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Vitamin D

Oxidative Stress, Immunity, and Aging


OXIDATIVE STRESS AND DISEASE
Series Editors

Lester Packer, PhD
Enrique Cadenas, MD, PhD
University of Southern California School of Pharmacy
Los Angeles, California

1. Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases,
edited by Luc Montagnier, René Olivier, and Catherine Pasquier
2. Understanding the Process of Aging: The Roles of Mitochondria, Free Radicals,
and Antioxidants, edited by Enrique Cadenas and Lester Packer
3. Redox Regulation of Cell Signaling and Its Clinical Application, edited by
Lester Packer and Junji Yodoi
4. Antioxidants in Diabetes Management, edited by Lester Packer, Peter Rösen,
Hans J. Tritschler, George L. King, and Angelo Azzi
5. Free Radicals in Brain Pathophysiology, edited by Giuseppe Poli, Enrique Cadenas,
and Lester Packer
6. Nutraceuticals in Health and Disease Prevention, edited by Klaus Krämer,
Peter-Paul Hoppe, and Lester Packer
7. Environmental Stressors in Health and Disease, edited by Jürgen Fuchs and
Lester Packer
8. Handbook of Antioxidants: Second Edition, Revised and Expanded, edited by
Enrique Cadenas and Lester Packer
9. Flavonoids in Health and Disease: Second Edition, Revised and Expanded,
edited by Catherine A. Rice-Evans and Lester Packer

10. Redox–Genome Interactions in Health and Disease, edited by Jürgen Fuchs,
Maurizio Podda, and Lester Packer
11. Thiamine: Catalytic Mechanisms in Normal and Disease States, edited by
Frank Jordan and Mulchand S. Patel
12. Phytochemicals in Health and Disease, edited by Yongping Bao and
Roger Fenwick
13. Carotenoids in Health and Disease, edited by Norman I. Krinsky, Susan T. Mayne,
and Helmut Sies
14. Herbal and Traditional Medicine: Molecular Aspects of Health, edited by
Lester Packer, Choon Nam Ong, and Barry Halliwell
15. Nutrients and Cell Signaling, edited by Janos Zempleni and Krishnamurti
Dakshinamurti
16. Mitochondria in Health and Disease, edited by Carolyn D. Berdanier
17. Nutrigenomics, edited by Gerald Rimbach, Jürgen Fuchs, and Lester Packer
18. Oxidative Stress, Inflammation, and Health, edited by Young-Joon Surh and
Lester Packer
19. Nitric Oxide, Cell Signaling, and Gene Expression, edited by Santiago Lamas and
Enrique Cadenas


20. Resveratrol in Health and Disease, edited by Bharat B. Aggarwal and
Shishir Shishodia
21. Oxidative Stress and Age-Related Neurodegeneration, edited by Yuan Luo and
Lester Packer
22. Molecular Interventions in Lifestyle-Related Diseases, edited by Midori Hiramatsu,
Toshikazu Yoshikawa, and Lester Packer
23. Oxidative Stress and Inflammatory Mechanisms in Obesity, Diabetes, and the
Metabolic Syndrome, edited by Lester Packer and Helmut Sies
24. Lipoic Acid: Energy Production, Antioxidant Activity and Health Effects, edited by
Mulchand S. Patel and Lester Packer

25. Dietary Modulation of Cell Signaling Pathways, edited by Young-Joon Surh,
Zigang Dong, Enrique Cadenas, and Lester Packer
26. Micronutrients and Brain Health, edited by Lester Packer, Helmut Sies,
Manfred Eggersdorfer, and Enrique Cadenas
27. Adipose Tissue and Inflammation, edited by Atif B. Awad and Peter G. Bradford
28. Herbal Medicine: Biomolecular and Clinical Aspects, Second Edition, edited by
Iris F. F. Benzie and Sissi Wachtel-Galor
29. Flavonoids and Related Compounds: Bioavailability and Function, edited by
Jeremy P. E. Spencer and Alan Crozier
30. Mitochondrial Signaling in Health and Disease, edited by Sten Orrenius, Lester
Packer, and Enrique Cadenas
31. Vitamin D: Oxidative Stress, Immunity, and Aging, edited by Adrian F. Gombart



Vitamin D

Oxidative Stress, Immunity, and Aging
Edited by

Adrian F. Gombart

Boca Raton London New York

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Contents
Series Preface.....................................................................................................................................ix
Preface...............................................................................................................................................xi
Editor.............................................................................................................................................. xiii

Contributors...................................................................................................................................... xv

SECTION I  Vitamin D: An Overview
Chapter 1 Vitamin D: A Fountain of Youth in Gene Regulation.................................................. 3
Peter W. Jurutka, G. Kerr Whitfield, Ryan Forster, Shane Batie,
Jamie Lee, and Mark R. Haussler
Chapter 2 Vitamin D Receptor: Genomic and Epigenomic Effects............................................ 37
Prashant K. Singh and Moray J. Campbell
Chapter 3 Vitamin D Analogs and Their Clinical Uses.............................................................. 65
Glenville Jones
Chapter 4 Extrarenal CYP27B1 and Vitamin D Physiology.......................................................99
Martin Hewison

