Iodine Intake

Conclusion

Chapter 12. Iodine and Thyroid Hormone Synthesis, Metabolism, and Action

Iodide Transport

Iodine Clearance

Direct Actions of Iodine

Thyroid Hormone Action

Chapter 13. Iodine and Adipocytokines: Cellular Aspects

Introduction

The Role of Iodine in the Human Body

Possible Mechanism of Iodine Action

Iodine and Adipocytokines

Summary Points

Part IV. Iron

Chapter 14. Iron: Basic Nutritional Aspects

Introduction

Physiological Functions of Iron

Adverse Consequences of Too Much Iron

Finding the Right Balance

Iron and Our Diet

Chapter 15. Hepcidin and the Hormonal Control of Iron Homeostasis

Introduction

Biology of Hepcidin

The Regulation of Hepcidin Expression

Conclusion

Chapter 16. Genetic Rodent Models of Systemic Iron Homeostasis

Introduction

Maternal–Fetal and Maternal–Neonatal Iron Physiology

Overview of Dietary Iron Absorption

Gastric Acidification

Iron Import Into Duodenum

Iron Export From Duodenum to Blood

Regulation of Hepcidin Expression

Distribution of Iron to Organs

Cellular Iron Utilization

Erythropoiesis

Recycling of Iron From Red Cells

Iron Export

Iron Toxicity

Areas for Additional Models and Future Research

Other Minerals

Summary Points

Key Facts

Mini Dictionary of Terms

Chapter 17. Iron, Cancer, and Hypoxia-Inducible Factor Signaling

Introduction

Oxygen-Sensing Transcription Factors

Oxygen and Iron Signaling

Hypoxia and Cancer

Iron and Cancer Risk

Mechanisms Regulating Intratumoral Iron

Mechanisms of Iron Action

Potential in Cancer Therapy

Other Minerals in Cancer

Summary Points and Key Facts

Mini Dictionary of Terms

Chapter 18. Iron Transporters and Iron Homeostasis

Overview of Whole-Body Iron Homeostasis and Scope of Review

Dietary Iron Absorption by the Enterocyte

Iron Metabolism in the Hepatocyte

Iron Metabolism in the Developing Erythroid Cell

Regulation of Iron Homeostasis by Hepcidin

Chapter 19. Regulation of Divalent Metal-Ion Transporter-1 Expression and Function

Introduction—Historical Perspective

One Divalent Metal-Ion Transporter-1 Function—Nutritional Iron Entry Into Duodenal Enterocytes

A Second DMT1 Function—Endosomal Export after Iron Acquisition in Most Cells via the Tf Cycle

Metal-Ion Transport by Divalent Metal-Ion Transporter-1

Reduction, Divalent Metal-Ion Transporter-1 Function, and Iron Metabolism

Divalent Metal-Ion Transporter-1 Isoforms, Interactions, and Modifications

Divalent Metal-Ion Transporter-1 Regulation—An Overview

DMT1 Regulation—Transcription

Divalent Metal-Ion Transporter-1 Regulation—Posttranscriptionally at the mRNA Level

Divalent Metal-Ion Transporter-1 Regulation—at the Translational Level

Divalent Metal-Ion Transporter-1 Regulation—Posttranslationally at the Protein Turnover Level

Summary Points

Mini Dictionary of Terms

Part V. Zinc

Chapter 20. Discovery of Zinc for Human Health and Biomarkers of Zinc Deficiency

Introduction

Discovery of Zinc as an Essential Element for Human Health

Therapeutic Impact of Zinc

Biomarkers of Zinc Deficiency in Egypt

Chapter 21. Zinc Signals and Immune Function

Signal Transduction in Mammalian Cells

The Immune System

The Physiological Role of Zinc in the Immune System

Zinc Signals

Sensors for Zinc Signals

Targets of Zinc Signals

Conclusion

Chapter 22. Posttranslational Mechanisms of Zinc Signaling

Introduction

Zinc-Transport Proteins

Posttranslational Modification of Zinc-Transport Proteins

Applications

How Other Minerals Are Affected

Key Facts

Mini Dictionary of Terms

Chapter 23. Zinc Transporters in Health and Disease

Introduction

ZIP and ZnT Transporter Biochemistry

ZIP and ZnT Transporters in Health and Disease

Zinc Transporters and Inherited Human Diseases

ZnT and ZIP Single Nucleotide Polymorphisms

Conclusions

Chapter 24. Genetic Study of Zinc Transporters and Zinc Signaling

Introduction: Zinc as an Essential Trace Element

Zinc Transporters and Signaling in Physiology and Pathogenesis

Zinc Transporters and the Zn Signaling Axis

Future Perspectives

Part VI. Magnesium

Chapter 25. Magnesium: Basic Nutritional Aspects

Introduction

Tissue Distribution

Biological Roles

Body Homeostasis

Requirements

Dietary Deficiency Occurrence

Status Assessment

Deficiency Signs

Dietary Sources

Toxicity

Chapter 26. Magnesium and the Immune Response

Introduction (Background)

General Structural Features of Mg²+ Permeable Ion Channels and Transporters

Mg²+ and Its Potential Roles in Immunoreceptor Signaling

SLC41A1 and SLC41A2—Relatives of the Bacterial MgtE Family

The Channel Kinases TRPM6 and TRPM7—Mg²+ Sensors and Master Regulators

Conclusions

Chapter 27. Magnesium Intake and Chronic Disease in Humans

Introduction

Magnesium and Diabetes Mellitus

Magnesium and Metabolic Syndrome

Magnesium and Hypertension

Magnesium and Cardiovascular Disease

Magnesium and Cancer

Magnesium and Interactions With Other Minerals

Conclusion

Summary Points

Chapter 28. Magnesium and Embryonic Development

Introduction

Chemical Properties of Magnesium and Its Function and Regulation in Cells

Magnesium’s Impact on Embryogenesis

Function of TRPM7 and TRPM6 Channels During Embryogenesis

TRPM7 Regulates Gastrulation During Vertebrate Embryogenesis

Conclusions

Chapter 29. Magnesium, Vascular Function, and Hypertension

Introduction

Magnesium Metabolism

Magnesium Absorption in the Gastrointestinal Tract

Magnesium Excretion by the Kidney

Compartmentalization of Magnesium in the Body

Regulation of Cellular Magnesium Homeostasis

Cellular Magnesium Influx

TRPM6 and TRPM7 Cation Channels

Magnesium Transporter Subtype 1

Magnesium Efflux from Cells

Magnesium and Vascular Function

Magnesium and Hypertension

TRPM7 and Cardiovascular Disease

Conclusions

Part VII. Manganese

Chapter 30. Nutritional, Genetic, and Molecular Aspects of Manganese Intoxication

Essentiality and Toxicity of Manganese

Mechanisms of Mn Transport to the Central Nervous System

Molecular Mechanisms of Mn Neurotoxicity

Genetic Aspects of Mn Intoxication

Concluding Remarks

Chapter 31. Manganese and Nutritional Immunity

Introduction

Manganese Distribution

Calprotectin: Chelating Manganese and Zinc at the Site of Infection

Natural Resistance-Associated Macrophage Protein-1/SLC11A1: Starving Bacteria in Macrophages

Bacterial Manganese Importers: Acquiring Manganese During Infection

Manganese-Requiring Processes in Bacterial Pathogens

Intersection of Manganese Nutritional Immunity With Other Minerals

Areas for Future Study

Conclusions

Summary Points

Key Facts

Mini Dictionary of Terms

Chapter 32. Manganese and Mitochondrial Function

Introduction

Manganese Speciation: Does Mn²+ or Mn³+ Cause the Most Damage?

Mitochondria

The Mitochondrial Role in Apoptosis

Manganese Inhibition of Oxidative Phosphorylation

Manganese-Induced Increases in Mitochondrial Production of Reactive Oxygen Species

Evidence for Induction of Apoptosis by Manganese

Conclusions

Part VIII. Molybdenum

Chapter 33. Molybdenum Cofactor in Humans: Health, Disease, and Treatment

Introduction

Deficiencies in Molybdenum Enzymes

Molybdenum Cofactor Deficiency

Treatment of Molybdenum Cofactor Deficiency

Association of Molybdenum With Other Metals and Disorders

Conclusion and Future Perspectives

List of Abbreviations

Part IX. Phosphorus

Chapter 34. Phosphorus: Basic Nutritional Aspects

Introduction

How Other Minerals Are Affected or Behave

Summary Points

Key Facts

Mini Dictionary of Terms

Chapter 35. Molecular Mechanisms of Adverse Health Effects Associated With Excess Phosphorus Intake

Introduction

Molecular Mechanisms Regulating Phosphorus Homeostasis

Excess Phosphorus Intake Associated With Disruption of Regulatory Mechanisms Related to Phosphorus Homeostasis and Disease Risk

Summary and Conclusions

Chapter 36. Transcriptional Regulation of Sodium-Phosphate Cotransporter Gene Expression

Introduction

Regulatory Factors of Type II NaPi Cotransporters

Transcriptional Regulation of SLC34A1/NaPi-IIa Gene

Transcriptional Regulation of SLC34A2/NaPi-IIb Gene

Transcriptional Regulation of the SLC34A3/NaPi-IIc Gene

Conclusion and Perspective

Part X. Selenium

Chapter 37. Selenium: Basic Nutritional Aspects

Nutritional Essentiality and Metabolism of Selenium

Incorporation of Selenium Into Selenoproteins

Physiological Functions of Selenoproteins

Regulation of Selenoprotein Expression

Pharmacological Effects of Selenium

Selenium Toxicity and Interactions With Vitamin E and Minerals

New Methods and Models and Future Perspectives

Chapter 38. Selenium and Cancer

Introduction

Selenium and Epidemiologic Studies

Selenocompounds or Selenoproteins?

