D Vitamin D- More Than a Bone-a-Fide Hormone
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MINIREVIEW
Vitamin D: More Than a Bone-a-Fide HormoneAMELIA L. M. SUTTON AND PAUL N. MACDONALD
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106
The vitamin D endocrine system is critical for the
proper development and maintenance of mineral
ion homeostasis and skeletal integrity. Beyond
these classical roles, recent evidence suggests
that the bioactive metabolite of vitamin D, 1,25-
dihydroxyvitamin D3, functions in diverse physio-
logical processes, such as hair follicle cycling,
blood pressure regulation, and mammary gland
development. This minireview explores the current
progress in unraveling the complexities of the vi-
tamin D endocrine system by focusing on four main
areas of research: the resolution of the vitamin D
receptor crystal structure, the molecular details of
1,25-dihydroxyvitamin D3-mediated transcription,
murine knockout models of key genes in the en-
docrine system, and alternative vitamin D recep-
tors and ligands. (Molecular Endocrinology 17:
777791, 2003)
VITAMIN D WAS discovered nearly a century agoas the nutrient that prevented rickets, a devastat-ing skeletal disease characterized by undermineral-
ized bones (1). Since that time, our concept of vitamin
D and, in particular, its most bioactive derivative, 1,25-
dihydroxyvitamin D3 [1,25-(OH)2D3], has evolved from
that of an essential micronutrient to that of a hormone
involved in a complex endocrine system that directs
mineral homeostasis, protects skeletal integrity, and
modulates cell growth and differentiation in a diverse
array of tissues. 1,25-(OH)2D3 acts in concert with PTHto tightly regulate the concentration of serum calcium
and phosphate, thereby maintaining proper skeletal
mineralization (Fig. 1). A major function of 1,25-
(OH)2D3 is to promote intestinal absorption of calcium
and phosphate. However, it also may have direct ef-
fects on the bone (2), in which continuous remodeling
must occur to sustain structural integrity. For example,
in vitro studies indicate that 1,25-(OH)2D3 stimulates
osteoblasts, the resident bone-forming cells, to termi-
nally differentiate and to deposit calcified matrix (3).
Conversely, when dietary sources are inadequate to
maintain normocalcemia, 1,25-(OH)2D3 may stimulate
calcium mobilization from the bone by promoting the
differentiation of precursor cells into mature, bone-resorbing osteoclasts (4).
The hormonal or bioactive form of vitamin D is1,25-(OH)
2D3.
It is generated from sequential hy-
droxylations of vitamin D3, a secosteroid precursorthat is obtained from the diet or produced in the skinupon exposure to UV light (5, 6). The first hydroxy-lation of vitamin D
3occurs at the C-25 position and
is catalyzed by vitamin D-25-hydroxylase in the liver
to produce 25-hydroxyvitamin D3 [25(OH)D3], themajor circulating form of vitamin D in mammals.25(OH)D
3is the substrate for a second hydroxylase,
the renal 25(OH)D3
-1-hydroxylase (1OHase),
resulting in the production of the most bioactivemetabolite, 1,25-(OH)2D3. A classic endocrine feed-back system operates to tightly control serum levelsof 1,25-(OH)
2D3
(5, 6). For example, renal 1OHaseactivity is stimulated by low serum calcium and
phosphorus levels and by PTH. The expression of1OHase is negatively regulated by high levels of1,25-(OH)
2D3
. Inactivation, or catabolism, of vitaminD metabolites is initiated by the ubiquitous enzyme
25-hydroxyvitamin D3-24-hydroxylase (24OHase) togenerate either 24,25(OH)2D3 or 1,24,25(OH)3D3.The 24-hydroxylated metabolites are further de-graded and eventually excreted as either calcitroicacid or 23-carboxyl derivatives. This catabolic pro-cess is also carefully regulated as 1,25-(OH)
2D3
stimulates 24OHase expression to prevent exces-
sive synthesis of the hormone.The biological effects of 1,25-(OH)2D3 are mediated
through the vitamin D receptor (VDR), a member of thenuclear receptor superfamily of ligand-activated tran-scription factors (7, 8). Binding of 1,25-(OH)
2D3
to VDRinitiates a cascade of macromolecular interactions ul-
Abbreviations: AF-2, Activation function-2; CBP, cAMPresponse element binding protein-binding protein; DBD,DNA-binding domain; 1,25-(OH)
2D3
, 1,25-dihydroxyvitaminD3
; 1OHase, 25(OH)D3
-1-hydroxylase; 25-(OH)D3
, 25-hydroxyvitamin D
3; 24-OHase, 25-(OH)D
3-24-hydroxylase;
DRIP, VDR-interacting protein; HAT, histone acetyltrans-ferase; HVDRR, hereditary vitamin D-resistant rickets; LBD,ligand-binding domain; LCA, lithocholic acid; NCoA, nuclearreceptor coactivator; RAR, retinoic acid receptor; RXR, reti-noid X receptor; SKIP, ski-interacting protein; SRC, steroidreceptor coactivator; TIF2, transcription intermediary fac-tor-2; TRAP, thyroid receptor-activating protein; VDR, vitaminD receptor; VDRE, vitamin D response element; VDRKO, VDRknockout.