SECTION II Oxidative Stress
Chapter 5 Vitamin D and Oxidative Stress................................................................................ 131
Huei-Ju Ting and Yi-Fen Lee
Chapter 6 Vitamin D3 Upregulated Protein 1 (VDUP1): Roles in Redox Signaling and
Stress-Mediated Diseases.......................................................................................... 151
Dong Oh Kim, Hyun-Woo Suh, Haiyoung Jung, Young Jun Park, and Inpyo Choi
Chapter 7 Vitamin D and Its Role in Photoprotection of the Skin............................................ 165
Clare Gordon-Thomson, Wannit Tongkao-on, and Rebecca S. Mason
Chapter 8 Vitamin D and Adipose Tissue: 1α, 25-Dihydroxyvitamin D3 (Calcitriol)
Modulation of Energy Metabolism, Reactive Oxygen Species, and
Inflammatory Stress.................................................................................................. 185
Antje Bruckbauer and Michael B. Zemel
vii


viii

Contents

Chapter 9 Membrane Receptors for Vitamin D Metabolites and the Role of Reactive
Oxygen Species......................................................................................................... 201
Ramesh C. Khanal and Ilka Nemere

SECTION III Immunity and Disease
Chapter 10 Vitamin D and Human Innate Immunity.................................................................. 223
Eun-Kyeong Jo, Dong-Min Shin, and Robert L. Modlin
Chapter 11 Vitamin D and Autoimmune Disease....................................................................... 239
Colleen E. Hayes, Corwin D. Nelson, and Justin A. Spanier
Chapter 12 Vitamin D: Anti-Inflammatory Actions to Prevent and Treat Diseases...................307
Jun Sun
Chapter 13 Vitamin D and Infection........................................................................................... 323
Jim Bartley and Carlos A. Camargo, Jr.

SECTION IV Aging
Chapter 14 Potential Role of Vitamin D and Fibroblast Growth Factor 23–Klotho System
in Aging..................................................................................................................... 351
Nasimul Ahsan, Syed K. Rafi, Beate Lanske, and Mohammed S. Razzaque
Chapter 15 Vitamin D and Cardiovascular Disease.................................................................... 363
Jared P. Reis and Pamela L. Lutsey
Chapter 16 Vitamin D, Aging, and Chronic Diseases................................................................. 385
Pentti Tuohimaa
Chapter 17 Vitamin D: Defending the Aging Nervous System...................................................407
Cédric Annweiler


Series Preface
Through evolution, oxygen—itself a free radical—was chosen as the terminal electron acceptor

for respiration; hence, the formation of oxygen-derived free radicals is a consequence of aerobic
metabolism. These oxygen-derived radicals are involved in oxidative damage to cell components
inherent in several pathophysiological situations. Conversely, cells convene antioxidant mechanisms
to counteract the effects of oxidants by either a highly specific manner (e.g., superoxide dismutases)
or in a less specific manner (e.g., through small molecules such as glutathione, vitamin E, vitamin
C, etc.). Oxidative stress—as classically defined—entails an imbalance between oxidants and antioxidants. However, the same free radicals that are generated during oxidative stress are produced
during normal metabolism and, as a corollary, are involved in both human health and disease by
virtue of their involvement in the regulation of signal transduction and gene expression, activation of
receptors and nuclear transcription factors, antimicrobial and cytotoxic actions of immune system
cells, as well as aging and age-related degenerative diseases.
In recent years, the research disciplines interested in oxidative stress have increased our knowledge of the importance of the cell redox status and the recognition of oxidative stress as a process
with implications for many pathophysiological states. From this multidisciplinary and interdisciplinary interest in oxidative stress emerges a concept that attests to the vast consequences of the complex
and dynamic interplay of oxidants and antioxidants in cellular and tissue settings. Consequently,
our view of oxidative stress is growing in scope and new future directions. Likewise, the term reactive oxygen species, adopted at some stage in order to highlight nonradical/radical oxidants, now
fails to reflect the rich variety of other species in free radical biology and medicine, encompassing
nitrogen-, sulfur-, oxygen-, and carbon-centered radicals. These reactive species are involved in the
redox regulation of cell functions, and, as a corollary, oxidative stress is increasingly viewed as a
major upstream component in cell signaling cascades involved in inflammatory responses, stimulation of cell adhesion molecules, and chemoattractant production and as an early component in agerelated neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases
and amyotrophic lateral sclerosis. Hydrogen peroxide is probably the most important redox signaling molecule that, among others, can activate NFκB, Nrf2, and other universal transcription factors
and is involved in the redox regulation of insulin and MAPK signaling. These pleiotropic effects of
hydrogen peroxide are largely accounted for by changes in the thiol/disulfide status of the cell, an
important determinant of the cell’s redox status with clear involvement in adaptation, proliferation,
differentiation, apoptosis, and necrosis.
The identification of oxidants in regulation of redox cell signaling and gene expression was a
significant breakthrough in the field of oxidative stress: the classical definition of oxidative stress
as an imbalance between the production of oxidants and the occurrence of antioxidant defenses
now seems to provide a limited depiction of oxidative stress, but it emphasizes the significance
of cell redox status. Because individual signaling and control events occur through discrete redox
pathways rather than through global balances, a new definition of oxidative stress was advanced
by Dean P. Jones as a disruption of redox signaling and control that recognizes the occurrence of

compartmentalized cellular redox circuits. These concepts are anticipated to serve as platforms for
the development of tissue-specific therapeutics tailored to discrete, compartmentalized redox circuits. This, in essence, dictates principles of drug development-guided knowledge of mechanisms
of oxidative stress. Hence, successful interventions will take advantage of new knowledge of compartmentalized redox control and free radical scavenging.
Virtually all diseases thus far examined involve free radicals. In most cases, free radicals are
secondary to the disease process, but in some instances, causality is established by free radicals.