Polymorphisms in Selenoproteins and Relevance to Cancer

Concluding Remarks

Chapter 39. Could Selenium Be a Double-Edged Sword?

Introduction

Sources of Animal-Based Se for Human Consumption

Selenoproteins and Their Functions

Mechanisms of Selenium/Selenoprotein Action

Nutritional Requirements of Selenium

Types of Selenium Supplements

Dual Role of Cancer Prevention and Promotion

Interaction of Selenium With Other Micro Nutrients

Conclusion

Part XI. Electrolytes

Chapter 40. Sodium: Basic Nutritional Aspects

Introduction

How Other Minerals Are Affected or Behave

Summary Points

Key Facts

Mini Dictionary of Terms

Chapter 41. Potassium Channel Mutations and Human Disease: Focus on Adrenal Hypertension

Introduction

Broad Classification of Potassium Channels

Known Mutated Potassium Channel Genes and Associated Phenotypes

Primary Aldosteronism: A Common Cause of Hypertension Associated With KCNJ5 Mutations

Conclusions

Part XII. Nonessentials

Chapter 42. Chromium: Basic Nutritional and Toxicological Aspects

Introduction

Chromium Is Not an Essential Element

Chromium(III) Toxicity and the Fate of Chromium Supplements In Vivo

Pharmacologically Active Chromium

Chapter 43. Nonessential Trace Minerals: Basic Nutritional and Toxicological Aspects

Introduction

Boron

Silicon

Conclusion

Chapter 44. Fluoride: Intake and Metabolism, Therapeutic and Toxicological Consequences

Chemical Origin and Properties

Sources of Fluoride Exposure

Fluoride Metabolism

Function

Excessive Intake and Toxicity

Adequate Daily Intake and Tolerable Upper Intake Level

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1800, San Diego, CA 92101-4495, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2017 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-0-12-802168-2

For information on all Academic Press publications visit our website at https://www.elsevier.com/

Publisher: Nikki Levy

Acquisition Editor: Megan Ball

Editorial Project Manager: Jaclyn Truesdell

Production Project Manager: Lisa Jones

Designer: Victoria Pearson

Typeset by TNQ Books and Journals

List of Contributors

Abedalrazaq Alkukhun,     Yale University School of Medicine, New Haven, CT, United States

Gregory Jon Anderson,     QIMR Berghofer Medical Research Institute, Australia

Tayze T. Antunes,     University of Ottawa, Ottawa, ON, Canada

Michael Aschner,     Albert Einstein College of Medicine, New York, NY, United States

Terry J. Aspray

Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom

Freeman Hospital, Newcastle upon Tyne, Tyne and Wear, United Kingdom

Thomas Bartnikas,     Brown University, Providence, Rhode Island, United States

Abdel A. Belaidi,     The University of Melbourne, Parkville, VIC, Australia

Roberto Bravo-Sagua,     Universidad de Chile, Santiago, Chile

Gregory A. Brent,     David Geffen School of Medicine at UCLA, Los Angeles, CA, United States

George J. Brewer,     University of Michigan, Ann Arbor, MI, United States

Mona S. Calvo,     U.S. Food and Drug Administration, Laurel, MD, United States

Bradley Allen Carlson,     National Institutes of Health, Bethesda, MD, United States

Wen-Hsing Cheng,     Mississippi State University, Mississippi, MS, United States

Sylvia Christakos,     Rutgers, The State University of New Jersey, New Jersey Medical School, Newark, NJ, United States

Mariana Cifuentes,     Universidad de Chile, Santiago, Chile

James F. Collins,     University of Florida, Gainesville, FL, United States

Puneet Dhawan,     Rutgers, The State University of New Jersey, New Jersey Medical School, Newark, NJ, United States

Ralph Marsland Duckworth

Teesside University, Middlesbrough, United Kingdom

Newcastle University, Newcastle-upon-Tyne, United Kingdom

Lynnette Robyn Ferguson,     University of Auckland, Auckland, New Zealand

David Michael Frazer,     QIMR Berghofer Medical Research Institute, Australia

Toshiyuki Fukada

Tokushima Bunri University, Tokushima, Japan

Showa University, Tokyo, Japan

RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

Kazuhisa Fukue,     Kyoto University, Kyoto, Japan

Priyanka V. Gangodkar,     GenePath Dx (Causeway Healthcare Private Limited), Pune, India

Eduardo Garcia-Fuentes

Institute of Biomedical Research of Malaga (IBIMA), Regional University Hospital, Malaga, Spain

CIBEROBN, Institute of Health Carlos III, Malaga, Spain

Michael D. Garrick,     University at Buffalo, Buffalo, NY, United States

John P. Geibel,     Yale University School of Medicine, New Haven, CT, United States

Fayez K. Ghishan,     University of Arizona, Tucson, AZ, United States

Vadim N. Gladyshev,     Harvard Medical School, Boston, MA, United States

A. Grubman,     The University of Melbourne, Parkville, VIC, Australia

Thomas E. Gunter,     University of Rochester, Rochester, NY, United States

Hajo Haase,     Berlin Institute of Technology, Berlin, Germany

Dolph Lee Hatfield,     National Institutes of Health, Bethesda, MD, United States

Ka He,     Indiana University, Bloomington, IN, United States

Carolina Herrera,     Brown University, Providence, Rhode Island, United States

Kayo Ikuta,     Tokushima University, Tokushima, Japan

Francisco J. Rios,     University of Glasgow, Glasgow, Scotland

Sami Judeeba,     Yale University School of Medicine, New Haven, CT, United States

Lillian J. Juttukonda,     Vanderbilt University Medical Center, Nashville, TN, United States

Taiho Kambe,     Kyoto University, Kyoto, Japan

Ichiro Kaneko,     Tokushima University, Tokushima, Japan

Yujian James Kang

Sichuan University, Chengdu, Sichuan, China

University of Louisville, School of Medicine, Louisville, KY, United States

Nishi Karunasinghe,     University of Auckland, Auckland, New Zealand

Anuradha V. Khadilkar,     Jehangir Medical Research Institute Jehangir Hospital, Pune, India

Pawel R. Kiela,     University of Arizona, Tucson, AZ, United States

Katerine S. Knust,     Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Rio de Janeiro, Brazil

Mitchell D. Knutson,     University of Florida, Gainesville, FL, United States

Yuko Komiya,     Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, United States

Daniel Laubitz,     University of Arizona, Tucson, AZ, United States

Sergio Lavandero

Universidad de Chile, Santiago, Chile

University of Texas Southwestern Medical Center, Dallas, TX, United States

Xin Gen Lei,     Cornell University, Ithaca, NY, United States

Angela M. Leung

UCLA David Geffen School of Medicine, Los Angeles, CA, United States

VA Greater Los Angeles Healthcare System, Los Angeles, CA, United States

Anna Milanesi,     David Geffen School of Medicine at UCLA, Los Angeles, CA, United States

Ken-ichi Miyamoto,     Tokushima University, Tokushima, Japan

Augusto C. Montezano,     University of Glasgow, Glasgow, Scotland

Stefano Mora,     IRCCS San Raffaele Scientific Institute, Milan, Italy

Armando Salim Munoz-Abraham,     Yale University School of Medicine, New Haven, CT, United States

Forrest Harold Nielsen,     USDA, ARS, Grand Forks Human Nutrition Research Center, Grand Forks, ND, United States

Thirayost Nimmanon

Cardiff University, Cardiff, United Kingdom

Phramongkutklao College of Medicine, Bangkok, Thailand

Yukina Nishito,     Kyoto University, Kyoto, Japan

Tanara Vieira Peres,     Albert Einstein College of Medicine, New York, NY, United States

Anne-Laure Perraud

National Jewish Health, Denver, CO, United States

University of Colorado Denver, Denver, CO, United States

Michael Pettiglio,     Brown University, Providence, Rhode Island, United States

Nikhil D. Phadke,     GenePath Dx (Causeway Healthcare Private Limited), Pune, India

Ananda S. Prasad,     Wayne State University School of Medicine, Detroit, MI, United States

Vijayababu M. Radhakrishnan,     University of Arizona, Tucson, AZ, United States

Marcela Reyes,     Universidad de Chile, Santiago, Chile

Loren Warren Runnels,     Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, United States

Carsten Schmitz

University of Colorado Denver, Denver, CO, United States

National Jewish Health, Denver, CO, United States

Guenter Schwarz,     University of Cologne, Cologne, Germany

Hiroko Segawa,     Tokushima University, Tokushima, Japan

Yatrik Madhukar Shah,     University of Michigan, Ann Arbor, MI, United States

Eric P. Skaar,     Vanderbilt University Medical Center, Nashville, TN, United States

Laura Soldati,     Università degli Studi of Milan, Milan, Italy

Michael Stowasser,     The University of Queensland, School of Medicine, Brisbane, QLD, Australia

Sawako Tatsumi,     Tokushima University, Tokushima, Japan

Kathryn M. Taylor,     Cardiff University, Cardiff, United Kingdom

Ryuta Tobe,     National Institutes of Health, Bethesda, MD, United States

Rhian M. Touyz,     University of Glasgow, Glasgow, Scotland

Cari Lewis Tsinovoi,     Indiana University, Bloomington, IN, United States

Petra Akiko Tsuji,     Towson University, Towson, MD, United States

Jaime Uribarri,     The Icahn School of Medicine at Mount Sinai, New York, NY, United States

Inés Velasco,     Hospital Riotinto, Huelva, Spain

Vaishali Veldurthy,     Rutgers, The State University of New Jersey, New Jersey Medical School, Newark, NJ, United States

Giuseppe Vezzoli,     IRCCS San Raffaele Scientific Institute, Milan, Italy

John Bertram Vincent,     The University of Alabama, Tuscaloosa, AL, United States

Tao Wang,     Sichuan University, Chengdu, Sichuan, China

Ran Wei,     Rutgers, The State University of New Jersey, New Jersey Medical School, Newark, NJ, United States

Marianne Wessling-Resnick,     Harvard T.H. Chan School of Public Health, Boston, MA, United States

A.R. White,     The University of Melbourne, Parkville, VIC, Australia

Ying Xiao,     Sichuan University, Chengdu, Sichuan, China

Xiang Xue,     University of Michigan, Ann Arbor, MI, United States

Hironori Yamamoto,     Jin-ai University, Fukui, Japan

Wen Yin,     Sichuan University, Chengdu, Sichuan, China

Wenjing Zhang,     Sichuan University, Chengdu, Sichuan, China

Fatemeh Vida Zohoori,     Teesside University, Middlesbrough, United Kingdom

Series Preface

In this series on Molecular Nutrition, the editors of each book aim to disseminate important material pertaining to molecular nutrition in its broadest sense. The coverage ranges from molecular aspects to whole organs, and the impact of nutrition or malnutrition on individuals and whole communities. It includes concepts, policy, preclinical studies, and clinical investigations relating to molecular nutrition. The subject areas include molecular mechanisms, polymorphisms, SNPs, genomic wide analysis, genotypes, gene expression, genetic modifications, and many other aspects. Information given in the Molecular Nutrition series relates to national, international, and global issues.

A major feature of the series that sets it apart from other texts is the initiative to bridge the transintellectual divide so that it is suitable for novices and experts alike. It embraces traditional and nontraditional formats of nutritional sciences in different ways. Each book in the series has both overviews and detailed and focused chapters.

Molecular Nutrition is designed for nutritionists, dieticians, educationalists, health experts, epidemiologists, and health-related professionals such as chemists. It is also suitable for students, graduates, postgraduates, researchers, lecturers, teachers, and professors. Contributors are national or international experts, many of whom are from world-renowned institutions or universities. It is intended to be an authoritative text covering nutrition at the molecular level.

Victor R. Preedy

Series Editor

Part I

Calcium

Outline

Chapter 1. Calcium-Sensing Receptor Polymorphisms and Human Disease

Chapter 2. Molecular Aspects of the Calcium-Sensing Receptor and Calcium Homeostasis

Chapter 3. New Developments in Our Understanding of the Regulation of Calcium Homeostasis by Vitamin D

Chapter 4. Calcium in Obesity and Related Diseases: The Calcium-Sensing Receptor as a Novel Mediator

Chapter 5. Calcium: Basic Nutritional Aspects

Chapter 6. Molecular Aspects of Calcium and Bone Mineralization

Chapter 1

Calcium-Sensing Receptor Polymorphisms and Human Disease

Giuseppe Vezzoli¹, Laura Soldati²,  and Stefano Mora¹     ¹IRCCS San Raffaele Scientific Institute, Milan, Italy     ²Università degli Studi of Milan, Milan, Italy

Abstract

The calcium-sensing receptor (CaSR) is a plasma membrane protein that senses calcium in the extracellular fluid. Cloned from bovine parathyroid cells in 1993, CaSR is mainly expressed in the parathyroid glands and renal distal tubules, and it enables these tissues to regulate parathyroid hormone secretion and calcium excretion, respectively, in response to changes in serum calcium. CaSR is also crucial for normal growth plate development and bone remodeling. Therefore CaSR is a key regulator of calcium homeostasis. Moreover, CaSR polymorphisms have been associated with serum calcium concentration and urine calcium excretion in humans. Polymorphisms decreasing CaSR expression in renal tubular cells may increase risk for developing calcium oxalate kidney stones. CaSR gene polymorphisms may modify the phenotype of healthy subjects and patients with primary and secondary hyperparathyroidism, and it may allow identification of individuals prone to developing kidney stones.

Keywords

Calcimimetics; Calcium-sensing receptor; Genetic polymorphisms; Hypercalcemia; Hyperparathyroidism; Hypocalcemia; Kidney stone disease

Introduction

Circulating calcium ions can directly modulate cell activity in humans by means of a plasma membrane receptor that is sensitive to extracellular calcium, the calcium-sensing receptor (CaSR). CaSR was firstly cloned from bovine parathyroid cells in 1993 (Brown et al., 1992) and then in human parathyroid cells and renal tubular cells (Aida et al., 1995; Garrett et al., 1995). CaSR is a 1078-amino–acid protein that belongs to the third class of G-protein-coupled receptor (GPCR) family. It is expressed as a disulfide-linked homodimer in caveolin-rich areas of the plasma membrane, although it may also form heterodimers with other members of the GPCR family (Kifor et al., 1998). As an environmental sensor, CaSR elicits the paracrine or autocrine adaptive responses of human cells to changes in local or serum calcium concentrations. This adaptive response is fundamental for the physiological effect of parathyroid and kidney cells in human calcium homeostasis. The parathyroid glands and renal distal tubules are the tissues with the highest expression of CaSR, and its presence enables them to regulate calcium excretion and parathyroid hormone (PTH) secretion in response to serum calcium changes (Fig. 1.1). CaSR stimulation by the increase of serum calcium is followed by the inhibition of calcium reabsorption in the renal tubules and PTH secretion to restore normal serum calcium levels (Riccardi and Brown, 2010; Riccardi and Kemp, 2012). CaSR was also shown to be essential for osteoblast-mediated bone remodeling (Dvorak et al., 2004). Therefore CaSR is a key factor in calcium homeostasis (Riccardi and Kemp, 2012).

The CaSR molecule includes a large bilobed Venus-flytrap–like extracellular domain of 612 amino acids, a seven-membrane–spanning domain of 250 amino acids, and a C-terminal intracellular domain of 216 amino acids (Riccardi and Kemp, 2012). Calcium binding to the negatively charged residues in the pocket of the CaSR extracellular domain induces a conformational change of the CaSR molecule that causes the transmembrane and intracellular domains to activate intracellular signaling. Calcium ions are the main CaSR agonists, but CaSR also responds to other divalent (Ba, Cd, Co, Mg) and trivalent (Gd, La) cations and to polycationic compounds such as polyamines, aminoglycosides (neomycin, gentamycin), and polypeptides (poly-L-arginine, β-amyloid) (Riccardi and Kemp, 2012). The signaling cascade induced by CaSR activation is tissue specific and mediated by G-proteins (Fig. 1.2) (Magno et al., 2011). However, CaSR has also been identified in many organs not directly involved in calcium homeostasis and is now considered as ubiquitously expressed in mammalian cells. It has been implicated in insulin secretion, adipocyte metabolism, smooth muscle cell activity, and gastric function (Table 1.1), although its effects in these tissues is not as crucial as that in calcium-regulating organs (Riccardi and Kemp, 2012).

The human CaSR gene (3q13.3–21) spans 103  kb and comprises eight exons with two promoters, P1 and P2, having unknown functional differences (Fig. 1.3) (Canaff and Hendy, 2002). Loss-of-function mutations cause familial hypocalciuric hypercalcemia (FHH; OMIM #145980) in heterozygous patients and severe neonatal hyperparathyroidism (SNH; OMIM #239200) in homozygous patients (Hofer and Brown, 2003; Pearce et al., 1995). In these patients, CaSR cannot inhibit PTH production and renal tubular calcium reabsorption appropriately and patient phenotype is characterized by hypercalcemia and low calcium excretion. Serum PTH and calcium are slightly or moderately high in FHH, but severely high in SNH. SNH patients also develop bone demineralization and failure to thrive in the first 6  months of life. Mutations of two other genes, GNA11 (19p13) and AP2S1 (19q13), may also cause FHH. Gain-of-function mutations of CaSR cause autosomal dominant hypercalcemia (ADH; OMIM #601198), a disorder characterized by high urinary calcium excretion and inappropriately low serum PTH and hypocalcemia. ADH in patients with highly activating mutations is associated with Bartter syndrome type 5 because of a urinary sodium and potassium leak resulting in renal hypokalemia (Vezzoli et al., 2006).

Figure 1.1  Renal and parathyroid response to increases in serum calcium concentrations. Calcium-sensing receptor (CaSR) is sensitive to tubular fluid calcium in the proximal tubule and in the collecting duct whereas it responds to serum calcium in the ascending limb and the cortical convoluted tubule.