ix


x

Series Preface

Thus, there is a delicate balance between oxidant and antioxidants in health and diseases. Their
proper balance is essential for ensuring healthy aging. Compelling support for the involvement of
free radicals in disease development originates from epidemiological studies showing that enhanced
antioxidant status is associated with reduced risk of several diseases. Of great significance is the
role that micronutrients play in modulation of cell signaling: this establishes a strong linking of diet
and health and disease centered on the abilities of micronutrients to regulate redox cell signaling
and modify gene expression.
Oxidative stress is an underlying factor in health and disease. In this series of books, the importance of oxidative stress and diseases associated with organ systems is highlighted by exploring the
scientific evidence and clinical applications of this knowledge. The series is intended for researchers
in the basic biomedical sciences and clinicians. The potential of such knowledge for healthy aging
and disease prevention warrants further knowledge about how oxidants and antioxidants modulate
cell and tissue function.
This series volume provides an update on the essential role of vitamin D in optimal human
nutritional requirements and in health and disease. Further insights are provided into the protective
role of vitamin D in autoimmune, infectious, and inflammatory diseases, oxygen metabolism, and
healthy aging. In recent years, many new tissue-specific actions of vitamin D have been recognized. This area of research is rapidly expanding. We congratulate Adrian Gombart, the editor-inchief, for providing this valuable addition to the science of vitamin D.
Enrique Cadenas

Lester Packer


Preface
Vitamin D insufficiency/deficiency is a worldwide public health problem in both developed and
developing countries. Vitamin D promotes and maintains healthy bones and teeth, but with the
near eradication of rickets in the early part of the twentieth century by the fortification of foods,
chronic insufficiency has gone largely unrecognized. However, with (1) the current reemergence of
nutritional rickets among infants, (2) recent evidence that low levels of circulating vitamin D are
associated with increased risk and mortality from cancer, and (3) evidence of the potential beneficial effects of vitamin D on multiple sclerosis, rheumatoid arthritis, diabetes, cardiovascular disease, aging, and microbial infections, there has been renewed interest in this vitamin. In 2007, Time
magazine cited the benefits of vitamin D in its list of Top 10 Medical Breakthroughs, and vitamin
D continues to receive extensive coverage in both the scientific and lay press. Although extensive
research has been done on vitamin D, the molecular and cellular mechanisms responsible for its
many benefits have not been fully elucidated. In November 2010, the Institute of Medicine issued
new Dietary Reference Intakes for vitamin D. The committee provided an exhaustive review of
studies on potential health outcomes and found that the evidence supported only a role for vitamin
D in bone health but not in other health conditions. However, it is clear that there is a preponderance
of studies demonstrating a biologically plausible role for vitamin D in more than bone health. The
focus of this book is on the role of vitamin D in oxidation, immunity, and aging. These topics are
receiving increased attention in the research community but have not been covered extensively in
past books.
This book is a state-of-the-art compilation of recent information and is divided into four sections
that cover studies of vitamin D in regulating numerous aspects of health, disease, and aging. Section
I, Vitamin D: An Overview, reviews literature regarding vitamin D and its genomic and nongenomic effects, the role of therapeutic analogs in treating disease, and the production of vitamin D by
the body. The areas reviewed in this section provide a background for the subsequent sections. The
chapters in Section II, Oxidative Stress, cover the role that vitamin D plays in modulating oxidative
stress, with areas of focus including cancer, stress-mediated diseases, photoprotection of the skin,
and energy metabolism. The chapters in Section III, Immunity and Disease, review evidence for
vitamin D in regulating the immune response and the importance that it plays in protecting against
autoimmune, infectious, and inflammatory diseases. The final section, Aging, focuses on the role

that the vitamin D pathway plays in the regulation of the aging process, including aspects of oxidative stress, senescence, and mortality. Furthermore, its role in protection against cardiovascular
disease and nervous system disorders is discussed.
The editor greatly appreciates the time and effort given by all of the authors, who have shared
their knowledge in writing outstanding, timely, and scholarly chapters on the biological actions of
vitamin D. The chapters in this book represent important contributions toward understanding the
mechanisms by which vitamin D promotes health. In addition, the information presented greatly
increases awareness of the importance that vitamin D plays during development, at birth, and
throughout the aging process. It will serve as a valuable reference to researchers in academia, nutrition, medicine, and industry.