Figure 1.2  Calcium ions bind to the negatively charged pocket of the calcium-sensing receptor (CaSR) extracellular domain and activate different signaling cascades mediated by G-proteins. Various protein-binding partners of the CaSR’s C-terminal region have been recognized, including filamin, potassium channels, dorfin, and β-arrestin.

CaSR activity may be modified by genetic polymorphisms (Table 1.2). Two nonsynonymous polymorphisms of the intracellular domain, rs1801725 and rs1402636, have a significant frequency in the general population and are associated with specific benign phenotypes. In addition, polymorphisms of noncoding regions of CaSR have been associated with specific phenotypes; among them, the rs6776158 polymorphism of promoter 1 may have particular relevance in human disorders.

Table 1.1

Functions of Different Cells Influenced by Extracellular Calcium Ions and Calcium-Sensing Receptor Activities (Riccardi and Kemp, 2012).

See the text for specification on renal effects.

Figure 1.3  Promoters and the most significant polymorphisms in the calcium-sensing receptor gene.

Table 1.2

Calcium-Sensing Receptor Gene Polymorphisms Involved in Human Disorders

a Frequency in Asian people.

Calcium-Sensing Receptor in the Parathyroid Glands

PTH has a pivotal role in calcium homeostasis because of its effects on renal tubular calcium reabsorption and bone metabolism. CaSR is the key of the negative feedback regulating PTH secretion according to the circulating calcium concentration. CaSR stimulation leads to adenylate cyclase inhibition, protein kinase C activation, and calcium mobilization from intracellular stores, which decrease PTH gene transcription and PTH secretion (Kifor et al., 1997). The opposite occurs during hypocalcemia, which may stimulate parathyroid cell proliferation to maintain high levels of PTH synthesis and secretion (Corbetta et al., 2002). In the case of chronic hypocalcemia, this mechanism may give rise to nodular proliferation of parathyroid cells in patients with chronic kidney disease or hypophosphatemic rickets (Yano et al., 2000a). Parathyroid adenomas isolated from patients with primary hyperparathyroidism are characterized by a reduced CaSR expression; therefore they are insensitive to the normal negative feedback regulation of PTH secretion that contributes to the shift to the right of the PTH–calcium curve and to the autonomous PTH secretion of the gland. The defect in CaSR expression was associated with decreased transcriptional activity of promoter 1 (Chikatsu et al., 2000). In addition, hyperplastic glands from patients with chronic kidney disease may have decreased expression of CaSR (Yano et al., 2000a).

Calcium-Sensing Receptor in the Kidney

CaSR has been detected in glomerular podocytes, mesangial cells, and in cells of all renal tubular segments from humans and laboratory animals (Riccardi and Brown, 2010; Riccardi and Kemp, 2012). The highest expression was observed in the thick ascending limb of the loop of Henle, where approximately 25% of calcium is filtered. Here, CaSR is located in the basolateral cell membrane and is activated by the increase in circulating calcium concentrations. Microperfusion studies in renal tubules from laboratory animals showed that CaSR inhibits paracellular calcium reabsorption in the ascending limb (Riccardi and Brown, 2010). This effect is sustained by CaSR-induced production of two microRNAs, miR-9 and miR-374, that upregulate claudin-14 expression and downregulate claudin-16 expression (Fig. 1.4). Claudin-16 and claudin-19 form tight junction channels, which drive paracellular reabsorption of calcium and magnesium, whereas claudin-14 inhibits permeability of this channel (Gong and Hou, 2014; Toka et al., 2012). To enhance calcium transport in the ascending limb, CaSR reduces the transepithelial electric gradient driving paracellular calcium reabsorption (Fig. 1.4) by inhibiting sodium-potassium-chloride cotransport (NKCC2) activity and potassium recycling channels (which generate the transepithelial electric gradient; Gamba and Friedman, 2009). In addition to passive reabsorption in the ascending limb, experiments in cultured canine cells showed that CaSR inhibits active calcium reabsorption in cortical convoluted tubules where CaSR is located in the basolateral cell membrane (Gamba and Friedman, 2009; Blankenship et al., 2001).

In the collecting duct, CaSR is expressed on the luminal membrane of tubular cells and is sensitive to the tubular fluid calcium concentration that results from CaSR-modulated calcium reabsorption in the ascending limb (Topala et al., 2009). Its activation may decrease cAMP production and aquaporin-2 trafficking, which downregulates the tubular response to antidiuretic hormone and water reabsorption. Furthermore, CaSR may stimulate urine acidification in this tubular segment by enhancing proton pump function (Bustamante et al., 2008; Casare et al., 2014). In the proximal tubule, CaSR is expressed on the luminal cell membrane and is sensitive to calcium present in the glomerular filtrate. Cell culture experiments showed that CaSR may antagonize the effect of PTH and promote phosphate reabsorption via the sodium-phosphate cotransporter (NPT2) by inhibiting cAMP production (Ba et al., 2004). Here, it also promotes proton secretion through the sodium-hydrogen exchanger (NHE3; Capasso et al., 2013).

Figure 1.4  Activities and signaling pathways mediated by calcium-sensing receptor (CaSR) in a cell of the ascending limb of the Henle’s loop ( upper panel ) and the cortical convoluted tubule ( lower panel ). 20- HETE , 20-hydroxyeicosatetraenoic acid; NFAT , nuclear factor of activated T cells; NKCC2 , sodium–potassium–chloride cotransporter; PKA , protein kinase A; PLA2 , the phospholipase A2; PMCA , plasma membrane calcium-transporting ATPase; ROMK , renal outer-medullary potassium channel; TRPV5 , transient receptor potential cation channel subfamily V member 5. miR-9 and miR-374 are two microRNA molecules that target claudin-14 gene transcription.

These findings demonstrate the integrated effects of CaSR in the kidney. CaSR activation by serum calcium results in a calciuretic effect in the ascending limb and cortical distal tract that increases the risk of calcium-phosphate precipitation in the tubular lumen. This risk is counterbalanced by the fact that tubular fluid calcium may activate CaSR in the collecting duct leading to water and proton excretion and increased calcium-phosphate solubility. Furthermore, activation of CaSR in the proximal tubule decreases phosphate load to the distal tubule (Renkema et al., 2009).

Calcium-Sensing Receptor in Bones

Extracellular calcium is important for chondrocyte differentiation and normal development of the growth plate. Calcium deficiency may cause rickets as a result of delayed chondrocyte differentiation and reduced matrix synthesis and mineralization (Thacher, 2003). Several studies showed that the extracellular calcium sensing of chondrocytes is mediated by CaSR (Chang et al., 1999, 2008). CaSR has been localized to maturing chondrocytes in the growth plate and its expression was found to increase as the cells hypertrophy, suggesting a role of the receptor in mediating terminal differentiation (Chang et al., 1999). Hypertrophic chondrocytes from homozygous knockout mice showed a markedly reduced expression of insulin growth-like factor (IGF)-1 and IGF1 receptor, suggesting that CaSR promotes chondrocyte differentiation by enhancing IGF1 production or signaling (Chang et al., 2008).

Studies performed on cells of the osteoblastic lineage demonstrated that changes in extracellular calcium affect cell proliferation, differentiation, and mineralization via the CaSR (Dvorak et al., 2004; Chattopadhyay et al., 2004). Despite the evidence that CaSR mediates extracellular calcium sensing in osteoblasts, its role in bone development in vivo has been controversial. The generation of a conditional CaSR knockout model, in which the receptor was broadly deleted across the osteoblast lineage (including preosteoblasts, proliferating, differentiating, and mature osteoblasts and osteocytes) showed an early lethality and skeletal abnormalities (retarded skeletal development and growth; Chang et al., 2008; Dvorak-Ewell et al., 2011). From the second week of life, conditional knockout mice suffered long bone fractures along with deficient skeletal mineralization. Microcomputed tomography studies showed markedly reduced mineral content in trabecular and cortical bone, decreased bone volume, and altered trabecular architecture (Dvorak-Ewell et al., 2011). Fluorescently labeled mineralizing surfaces exhibited a markedly delayed and disorganized mineralization of trabecular and cortical bone. Conversely, osteoclast numbers and activity were increased in the knockout mice, and receptor activator of nuclear factor kappa-B ligand (RANK-L) mRNA expression was increased in femoral cortices. Cultured osteoblasts isolated from the knockouts showed an elevated osteoclastogenesis potential, stimulating the development of osteoclasts that were up to fivefold more positive to tartrate-resistant acid phosphatase compared with cells from control mice (Dvorak-Ewell et al., 2011). During synthesis, CaSR seems to result in bone anabolism in the presence of calcium by promoting commitment, survival, and differentiation of early osteoblasts and by suppressing local RANK-L–dependent osteoclastogenesis.

During bone resorption, the local calcium concentration reaches 8–40  mM, and the surrounding cells are exposed to these high calcium levels. This stimulus is sensed by CaSR present in cells populating the bone marrow. If stimulated with calcium, mesenchymal stromal cells derived from rat bone marrow migrate and proliferate in a concentration-dependent manner and overexpress osteogenic markers (Gonzales-Vazquez et al., 2014). During fetal life in mice, the unusual calcium level in bone interstitial fluid also triggers hematopoietic stem cells to home to the endosteal niche within the bone marrow after migration from liver or spleen. Hematopoietic stem cells that express CaSR were able to home to bone marrow whereas those from CaSR knockout mice could not (Adams et al., 2006). This mechanism leads to the preferential localization of adult mammalian hematopoiesis in bones.