xi



Editor
Adrian F. Gombart earned his PhD in microbiology from the University of Washington. For many
years, he was an assistant and associate professor at Cedars-Sinai Medical Center and the David
Geffen School of Medicine at the University of California, Los Angeles, in the Department of
Biomedical Sciences and Division of Hematology and Oncology. He is currently a principal investigator in the Linus Pauling Institute and an associate professor in the Department of Biochemistry
and Biophysics, Oregon State University. Dr. Gombart is a member of the American Society for
Hematology and the Society for Leukocyte Biology.
Dr. Gombart’s research interests focus on the role of vitamin D in the innate immune response
against infection. His laboratory studies the regulation of antimicrobial peptide gene expression
by vitamin D and other nutritional compounds. Dr. Gombart was recognized for his contributions
to the field of vitamin D and immunity with a Young Investigator Award from the Vitamin D
Workshop in 2005.

xiii

Contributors
Nasimul Ahsan
Department of Medicine
Fayetteville VA Medical Center
Fayetteville, North Carolina
Cédric Annweiler
Department of Neuroscience
Angers University Hospital
University of Angers
Angers, France
and
Division of Geriatric Medicine
Department of Medicine
The University of Western Ontario
London, Ontario, Canada
Jim Bartley
Department of Surgery
University of Auckland
Auckland, New Zealand
Shane Batie
Division of Mathematical and Natural Sciences
Arizona State University
Phoenix, Arizona
Antje Bruckbauer
NuSirt Sciences, Inc.
Knoxville, Tennessee
Carlos A. Camargo, Jr.
Department of Emergency Medicine
Massachusetts General Hospital
Harvard Medical School

Boston, Massachusetts

Inpyo Choi
Cell Therapy Research Center
Korea Research Institute of Bioscience and
Biotechnology
and
Department of Functional Genomics
University of Science and Technology
Yuseong, Republic of Korea
Ryan Forster
Basic Medical Sciences
University of Arizona College of Medicine
Phoenix, Arizona
Clare Gordon-Thomson
School of Medical Science (Physiology) and
Bosch Institute
Sydney Medical School
University of Sydney
Sydney, Australia
Mark R. Haussler
Basic Medical Sciences
University of Arizona College of Medicine
Phoenix, Arizona
Colleen E. Hayes
Department of Biochemistry
College of Agricultural and Life Sciences
University of Wisconsin–Madison
Madison, Wisconsin
Martin Hewison

Department of Orthopaedic Surgery and
Molecular Biology Institute
David Geffen School of Medicine
University of California, Los Angeles
Los Angeles, California

Moray J. Campbell
Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Buffalo, New York

xv


xvi

Eun-Kyeong Jo
Department of Microbiology
Infection Signaling Network Research Center
College of Medicine
Chungnam National University
Daejeon, South Korea
Glenville Jones
Department of Biomedical and Molecular
Sciences
and
Department of Medicine
Queen’s University
Kingston, Ontario, Canada
Haiyoung Jung

Cell Therapy Research Center
Korea Research Institute of Bioscience and
Biotechnology
Yuseong, Republic of Korea
Peter W. Jurutka
Division of Mathematical and Natural Sciences
Arizona State University
and
Basic Medical Sciences
University of Arizona College of Medicine
Phoenix, Arizona
Ramesh C. Khanal
Department of Nutrition and Food Sciences
and
Center for Integrated BioSystems
Utah State University
Logan, Utah
Dong Oh Kim
Cell Therapy Research Center
Korea Research Institute of Bioscience and
Biotechnology
and
Department of Functional Genomics
University of Science and Technology
Yuseong, Republic of Korea
Beate Lanske
Department of Developmental Biology
Harvard School of Dental Medicine
Boston, Massachusetts

Contributors

Jamie Lee
Division of Mathematical and Natural Sciences
Arizona State University
Phoenix, Arizona
Yi-Fen Lee
Department of Urology
University of Rochester Medical Center
Rochester, New York
Pamela L. Lutsey
Division of Epidemiology and Community
Health
School of Public Health
University of Minnesota
Minneapolis, Minnesota
Rebecca S. Mason
School of Medical Science (Physiology) and
Bosch Institute
Sydney Medical School
University of Sydney
Sydney, Australia
Robert L. Modlin
Division of Dermatology
Department of Microbiology, Immunology, and
Molecular Genetics
University of California, Los Angeles
Los Angeles, California
Corwin D. Nelson
Department of Biochemistry

College of Agricultural and Life Sciences
University of Wisconsin–Madison
Madison, Wisconsin
Ilka Nemere
Department of Nutrition and Food Sciences
and
Center for Integrated BioSystems
Utah State University
Logan, Utah
Young Jun Park
Cell Therapy Research Center
Korea Research Institute of Bioscience and
Biotechnology
Yuseong, Republic of Korea


xvii

Contributors

Syed K. Rafi
Department of Anatomy and Cell Biology
University of Kansas Medical Center
Kansas City, Kansas

Jun Sun
Department of Biochemistry
Rush University
Chicago, Illinois

Mohammed S. Razzaque
Department of Oral Medicine, Infection and
Immunity
Harvard School of Dental Medicine
Boston, Massachusetts