Calcium-Sensing Receptor Gene Polymorphisms and Calcium–Phosphate Homeostasis

Approximately 200 nonsynonymous, single-nucleotide polymorphisms of the human CaSR gene have been detected. Two apparently benign nonsynonymous polymorphisms—rs1801725 (G  >  T; Ala986Ser) and rs1042636 (A  >  G; Arg990Gly)—have been most extensively studied in humans because of their significant minor allele frequency in the European population (∼20% for rs1801725% and 5% for rs1402636; Cole et al., 1998). Another polymorphism, rs1801726 (C  >  G, Gln1011Glu), has a minor allele frequency of approximately 2% (Scillitani et al., 2004). These polymorphisms are located in exon 7, a region that codes for the CaSR intracellular domain. Other polymorphisms not changing amino acid sequence have been studied in humans. rs17251221 (A  >  G) is located in intron 4 and is in linkage with rs1801725 (Kapur et al., 2010); rs6776158 (A  >  G) is a polymorphism of the CaSR gene promoter 1 with a minor allele frequency of approximately 27%; it is in linkage with rs1501899 (G  >  A), located in intron 1, and rs7652589 (G  >  A) located upstream promoter 1 in the 5′-untranslated region (Fig. 1.3; Vezzoli et al., 2013). These polymorphisms were in the first haplotype block of the CaSR gene including the 3′ untranslated region and exon 1; another haplotype block includes the coding part of the gene (Yun et al., 2007).

In 1999 a Canadian study documented the association of serum calcium with the genotype at rs1801725; the minor allele T was associated with higher serum calcium values that rose with the number of copies of the allele (Cole et al., 1998). These findings were generally confirmed in subsequent studies (Scillitani et al., 2004), including two genome-wide association studies (GWASs) that identified rs1801725 and rs17251221 as the CaSR polymorphisms having the most significant association with serum calcium. Their minor allele was associated with higher serum calcium and explained 0.54% of serum calcium variance. One of these GWASs found that rs1402636 also contributed to serum calcium levels because the minor allele G was associated with lower serum calcium (Kapur et al., 2010). Although the phenotype suggested a functional effect, no alterations of CaSR activity were observed when the minor allele at rs1801725 was tested in embryonic renal cells transfected with the CaSR gene (Vezzoli et al., 2007).

An effect on calcium excretion could be exerted by rs1402636 because the minor allele G was associated with idiopathic hypercalciuria and greater calcium excretion in patients with primary hyperparathyroidism (Vezzoli et al., 2007; Corbetta et al., 2006). Functional tests in embryonic renal cells transfected with the CaSR showed an activating effect of the G minor allele at rs1402636; it may increase CaSR sensitivity to calcimimetic, cell calcium oscillations and activity of the endoplasmic reticulum (ER) calcium pump that leads to calcium accumulation in the ER (Terranegra et al., 2010; Ranieri et al., 2013). This allele could encode a more efficient CaSR that depresses tubular calcium reabsorption in the ascending limb and decreases serum calcium in individuals with this genotype (Kapur et al., 2010; Scillitani et al., 2004).

Calcium-Sensing Receptor Gene Polymorphisms and Disorders of the Parathyroid Glands

CaSR gene polymorphisms were shown to possibly influence the phenotype of patients with primary hyperparathyroidism in different case series. Concentrations of serum ionized calcium and PTH were higher in patients carrying the minor alleles at polymorphisms of the regulatory region (rs7652589 and rs1501899), which are in linkage disequilibrium with rs6776158 of promoter 1, causing decreased CaSR expression (Vezzoli et al., 2011). In addition, kidney stone risk was higher in patients carrying the minor allele of these polymorphisms (Vezzoli et al., 2011). Calcium excretion and stone risk were instead promoted by rs1402636, which increases CaSR activity (Corbetta et al., 2006; Scillitani et al., 2007). The contemporary presence of the minor allele at polymorphisms rs1501899 and rs1402636 led to an increase of stone risk despite their apparently different effects on CaSR function (Vezzoli et al., 2014). Moreover, rs1402636 may influence the phenotype in uremic patients with secondary hyperparathyroidism. The minor allele of this polymorphism may be associated with lower serum PTH levels (Yano et al., 2000b) and suppression of PTH levels in response to the increase of postdialysis serum calcium (Yokoama et al., 2002), suggesting greater control of PTH secretion in patients carrying the minor allele.

Calcium-Sensing Receptor Gene Polymorphisms and Disorders of the Kidney

CaSR has been implicated in the control of glomerular function because calcimimetics can stabilize the cytoskeleton of podocytes and decrease glomerular sclerosis, proteinuria, blood pressure, and the progression of renal failure in rat kidney (Ogata et al., 2003). However, no data are available relating CaSR to renal filtration in humans. On the contrary, various CaSR genotypes, including rs1402638, rs6776158, rs1501899, and rs7652589, were hypothesized to play a role in calcium nephrolithiasis. Minor allele G at rs1402638 was shown to have an activating effect and was associated with hypercalciuria in calcium stone formers and osteoporotic women without kidney stones (Vezzoli et al., 2002). These findings may be explained by an increased inhibitory effect of the CaSR polymorphic variant on calcium reabsorption in the ascending limb (Vezzoli et al., 2007). Hypercalciuria may predispose individuals to urinary stones, and the polymorphism rs1402636 was associated with stones in some populations but not in others (O’Seaghdha et al., 2010). Minor alleles at polymorphisms of the regulatory region of the CaSR, rs6776158, rs1501899, and rs7652589, were associated with idiopathic calcium stones (Vezzoli et al., 2010, 2013) and with stone formers with primary hyperparathyroidism (Vezzoli et al., 2011). Furthermore, the minor allele at rs6776158 was shown to reduce promoter 1 transcriptional activity in two renal cell models. Accordingly, CaSR expression in medulla samples was decreased in patients carrying the minor allele at rs6776158 (Vezzoli et al., 2013). This polymorphism was not associated with calcium excretion and could promote stones by decreasing the protective effects of CaSR on calcium–phosphate precipitation within the tubular lumen or the interstitium. Taken together, these findings suggest a complex effect of CaSR on calcium stone risk; rs1402636 may predispose hypercalciuric subjects to stones by amplifying their tendency for high calcium excretion whereas rs6776158 may predispose subjects to stones by decreasing protection against intratubular calcium precipitation.

Calcium-Sensing Receptor Gene Polymorphisms and Bone Mineral Density

Several studies investigated the association between CaSR polymorphisms and bone mineral density (BMD). Many studies included the genotyping of the rs1801725 (Arg986ser) polymorphism in different cohorts. A study performed on 97 Swedish girls and young women found a negative association with BMD measured at the lumbar spine (Lorentzon et al., 2001). Subjects carrying the minor allele T had lower BMD measurements, but the relationship was biased by the simultaneous finding of a lower degree of physical activity within the same group of subjects. Conversely, other studies did not find an association between the CaSR rs1801725 polymorphism and BMD measured at the lumbar spine or the femur in large groups of postmenopausal Caucasian women (Takacs et al., 2002), or measured at the distal forearm in healthy Finnish adults (Laaksonen et al., 2009). Another study found no association of three polymorphisms located in exon 7 of CaSR (rs18017125, rs1402636, and rs18017126) with BMD measured at the lumbar spine, left forearm, and left total hip in 110 dizygotic female twins (Harding et al., 2006). Moreover, a recent GWAS did not show any association between BMD and the CaSR gene locus in 2211 women and 1633 men. These authors measured BMD at the lumbar spine, femoral neck, trochanter, and whole body and recorded calcaneal quantitative ultrasound (Gupta et al., 2011). Conversely, a recent study in 266 patients with primary hyperparathyroidism showed that the minor allele T at the rs1801725 polymorphism was more frequent in patients who suffered from vertebral fractures regardless of gender, BMD measurements, or serum calcium concentration. Furthermore, carriers of the minor allele at rs1801725 plus two of the following three factors—age, serum calcium concentration, and spinal BMD—showed a risk of fracture that was 4.7-fold higher than homozygotes for the more common allele (Eller-Vainicher et al., 2014).

Conclusions

Findings reported here indicate that environmental calcium may influence development and function of different organs, especially those involved in calcium homeostasis. CaSR mediates the effect of environmental calcium, and its polymorphic variants may contribute to individual variability. CaSR polymorphisms may be a determinant of serum calcium and calcium excretion and may predispose to calcium nephrolithiasis. Two mechanisms may explain CaSR polymorphism contribution to stone risk: (1) variants decreasing CaSR expression in the kidney may reduce its protective effect against calcium-phosphate precipitation and (2) variants associated with hypercalciuria may trigger calcium–phosphate precipitation in the tubular lumen. It has been hypothesized that the protective effect of CaSR in relation to kidney stones could reflect its protective effect against soft tissue and vascular calcification (Vezzoli et al., 2013). Therefore CaSR polymorphisms may influence individual phenotype in terms of serum calcium and calcium excretion. These changes may increase risk for bone fracture and kidney stones as observed in patients with primary and secondary hyperparathyroidism, although the effect of CaSR polymorphisms on fracture risk and BMD remains controversial. According to the current data, clinicians could use CaSR polymorphisms as markers to explain hypercalciuria, hypercalcemia, and predisposition to kidney stones, or to predict the response to calcimimetic drugs that are used to cure secondary hyperparathyroidism in patients undergoing dialysis and to correct hypercalcemia in patients with primary hyperparathyroidism.