Huei-Ju Ting
Department of Urology
University of Rochester Medical Center
Rochester, New York

Jared P. Reis
Division of Cardiovascular Sciences
National Heart, Lung, and Blood Institute
National Institutes of Health
Bethesda, Maryland
Dong-Min Shin
Department of Microbiology
Infection Signaling Network Research Center
College of Medicine
Chungnam National University
Daejeon, South Korea
Prashant K. Singh
Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Buffalo, New York
Justin A. Spanier
Department of Biochemistry
College of Agricultural and Life Sciences
University of Wisconsin–Madison

Madison, Wisconsin
Hyun-Woo Suh
Cell Therapy Research Center
Korea Research Institute of Bioscience and
Biotechnology
Yuseong, Republic of Korea

Wannit Tongkao-on
School of Medical Science (Physiology) and
Bosch Institute
Sydney Medical School
University of Sydney
Sydney, Australia
Pentti Tuohimaa
Medical School
University of Tampere
and
Centre for Laboratory Medicine
Tampere University Hospital
Tampere, Finland
G. Kerr Whitfield
Basic Medical Sciences
University of Arizona College of Medicine
Phoenix, Arizona
Michael B. Zemel
Department of Nutrition
The University of Tennessee
Knoxville, Tennessee

Section I
Vitamin D: An Overview



1

Vitamin D: A Fountain of
Youth in Gene Regulation
Peter W. Jurutka, G. Kerr Whitfield, Ryan Forster,
Shane Batie, Jamie Lee, and Mark R. Haussler

CONTENTS
1.1 Vitamin D Bioactivation and Its Endocrine/ Mineral Feedback Control...................................3
1.2 Biological Responses to the 1,25D Hormone Are Widespread................................................. 5
1.3Structure–Function of VDR and Mechanisms of Gene Regulation..........................................7
1.4 VDR Binds Nonvitamin D Ligands......................................................................................... 12
1.5 VDR-Mediated Control of Networks of Vital Genes.............................................................. 13
1.5.1 Detoxification of Endobiotics and Xenobiotics........................................................... 16
1.5.2 Phosphate Homeostasis Attenuates Senescence.......................................................... 17
1.5.3 VDR Ligands Promote Health Span via the Delay of Chronic Diseases of Aging........... 20
1.6 Conclusion and Perspectives.................................................................................................... 23
References.........................................................................................................................................26

1.1 VITAMIN D BIOACTIVATION AND ITS ENDOCRINE/ MINERAL
FEEDBACK CONTROL
The hormonal precursor and parent compound, vitamin D3, either can be obtained in the diet or
formed from 7-dehydrocholesterol in skin (epidermis) via a nonenzymatic, UV light-dependent
reaction (Figure 1.1). Vitamin D3 is then transported to the liver, where it is hydroxylated at the C-25

position of the side chain to produce 25-hydroxyvitamin D3 (25D), which is the major circulating
form of vitamin D3. The final step in the production of the hormonal form occurs mainly, but not
exclusively, in the kidney via a tightly regulated 1α-hydroxylation reaction (Figure 1.1). The cytochrome P450-containing (CYP) enzymes that catalyze 25- and 1α-hydroxylations are microsomal
CYP2R1 (Cheng et al. 2003) and mitochondrial CYP27B1, respectively. As depicted in Figure
1.1, 1,25-dihydroxyvitamin D3 (1,25D) circulates, bound to plasma vitamin D binding protein, to
various target tissues to exert its endocrine actions, which are mediated by the vitamin D receptor
(VDR). Many of the long-recognized functions of 1,25D involve the regulation of calcium and phosphate metabolism, raising the blood levels of these ions to facilitate bone mineralization, as well as
activating bone resorption as part of the remodeling cycle (Haussler et al. 2010).
In addition to affecting bone mineral homeostasis by functioning at the small intestine and bone,
1,25D also acts through its VDR mediator to influence a number of other cell types. These extraosseous actions of 1,25D-VDR include differentiation of certain cells in skin (Bikle and Pillai 1993) and
in the immune system (Mora et al. 2008; Figure 1.1). Interestingly, the skin and the immune system
are now recognized as extrarenal sites of CYP27B1 action to produce 1,25D locally for autocrine
and paracrine effects (Adams et al. 1985; Omdahl et al. 2002), creating intracrine systems (Figure
1.1) for extraosseous 1,25D-VDR functions distinct from the renal endocrine actions of 1,25D-VDR
on the small intestine and skeleton. Apparently, higher circulating 25D levels are required for optimal intracrine actions of 1,25D (Figure 1.1). This insight stems from the importance of attaining
3


4

Vitamin D: Oxidative Stress, Immunity, and Aging

Kidney

24-hydroxylated
D3 metabolites
1,25
CYP24A1 +

Skin

Low Ca2+

+

Liver
CYP2R1

Circulating
25(OH)D3

PTH
synthesis
1,25 –
VDR

Parathyroid

PTH

1α,25(OH)2D3
25(OH)D3
+ CYP27B1 –
FGF23
High PO43–

Intracrine action

Extrarenal (local) 1,25(OH)2D3 production:
skin, immune system, colon, vasculature, etc.