Summary Points

• CaSR is a 1078-amino–acid protein that belongs to the third class of the GPCR family, expressed as a disulfide-linked homodimer in caveolin-rich areas of the plasma membrane.

• CaSR is an environmental sensor that elicits paracrine or autocrine adaptive responses of human cells to local or serum variations of extracellular calcium concentration.

• CaSR is mainly expressed in the parathyroid glands and renal distal tubules.

• CaSR enables the parathyroid glands and renal tubules to regulate PTH secretion and calcium excretion in response to serum calcium changes.

• Two apparently benign nonsynonymous CaSR polymorphisms, rs1801725 and rs1042636, have been most extensively studied in humans.

• The genotype at rs1801725 was associated with serum calcium that was higher in carriers of the minor allele.

• The minor allele at rs1402636 upregulates in vitro CaSR activity and was associated with idiopathic hypercalciuria.

• The genotype at rs1402636 may modify the response to calcimimetic drugs.

• Polymorphisms associated with decreased CaSR expression—rs6776158, rs1501899, and rs7652589—were associated with idiopathic calcium stones.

• The phenotype of patients with primary hyperparathyroidism may be modified by CaSR polymorphisms.

• The minor allele at rs1801725 could have a negative effect on BMD values at the lumbar spine and fracture risk.

• Clinicians could use CaSR polymorphisms as markers to explain hypercalciuria, hypercalcemia, or kidney stone production, or to predict the response to calcimimetic drugs.

Key Facts

• CaSR is the extracellular calcium sensor that enables parathyroid glands and renal distal tubules to regulate calcium excretion and PTH secretion in response to serum calcium changes.

• CaSR gene polymorphisms were found to be associated with serum calcium and urine calcium excretion.

• CaSR activity in the renal tubules protects against calcium–phosphate precipitation in the tubular fluids.

• Polymorphisms decreasing CaSR expression in renal tubular cells may predispose individuals to calcium kidney stones.

• CaSR polymorphisms may modify the phenotype of healthy subjects and patients with primary and secondary hyperparathyroidism.

Mini Dictionary of Terms

Bone mineral density (BMD)   Variable used to express the amount of mineral matter per square centimeter at different bone segments, usually forearm, lumbar spine, and femur.

Calcimimetics   Drugs that are allosteric activators of CaSR, thereby decreasing PTH secretion and its hypercalcemic effect.

Caveolin   A membrane protein associated with microdomains in the plasma membrane that are rich in signal-transducing proteins.

Hyperparathyroidism   Condition of excessive PTH secretion from the parathyroid glands. Patients with primary hyperparathyroidism are characterized by hypercalcemia whereas those with secondary hyperparathyroidism display hypocalcemia or normocalcemia.

Osteoclasts   Bone cells that express RANK-ligand and are responsible for bone resorption.

Osteoblasts   Bone cells responsible for bone production and mineralization. They activate bone remodeling by producing RANK.

Polymorphism   Difference in DNA sequence among individuals that may contribute to individual variability and predisposition for diseases.

RANK   Receptor activator of nuclear factor kappa-B; produced by osteoblasts, it plays a critical role in triggering osteoclast activity.

Tight junction   A junctional element connecting adjacent epithelial cells. It is a barrier to maintain cell polarity.

References

Adams G.B, Chabner K.T, Alley I.R, Olson D.P, Szczepiorkowski Z.M, Poznansky M.C, Kos C.H, Pollak M.R, Brown E.M, Scadden D.T. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature. 2006;439:599–603.

Aida K, Koishi S, Tawata M, Onaya T. Molecular cloning of a putative Ca-sensing receptor cDNA from human kidney. Biochem. Biophys. Res. Commun. 1995;214:524–529.

Ba J, Brown D, Friedman P.A. Calcium-sensing receptor regulation of PTH-inhibitable proximal tubule phosphate transport. Am. J. Physiol. Ren. Physiol. 2004;285:F1233–F1243.

Blankenship K.A, Williams J.J, Lawrence M.S, McLeish K.R, Dean W.L, Arthur J.M. The calcium-sensing receptor regulates calcium absorption in MDCK cells by inhibition of PMCA. Am. J. Physiol. Ren. Physiol. 2001;280:F815–F822.

Brown E.M, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger M.A, Lytton J, Hebert S. Cloning and characterization of an extracellular Ca-sensing receptor from bovine parathyroid. Nature. 1992;366:575–580.

Bustamante M, Hasler U, Leroy V, deSeigneux M, Dimitrov M, Mordasini D, Rousselot M, Martin P.Y, Feraille E. Calcium-sensing receptor attenuates AVP-induced aquaporin-2 expression via a calmodulin-dependent mechanism. J. Am. Soc. Nephrol. 2008;19:109–116.

Canaff L, Hendy G.N. Human calcium-sensing receptor gene. J. Biol. Chem. 2002;277:30337–30350.

Capasso G, Geibel P.J, Damiano S, Jaeger P, Richards W.G, Geibel J.P. The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule. Kidney Int. 2013;84:277–284.

Casare F, Milan D, Fernandez R. Stimulation of calcium-sensing receptor increases biochemical H-ATPase activity in mouse cortex and outer medullary regions. Can. J. Physiol. Pharmacol. 2014;92:181–188.

Chang W, Tu C, Chen T.H, Komuves L, Oda Y, Pratt S.A, Miller S, Shoback D. Expression and signal transduction of calcium-sensing receptors in cartilage and bone. Endocrinology. 1999;140:5883–5893.

Chang W, Tu C, Chen T.H, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci. Signal. 2008;1:ra1.

Chattopadhyay N, Yano S, Tfelt-Hansen J, Rooney P, Kanuparthi D, Bandyopadhyay S, Ren X, Terwillinger E, Brown E.M. Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts. Endocrinology. 2004;145:3451–3462.

Chikatsu N, Fukumoto S, Takeuchi Y, Suzawa M, Obara T, Matsumoto T, Fujita T. Cloning and characterization of two promoters for the human calcium-sensing receptor (CaSR) and changes of CaSR expression in parathyroid adenomas. J. Biol. Chem. 2000;275:7553–7557.

Cole D.E.C, Peltekova V.D, Rubin L.A, Hawker G.A, Vieth R, Liew C.C, Hwang D.M, Evrovski J, Hendy G.N. A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet. 1998;353:112–115.

Corbetta S, Lania A, Filopanti M, Vicentini L, Ballarè E, Spada A. Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells. J. Clin. Endocrinol. Metab. 2002;87:2201–2205.

Corbetta S, Eller-Vainicher C, Filopanti M, Saeli P, Vezzoli G, Arcidiacono T, Loli P, Syren M.L, Soldati L, Beck-Peccoz P, Spada A. R990G polymorphism of the calcium-sensing receptor and renal calcium excretion in patients with primary hyperparathyroidism. Eur. J. Endocrinol. 2006;155:687–692.

Dvorak M.M, Siddiqua A, Ward D.T, Carter D.H, Dallas S.L, Nemeth E.F, Riccardi D. Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc. Natl. Acad. Sci. U.S.A. 2004;101:5140–5145.

Dvorak-Ewell M.M, Chen T.H, Liang N, Garvey C, Liu B, Tu C, Chang W, Bikle D.D, Shoback D.M. Osteoblast extracellular Ca2+sensing receptor regulates bone development, mineralization, and turnover. J. Bone Min. Res. 2011;26:2935–2947.

Eller-Vainicher C, Battista C, Guarnieri V, Muscarella S, Palmieri S, Salcuni A.S, Guglielmi G, Corbetta S, Minisola S, Spada A, Hendy G.N, Cole D.E, Chiodini I, Scillitani A. Factors associated with vertebral fracture risk in patients with primary hyperparathyroidism. Eur. J. Endocrinol. 2014;171:399–406.

Gamba G, Friedman P.A.F. Thick ascending limb: the Na+:K+:2Cl− co-transporter, NKCC2, and the calcium-sensing receptor, CaSR. Eur. J. Physiol. 2009;458:61–76.

Garrett J.E, Capuano I.V, Hammerland L.G, Hung B.C, Brown E.M, Hebert S.C, Nemeth E.F, Fuller F. Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J. Biol. Chem. 1995;270:12919–12925.

Gong Y, Hou J. Claudin-14 underlies Ca-sensing receptor–mediated Ca metabolism via NFAT-microRNA–based mechanisms. J. Am. Soc. Nephrol. 2014;25:745–760.

Gonzales-Vazquez A, Planell J.A, Engel E. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells. Acta Biomater. 2014;10:2824–2833.

Gupta M, Cheung C.L, Hsu Y.H, Demissie S, Cupples L.A, Kiel D.P, Karasik D. Identification of homogeneous genetic architecture of multiple genetically correlated traits by block clustering of genome-wide associations. J. Bone Min. Res. 2011;26:1261–1271.