Extraosseous effects of 1,25(OH)2D3-VDR:
Immunoregulation, antimicrobial defense,
xenobiotic detoxification, anti-inflammatory,
anti-cancer actions, cardiovascular benefits

C

HO

VDR

Vitamin D3
Diet

Vitamin D
catabolism

+

1,25
VDR

FGF23

A

D

OH

OH

1,25(OH)2D3
Circulating
1α,25(OH)2D3 1,25
Plasma
VDR

D Binding
Protein (DBP)

Vitamin D
receptor

+ synthesis
Osteocyte

Endocrine action
Intestinal calcium and
phosphate absorption
Bone remodeling

FIGURE 1.1  Vitamin D acquisition, regulation of metabolic activation/catabolism, and receptor-mediated
endocrine and intracrine actions of the 1,25D hormone.

adequate levels of circulating 25D revealed in a multitude of epidemiologic associations between
low 25D levels and chronic disease, coupled with statistically significant protection against a host of
pathologies by much higher circulating 25D (Bikle 2009). Thus, as depicted schematically in Figure
1.1, locally produced 1,25D appears to be capable of benefitting the vasculature to reduce the risk
of heart attack and stroke, controlling the adaptive immune system to lower the incidence of autoimmune disease while boosting the innate immune system to fight infection, effecting xeno­biotic

detoxification, and exerting antiinflammatory and anticancer pressure on epithelial cells prone to
fatal malignancies.
The parathyroid gland also expresses VDR (Brumbaugh et al. 1975; Wecksler et al. 1977), and
when the receptor is liganded with 1,25D, parathyroid hormone (PTH) synthesis is suppressed by a
direct action on gene transcription (De May et al. 1992). This negative feedback loop, which curtails
the stimulation of CYP27B1 by PTH under low calcium conditions (Figure 1.1), serves to limit the
bone-resorbing effects of PTH in anticipation of 1,25D-mediated increases in both intestinal calcium absorption and bone resorption, thus preventing hypercalcemia. More recent understanding
of the homeostatic control of phosphate has emerged, emanating originally from characterization
of unsolved familial hypo- or hyperphosphatemic disorders, which we now know are caused by
deranged levels of bone-derived FGF23 (Bergwitz and Juppner 2010). In short, FGF23 has materialized as a dramatic new phosphate regulator and a second phosphaturic hormone after PTH. We
(Kolek et al. 2005) and others (Quarles 2008) proved that 1,25D induces the release of FGF23 from
bone, specifically from osteocytes of the osteoblastic lineage (Figure 1.1), which is a process that is
independently stimulated by high circulating phosphate levels (Figure 1.1). Thus, in a striking and
elegant example of biological symmetry, PTH is repressed by 1,25D and calcium, whereas FGF23
is induced by 1,25D and phosphate, protecting mammals against hypercalcemia and hyperphosphatemia, respectively, either of which can elicit ectopic calcification.
As illustrated in Figure 1.1, using the kidney as an example, an important mechanism by which
the 1,25D-VDR-mediated endocrine or intracrine signal is terminated in all target cells is the
catalytic action of CYP24A1, which is an enzyme that initiates the process of 1,25D catabolism
(St-Arnaud 2010). The CYP24A1 gene is transcriptionally activated by 1,25D (Ohyama et al. 1994a;


Vitamin D: A Fountain of Youth in Gene Regulation

5

Zierold et al. 1994b), as well as by FGF23 (Figure 1.1). In addition, the 1α-hydroxylase (1α-OHase)
CYP27B1 gene is repressed by FGF23 and 1,25D, with the latter regulation affected by epigenetic
demethylation (Kim et al. 2009) in a short negative feedback loop to limit the production of 1,25D
(Murayama et al. 1999). Therefore, the vitamin D endocrine system is elegantly governed by feedback controls of vitamin D bioactivation, which interpret bone mineral ion status, and via feedforward induction of 1,25D catabolism to prevent the pathologies of hypervitaminosis D. The vitamin
D intracrine system, in contrast, appears to be dependent more on the availability of ample 25D

substrate to generate local 1,25D to lower the risk of chronic diseases of the epithelial (e.g., skin and
colon), immune, cardiovascular, and possibly nervous systems.

1.2  BIOLOGICAL RESPONSES TO THE 1,25D HORMONE ARE WIDESPREAD
We are the first group to propose that 1,25D, either alone or in combination with bona fide antiaging
gene products like klotho, is a mediator of healthful aging (Haussler et al. 2010). This hypothesis
could explain epidemiologic/association studies that suggest that 25D, in the newly recognized optimal high-normal range in blood, confers a lower risk of virtually all of the fatal diseases of aging
such as heart attack, stroke, and cancers. Thus, as depicted in Figure 1.2, the endocrine/intracrine
actions of vitamin D/klotho protect the vascular system, as well as epithelial cells subject to fatal cancers (Mordan-McCombs et al. 2007; breast, prostate, colon, and skin), the immune system (Liu et al.
2006a; Mora et al. 2008; Raghuwanshi et al. 2008; Figure 1.1), and possibly the central nervous system (Keisala et al. 2009). With respect to the chronic diseases of aging, it is now becoming clear that
the kidney represents the nexus of control, and we contend that klotho is a third renal hormone after
1,25D and erythropoietin. Therefore, in this chapter, we emphasize the importance of renal health
during aging and unveil the kidney as a focal point for the prevention of chronic diseases (Figure 1.2).
Figure 1.2 summarizes and integrates the endocrine regulation and actions of 1,25D-VDR in
exerting the bone mineral homeostatic, immune, cardiovascular, and anticancer effects. While the
predominant action of 1,25D-VDR is promoting intestinal calcium and phosphate absorption to prevent osteopenia, the signal for this function is PTH reacting to low calcium, whereas the hormonal
agent that feedback controls these events to preclude ectopic calcification is FGF23. In this fashion,
bone resorption and mineralization remain coupled to protect the integrity of the mineralized skeleton. FGF23 functions acutely in concert with PTH and chronically when PTH is suppressed by
calcium and 1,25D (Figure 1.2). In fact, FGF23 directly represses PTH (Ben-Dov et al. 2007; Figure
1.2) to abolish the activation of CYP27B1 by PTH while, at the same time, appropriating from PTH
the role of phosphate elimination. Like PTH, FGF23 inhibits renal Npt2a and Npt2c to elicit phosphaturia (Shimada et al. 2004a; Figure 1.2). In contrast to PTH, which is downregulated by 1,25D
in parathyroid glands, FGF23 is upregulated by 1,25D in osteocytes (Bergwitz and Juppner 2010;
Kolek et al. 2005; Liu et al. 2006b), which is a major source of FGF23 endocrine production by bone.
As illustrated in Figure 1.2 (lower right), hyperphosphatemia enhances osteocytic FGF23 production independently of 1,25D, rendering FGF23 the perfect phosphaturic counter-1,25D hormone
because it inhibits renal phosphate reabsorption and 1,25D biosynthesis via inhibition of CYP27B1
while enhancing 1,25D degradation by inducing CYP24A1 in all tissues (Figure 1.2). In this fashion,
FGF23 allows osteocytes to communicate with the kidney to govern circulating 1,25D, as well as
phosphate levels, thereby preventing excess 1,25D function and hyperphosphatemia. FGF23 signals
via renal FGFR/klotho coreceptors to promulgate phosphaturia (Razzaque 2009), repress CYP27B1
(Perwad et al. 2007), and induce CYP24A1 (Razzaque 2009; Shimada et al. 2004a; Figure 1.2).

FGF23 regulation is complex and multifactorial, including the suppressive proteins PHEX and
dentin matrix acidic phosphoprotein 1 (DMP-1; Figure 1.2). 1,25D represses PHEX expression in
UMR-106 osteocyte-like cells, which is in accordance with the induction of FGF23 in that the
PHEX suppressor is attenuated to permit maximal induction of FGF23 by 1,25D (Hines et al.
2004). It is conceivable that the mechanism of FGF23 induction by 1,25D is, in part or entirely,
a consequence of PHEX repression, yet the PHEX substrate which ultimately regulates FGF23


6

Vitamin D: Oxidative Stress, Immunity, and Aging

Vasculature

Catabolism to
1,24,25D, etc.

Cardiovascular influences

Epithelial cells
Breast
Prostate
Skin
Colon

CY

Secreted
klotho

Parathyroid
gland
PTH gene

Ca2+ receptor
(sensor)

e
rin on
rac rsi
Intonve
c

Kidney
PTH

1,25D

Net result =
Osteopenia

T-cells
B-cells
Macrophages

1,25D
RXR-VDR
DMP-1 gene

1,25D

CYP27B1
FGFR
Klotho

Ca2+ PO43–
1,25D
RXR-VDR Npt2c
Npt2a

Cell surface
generated
signals

FGF23
FGF23 gene
Degraded
FGF23

Blood Mineralization
Ca-PO4
Absorption

Resorption

1,25D
RXR-VDR
TRPV6

Ca2+

PHEX

PHEX gene

DMP-1

Reabsorption
Low Ca2+
in blood

23

Immune modulation

25OHD

1,25D
RXR-VDR

Immune cells

D

5
1,2

F
FG

Anticancer/detox effects
FGF23

A1

4
P2

Osteocyte

Osteoblast

PO43–

HTTs

PO43– sensor
postulated

Intermediary
transfactors
1,25D
RXR-VDR

Bone RANKL

Ca2+

Osteoclast

High PO43–
in blood

Net result =
Ectopic calcification

Npt2b

PO43–

Small intestine

FIGURE 1.2  (See color insert.) Parathyroid, kidney, and bone comprise an endocrine trio for the regulation
of phosphate and calcium metabolism to prevent osteopenia/osteoporosis and ectopic calcification (shaded in
light blue). Renal hormones 1,25D (shaded in light blue) and klotho (shaded in dark blue) reach beyond bone
mineral homeostasis to delay other chronic disorders of aging besides osteoporosis, such as cardiovascular
disease, epithelial cell cancers, and autoimmune disease.