Harding B, Curley A.J, Hannan F.M, Christie P.T, Bowl M.R, Turner J.J.O, Barbert M, Gillham-Nasenyat I, Hamson G, Spector T.D, Thakker R.V. Functional characterization of calcium sensing receptor polymorphisms and absence of association with indices of calcium homeostasis and bone mineral density. Clin. Endocrinol. 2006;65:598–605.

Hofer A.M, Brown E.M. Extracellular calcium sensing and signaling. Nature. 2003;4:530–538.

Kapur K, Johnson T, Beckmann N.D, Sehmi J, Tanaka T, Kutalik Z, Styrkarsdottir U, Zhang W, Marek D, Gudbjartsson D.F, Milaneschi Y, Holm H, DiIorio A, Waterworth D, Li Y, Singleton A.B, Bjornsdottir U.S, Sigurdsson G, Hernandez D.G, DeSilva R, Elliott P, Eyjolfsson G.I, Guralnik J.M, Scott J, Thorsteinsdottir U, Bandinelli S, Chambers J, Stefansson K, Waeber G, Ferrucci L, Kooner J.S, Mooser V, Vollenweider P, Beckmann J.S, Bochud M, Bergmann S. Genome-wide meta-analysis for serum calcium identifies significantly associated SNPs near the calcium-sensing receptor (CASR) gene. PLoS Genet. 2010;6:e1001035.

Kifor O, Diaz R, Butters R, Brown E.M. The Ca-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J. Bone Min. Res. 1997;12:715–722.

Kifor O, Diaz R, Butters R, Kifor I, Brown E.M. The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. J. Biol. Chem. 1998;273:21708–21713.

Laaksonen M.M.L, Outila T.A, Kärkkäinen M.U.M, Kemi V.E, Rita H.J, Perola M, Valsta L.M, Lamberg-Allardt C.J.E. Associations of vitamin D receptor, calcium-sensing receptor and papathyroid hormone gene polymorphisms with calcium homeostasis and peripheral bone density in adult Finns. J. Nutr. Nutr. 2009;2:55–63.

Lorentzon M, Lorentzon R, Lerner U.H, Nordstrom P. Calcium sensing recepto polymorphysm, circulating calcium concentrations and bone mineral density in healthy adolescent girls. Eur. J. Endocrinol. 2001;144:257–261.

Magno A.L, Ward B.K, Ratajczak T. The calcium-sensing receptor: a molecular perspective. Endocr. Rev. 2011;32:3–30.

O’Seaghdha C.M, Yang Q, Glazer N.L, Leak T.S, Dehghan A, Smith A.V, Kao W.H.L, Lohman K, Hwang S.J, Johnson A.D, Hofman A, Uitterlinden A.G, Chen Y.D.I, The GEFOS Consortium, Brown E.M, Siscovick D.S, Harris T.B, Psaty B.M, Coresh J, Gudnason V, Witteman J.C, Liu Y.M, Kestenbaum B.R, Fox C.S, Kottgen A. Common variants in the calcium-sensing receptor gene are associated with total serum calcium levels. Hum. Mol. Genet. 2010;19:4296–4303.

Ogata H, Ritz E, Odoni G, Amman K, Orth S.R. Beneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factors. J. Am. Soc. Nephrol. 2003;14:959–967.

Pearce S.H.S, Trump D, Wooding C, Besser G.M, Chew S.L, Grant D.B, Heath D.A, Hughes I.A, Paterson C.R, Whyte M.P, Thakker R.V. Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J. Clin. Invest. 1995;96:2683–2692.

Ranieri M, Tamma G, Di Mise A.M, Vezzoli G, Soldati L, Svelto M, Valenti G. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR) are associated with increased ER to cytosol calcium gradient. PLoS One. 2013;8:e79113.

Renkema K.Y, Velic A, Dijkman H.B, Verkaart S, van der Kemp A.W, Nowk M, Timmermans K, Doucet A, Wagner C.A, Bindels R.J, Hoenderop J.G. The calcium-sensing receptor promotes urinary acidification to prevent nephrolithiasis. J. Am. Soc. Nephrol. 2009;20:1705–1713.

Riccardi D, Brown E.M. Physiology and pathophysiology of the calcium-sensing receptor in the kidney. Am. J. Physiol. Ren. Physiol. 2010;298:F485–F499.

Riccardi D, Kemp P.J. The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease. Annu. Rev. Physiol. 2012;74:271–297.

Scillitani A, Guarnieri V, De G.S, Muscarella L.A, Battista C, D’Agruma L, Bertoldo F, Florio C, Minisola S, Hendy G.N, Cole D.E.C. Blood ionized calcium is associated with clustered polymorphisms in the carboxyl-terminal tail of the calcium-sensing receptor. J. Clin. Endocrinol. Metab. 2004;89:5634–5638.

Scillitani A, Guarnieri V, De G.S, Battista C, DeGironimo S, Muscarella L.A, Chiodini J, Cignarelli M, Minisola S, Bertoldo F, Francucci C.M, Malavolta N, Piovesan A, Mascia M.L, Muscarella S, Hendy G.N, D’Agruma L, Cole D.E.C. Primary hyperparathyroidism and the presence of kidney stones are associated with different haplotypes of the calcium-sensing receptor. J. Clin. Endocrinol. Metab. 2007;92:277–283.

Takacs I, Speer G, Banjnok E, Tabak A, Nagy Z, Horvath C, Kovacs K, Lakatos P. Lack of association between calcium-sensing receptor gene A986S polymorphism and bone mineral density in Hungarian postmenopausal women. Bone. 2002;30:849–852.

Terranegra A, Ferraretto A, Dogliotti E, Scarpellini M, Corbetta S, Barbieri A.M, Spada A, Arcidiacono T, Rainone F, Aloia A, Cusi D, Vezzoli G, Soldati L. Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor. J. Mol. Endocrinol. 2010;25:245–256.

Thacher T.D. Calcium-deficiency rickets. Endocr. Dev. 2003;6:105–125.

Toka H.R, Al-Romaih K, Koshy J.M, DiBrartolo S, Kos C.H, Quinn S.J, Curhan G.C, Mount D.B, Brown E.M, Pollak M.R. Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormone–independent hypocalciuria. J. Am. Soc. Nephrol. 2012;23:1879–1890.

Topala C.N, Schoeber J.P.H, Searchfield L.E, Riccardi D, Hoenderop J.G.J, Bindels R.J.M. Activation of the Ca-sensing receptor stimulates the activity of the epithelial Ca channel TRPV5. Cell Calcium. 2009;45:331–339.

Vezzoli G, Tanini A, Ferrucci L, Soldati L, Bianchin C, Franceschelli F, Malentacchi C, Porfirio B, Adamo D, Terranegra A, Falchetti A, Cusi D, Bianchi G, Brandi M.L. Influence of calcium-sensing receptor gene on urinary calcium excretion in stone-forming patients. J. Am. Soc. Nephrol. 2002;13:2517–2523.

Vezzoli G, Arcidiacono T, Paloschi V, Terranegra A, Biasion R, Weber G, Mora S, Syren M.L, Coviello D, Cusi D, Bianchi G, Soldati L. Autosomal dominant hypocalcemia with mild type 5 Bartter syndrome. J. Nephrol. 2006;19:525–528.

Vezzoli G, Terranegra A, Arcidiacono T, Biasion R, Coviello D, Syren M.L, Paloschi V, Giannini S, Mignogna G, Rubinacci A, Ferraretto A, Cusi D, Bianchi G, Soldati L. R990G polymorphism of calcium-sensing receptor does produce a gain-of-function and predispose to primary hypercalciuria. Kidney Int. 2007;71:1155–1162.

Vezzoli G, Terranegra A, Arcidiacono T, Gambaro G, Milanesi L, Mosca E, Soldati L. Calcium kidney stones are associated with a haplotype of the calcium-sensing receptor gene regulatory region. Nephrol. Dial. Transpl. 2010;25:2245–2252.

Vezzoli G, Scillitani A, Corbetta S, Terranegra A, Dogliotti E, Guarnieri V, Arcidiacono T, Paloschi V, Rainone F, Eller-Vainicher C, Borghi L, Nouvenne A, Guerra A, Meschi T, Allegri F, Cusi D, Spada A, Cole D.E.C, Hendy G.N, Spotti D, Soldati L. Polymorphisms at the regulatory regions of the calcium-sensing receptor gene influence stone risk in primary hyperparathyroidism. Eur. J. Endocrinol. 2011;164:421–427.

Vezzoli G, Terranegra A, Aloia A, Arcidiacono T, Milanesi L, Mosca E, Mingione A, Spotti D, Cusi D, Hou J, Hendy G.N, Soldati L. Decreased transcriptional activity of calcium-sensing receptor gene promoter 1 is associated with calcium nephrolithiasis. J. Clin. Endocrinol. Metab. 2013;98:3839–3847.

Vezzoli G, Scillitani A, Corbetta S, Terranegra A, Dogliotti E, Guarnieri V, Arcidiacono T, Macrina L, Mingione A, Brasacchio C, Eller-Vainicher C, Cusi D, Spada A, Cole D.E, Hendy G.N, Spotti D, Soldati L. Risk of nephrolithiasis in primary hyperparathyroidism is associated with two polymorphisms of the calcium-sensing receptor gene. J. Nephrol. 2014;40:499–506.