transcription is not known. Although DMP-1 is apparently not a PHEX substrate, loss of function
mutations in DMP1 cause a phenotype identical to XLH, with excess FGF23 producing hypophosphatemia (Quarles 2008). This suggests that DMP1, like PHEX, normally represses FGF23 expression in osteocytes, although this is inconsistent with the observation (Farrow et al. 2009) that 1,25D
induces DMP1 in UMR-106 cells. Fascinatingly, it has been shown recently (Martin et al. 2011) that
PHEX and DMP1 regulate FGF23 expression in osteocytes through a common pathway involving
FGF receptor (FGFR) signaling, intimating that PHEX and DMP-1 regulate FGF23 expression by
impacting an autocrine loop in the osteocyte whereby FGF23 governs its own synthesis (not shown
in Figure 1.2). FGF23 synthesis is also governed by high phosphate (Ito et al. 2005), possibly via
an undiscovered transcription factor (analogous to signaling through Gq by the calcium sensing


Vitamin D: A Fountain of Youth in Gene Regulation

7

receptor in the parathyroid and other tissues) to induce the FGF23 gene. Such factors that play a specific role in transduction of the phosphate signal are herein termed “hyperphosphatemia transducing
transfactors” (Figure 1.2). The targeting of these factors will be of great interest to those attempting to modulate FGF23 in patients such as those in renal failure who may benefit from reduced
FGF23 secretion (Fukumoto 2010; Juppner et al. 2010). Finally, identification of these factors will
also increase our comprehension of the control of FGF23, and we may, for the first time, be able to
integrate the 1,25D and phosphate arms of FGF23 regulation with other known osteocyte players
such as PHEX and DMP1 (Figure 1.2), as well as renal phosphate transporters, Npt2a/c. Indeed,
Demay and colleagues (Miedlich et al. 2010) have recently shown, by ablation of renal phosphate
transporter Npt2a, that phosphate is the central regulator of the FGF23 gene and is capable of prevailing over vitamin D because 1,25D fails to induce FGF23 when hypophosphatemia and elevated
1,25D occur concurrently.
Intestinal calcium absorption is mediated by 1,25D-VDR induction of TRPV6 (Barthel et al.
2007; Meyer et al. 2006), which supplies dietary calcium via transport to build the mineralized
skeleton (Figure 1.2). Indeed, TRPV6 null mice have 60% decreased intestinal calcium absorption, decreased bone mineral density (BMD), and, strikingly, 20% of animals exhibit alopecia and
dermatitis (Bianco et al. 2007) similar to VDR knockout mice (Li et al. 1997). Since the skin phenotype in VDR null mice is not ameliorated by the high-calcium rescue diet (Amling et al. 1999),
we speculate that TRPV6 may mediate calcium entry into keratinocytes to elicit differentiation and
hair cycling. Because calcium is protective against colon cancer (Garland et al. 1985), while hair
plus a full-stratum corneum reduce UV-induced skin damage and cancer, VDR-induced TRPV6
could also function in colon and skin to lower the risk of neoplasia in these two epithelial cell types
(Figure 1.2).
Although 1,25D also enhances intestinal phosphate absorption via the induction of Npt2b (Katai
et al. 1999), because phosphate is abundant in the diet and constitutively absorbed by the small
intestine, the phosphate absorption effect of 1,25D may not be as physiologically important as the
profound effect of 1,25D to trigger calcium transport.
In the osteoblast, RANKL constitutes one of the most dramatically 1,25D-upregulated bone
genes, the product of which affects 1,25D-VDR-mediated bone resorption through osteoclastogenesis (Figure 1.2). We have shown that RANKL is induced more than 5000-fold by 1,25D in mouse
ST-2 stromal cells in culture (Haussler et al. 2010). OPG, which is the soluble decoy receptor for
RANKL that tempers its activity, is simultaneously repressed by 86% (Haussler et al. 2010) to
amplify the bioeffect of displayed (or secreted) RANKL. Thus, like PTH, 1,25D is a potent boneresorbing, hypercalcemic hormone, and although chronic excess of either hormone elicits severe
osteopenic pathology, physiologic bone remodeling can be argued to strengthen the skeleton. In

other words, like a well-mineralized bone, an appropriately remodeled bone is a healthy bone and
is less susceptible to fractures and the eventual ravages of senile osteoporosis. Beyond bone, renal
1,25D and, especially, locally generated extrarenal 1,25D benefit the cardiovascular system in
which VDR is expressed in endothelial cells, smooth muscle cells, and cardiac myocytes. Finally,
kidney- and locally derived 1,25D also influences many cells in the immune system to modulate its
functions, as well as to exert anticancer actions in virtually all epithelial cells (Figure 1.2).

1.3 STRUCTURE–FUNCTION OF VDR AND MECHANISMS
OF GENE REGULATION
Various domains of the 427 amino acid human VDR are highlighted on a linear schematic of the
protein (Figure 1.3a), with the two major functional units being the N-terminal zinc finger DNA
binding domain (DBD), and the C-terminal ligand binding (LBD)/heterodimerization domain.
To date, the Protein Data Bank (PDB) database contains over 50 x-ray crystal structures for the
VDR LBD and four of the DBD bound as a homo- or heterodimer on VDRE DNA sequences. The