Yano S, Sugimoto T, Tsukamoto T, Chihrara K, Kobayashi A, Kitazawa S, Maeda S, Kitazawa R. Association of decreased calcium-sensing receptor expression with proliferation of parathyroid cells in secondary hyperparathyroidism. Kidney Int. 2000;58:1980–1986.

Yano S, Sugimoto T, Kanzawa M, Tsukamoto T, Hattori T, Hattori S, Chihara K. Association of polymorphic alleles of the calcium-sensing receptor gene with parathyroid hormone secretion in hemodialysis patients. Nephron. 2000;85:317–323.

Yokoama K, Shigematsu T, Tsukada T, Hara S, Yamada A, Kowagushi Y, Hosoya T. Calcium-sensing receptor gene polymorphism affects the parathyroid response to moderate hypercalcemic suppression in patients with ebd-stage renal disease. Clin. Nephrol. 2002;57:131–135.

Yun F.H.J, Wong B.Y.L, Chase M, Shuen A.Y, Canaff L, Thongthai K, Siminovitch K, Hendy G.N, Cole D.E.C. Genetic variation at the calcium-sensing receptor (CASR) locus: implications for clinical molecular diagnostics. Clin. Biochem. 2007;40:551–561.

Chapter 2

Molecular Aspects of the Calcium-Sensing Receptor and Calcium Homeostasis

Abedalrazaq Alkukhun, Armando Salim Munoz-Abraham, Sami Judeeba,  and John P. Geibel     Yale University School of Medicine, New Haven, CT, United States

Abstract

The calcium-sensing receptor (CaSR) was first cloned and characterized by Brown et al. more than 20  years ago. Since then, it has become recognized as a key modulator of many physiological functions. In the parathyroid gland, depending on the extracellular calcium levels, the CaSR modulates parathyroid hormone secretion through a feedback loop that coordinates calcium homeostasis in multiple interrelated systems. In bone, CaSR modulates bone mineralization and regulates osteoblast and osteoclast proliferation and function. In the kidney, CaSR regulates fluid and electrolyte balance. In the gastrointestinal tract the CaSR plays important roles in digestion, immune barrier function, gastric acid secretion, regulation of bile flow, pancreatic fluid control, and interaction with intestinal flora, and it exhibits an antidiarrheal effect in the colon. In the skin, the receptor is involved in regulation of vitamin D and immune response.

Keywords

Bone; Calcilytics; Calcimimetic; Gastrointestinal tract; Kidney; Parathyroid gland

Introduction

Since the first description of the calcium-sensing receptor (CaSR) was published more than 20  years ago (Alfadda et al., 2014), significant progress in understanding its diverse physiologic roles has been achieved. Many homeostatic pathways have been illustrated based on CaSR function, and now it is recognized as a key modulator of the physiology and pathophysiology of multiple organ systems (Alfadda et al., 2014). Ongoing research on the CaSR at the cellular and molecular level has revealed new functions of the receptor as new disease models are analyzed. Moreover, new therapies are being developed to treat a wide variety of diseases based on modulation of the CaSR. In this chapter we describe the role of the CaSR in various organ systems and outline how it modulates various physiological and pathophysiological functions.

Structural Components of the Calcium-Sensing Receptor

The CaSR was originally cloned from bovine parathyroid glands (Brown et al., 1993). It is now well known that the receptor is expressed in the kidney, parathyroid gland, thyroid gland, gastrointestinal (GI) tract, bone, and skin, among other tissues (Brown, 2007b; see Fig. 2.1).

The CaSR is a Class C, G-protein–coupled receptor that comprises three main structures: an extracellular domain (ECD), a seven-transmembrane–spanning domain, and an intracellular domain (Alfadda et al., 2014). The receptor has a large ECD that includes a Venus flytrap (VFT) sequence that contains dimerization and orthosteric sites for binding of endogenous agonists (Alfadda et al., 2014). The ECD contains 612 amino acid residues, and the majority of the disease-causing mutations are located in this domain (Alfadda et al., 2014). The ECD is linked to a transmembrane domain (TMD) rich in cysteine residues (Alfadda et al., 2014) that composes the majority of the protein. These cysteine residues form disulfide bridges within the TMD and with the VFT, which are required for the proper physiological function of the receptor (Alfadda et al., 2014). The CaSR ECD is formed by two lobes (LB1 and LB2) that are separated by a cavity delineating the ligand-binding site representing the VFT. Calcium (Ca²+) molecules bind in the cleft between the two lobes of each VFT, causing the lobes to close in on one another and the VFT to rotate, leading to receptor activation. The CaSR is a homodimer that is capable of coupling to multiple G-proteins, such as Gq/11. The CaSR regulates different pathways in different tissues where it is expressed. It can stimulate phospholipases, mitogen-activated protein kinases (MAPKs), ion transport, fluid secretion, and bicarbonate and acid transport (Alfadda et al., 2014; Brown, 2007a; Magno et al., 2011).

Calcium-Sensing Receptor Function in the Parathyroid Gland

The role of the CaSR in the parathyroid gland was one of the first physiological functions discovered. Since the successful cloning of the CaSR from parathyroid cells, it has been shown to regulate parathyroid hormone (PTH) secretion and thus influence calcium homeostasis (Brown, 2007a). Subsequently, CaSR has been linked to many inherited diseases with mutant forms of the receptor perturbing the balance between PTH secretion and extracellular Ca²+ levels (Brown et al., 1993).

The CaSR may be responsible for maintaining a constant level of ionized calcium (Ca²+) within the extracellular fluids surrounding chief cells of the parathyroid gland. In the parathyroid gland, CaSR regulates the production and secretion of PTH depending on whether Ca²+ is low or high (Brown, 2007a). When extracellular Ca²+ concentrations are low, PTH secretion increases due to downregulation of the CaSR. PTH produces two actions: (1) enhanced bone resorption to increase the availability of Ca²+ in serum and (2) increased renal production of 1,25-dihydroxyvitamin D3, which stimulates Ca²+ absorption in the GI tract. Concurrently, the CaSR located in the thyroid gland prevents calcitonin secretion. Calcitonin is a hormone responsible for stimulating renal Ca²+ excretion (Magno et al., 2011).

Figure 2.1  Schematic representation of known calcium-sensing receptor modulated organs. PTH , parathyroid hormone.

When extracellular Ca²+ increases, CaSR expression increases, which decreases PTH released into the circulation by acting at multiple levels (Brown, 2007a). The CaSR in the parathyroid gland has three main regulatory actions: (1) PTH synthesis, (2) PTH secretion, and (3) cell proliferation (Brown, 2007a). These actions of the CaSR are mainly produced by a negative feedback regulation that extends to the gene level. The CaSR expressed in the parathyroid regulates multiple signaling pathways, including MAPK, phospholipase C (PLC), phospholipase A2, and phospholipase D (Huang et al., 2007; Kifor et al., 2001). Kifor et al. (2001) described a high extracellular Ca²+ environment that produces CaSR activation (in a bovine parathyroid model) via protein kinase C (PKC), phospholipase A2, and phospholipase DGq/11-mediated activation of phosphatidylinositol-specific PLC that leads to intracellular Ca²+ mobilization and PKC activation. This chain of events stimulates a MAPK cascade that activates extracellular signal-regulated kinases (ERK1/2) and calcium-dependent phospholipase A2, causing the release of arachidonic acid (Kifor et al., 2001). Arachidonic acid is then metabolized into biologically active lipid mediators such as hydroperoxyeicosatetraenoic acid and hydroxyeicosatetranoic acid, which suppress PTH secretion (Kifor et al., 2001; Marie, 2010). This reduced PTH production is thought to occur via downregulation of PTH mRNA expression and reduced proliferation of parathyroid cells by the increased intracellular Ca²+ concentration (Magno et al., 2011; see Fig. 2.2).

The role of CaSR in Ca²+ homeostasis can be further explained by examining the consequences of receptor inactivation or stimulation in the parathyroid gland. Receptor inactivation in CaSR knockout mice, in which exaggerated PTH secretion occurs even with hypercalcemia, is usually fatal. Unrestrained PTH secretion may be driven by a higher set point at which Ca²+ triggers PTH release. Marked parathyroid hyperplasia is also observed in the knockout mice, exemplifying another pathway by which CaSR regulates parathyroid gland function and PTH secretion (Brown, 2007b). In humans, inactivating CaSR mutations occur in familial hypocalciuric hypercalcemia (FHH), an autosomal-dominant disease (heterozygous). In FHH, PTH release increases because the parathyroid gland cannot properly sense extracellular Ca²+ levels (Brown, 2007a; Magno et al., 2011). Also in this disease, renal Ca²+ reabsorption is excessive, causing hypocalciuria and thus exacerbating the hypercalcemia. Other findings include hypermagnesemia. FHH is relatively benign and asymptomatic in comparison to neonatal severe hyperparathyroidism (NSHPT), which is the homozygous form of FHH. In NSHPT, there is complete loss of CaSR function. It presents with severe hypercalcemia (>15  mg/dL), hyperparathyroidism, and skeletal demineralization. NSHPT can be fatal if it is not properly detected and treated (Brown, 2007b; Magno et al., 2011). Furthermore, autosomal-dominant hypocalcemia (ADH) occurs from activating CaSR mutations. ADH is characterized by an increased sensitivity of parathyroid cells to extracellular Ca²+ levels. This disorder leads to a spectrum of disease phenotypes, ranging from less (e.g.,