0888-8809/03/$15.00/0 Molecular Endocrinology 17(5):777791Printed in U.S.A. Copyright 2003 by The Endocrine Society
doi: 10.1210/me.2002-0363
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timately leading to transcription of select target genes
(9). 1,25-(OH)2
D3
associates with the VDR and pro-
motes its heterodimerization with retinoid X receptor
(RXR), a common heterodimeric partner for other class
II nuclear receptors (10). The liganded VDR-RXR het-
erodimer is the functionally active transcription factor
in 1,25-(OH)2D3-mediated transcription. The het-
erodimer binds with high affinity to vitamin D response
elements (VDREs) in the promoters of target genes.
VDREs are characterized by two direct hexameric re-
peats with an intervening spacer of three nucleotides
(DR-3 elements). Thus, 1,25-(OH)2
D3
target gene se-
lectivity is conferred, in part, through ligand binding,
VDR-RXR heterodimerization, and high-affinity bind-
ing to DR-3 VDREs. Beyond these initial steps, the
precise molecular mechanisms involved in target gene
activation by VDR are less evident. Recent attention
has turned to so-called coactivator proteins that inter-
act directly with VDR and other nuclear receptors in a
ligand-dependent manner (11). These coactivators par-
ticipate in an intricate multiprotein complex together with
the basal transcriptional machinery and histone modi-
Fig. 1. Metabolism and Mineral Homeostatic Functions of the Vitamin D Endocrine System
Bioactive 1,25-(OH)2
D3
is generated by sequential hydroxylations of its precursor vitamin D3
in the liver and the kidney.
1,25-(OH)2
D3
operates in a negative feedback loop by inducing expression of the catabolic enzyme 24-OHase and by inhibiting
expression of the anabolic enzyme 1OHase. In response to low serum calcium, PTH is produced and stimulates 1OHase
expression in the kidney and promotes calcium mobilization from the bone and reabsorption from the kidney. 1,25-(OH)2
D3
, in
turn, induces calcium absorption in the intestine and calcium release from the skeleton.
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fiers to stimulate expression of 1,25-(OH)2D3-regulated
genes.
Over the past five years, remarkable strides have
been made in clarifying the physiological functions
and the concomitant therapeutic potential of vitamin D
and its derivatives. Structural studies provide new in-
sight into the ligand-binding pocket of VDR com-plexed to 1,25-(OH)
2D3 and several potent synthetic
analogs and, thereby, present a scaffold on which to
build future VDR-targeted therapies. A more complete
molecular picture of VDR-mediated transcription is
emerging as the details of coactivator action are un-
raveled. Murine knockout models of VDR as well as
key enzymes involved in vitamin D metabolism reveal
the essential roles of vitamin D in vivo. Finally, inves-
tigators have begun to identify novel ligands and al-
ternative VDRs, from synthetic analogs to potential
membrane receptors, that signal new directions for the
field. Although the range of recent advances extends
far, we chose to focus this minireview on these four
areas of vitamin D research. Together, these areas of
progress have not only affirmed classic paradigms in
vitamin D physiology, but they also have opened up
new avenues of exploration for future research.
VDR CRYSTAL STRUCTURE
The VDR shares discrete structural and functional do-
mains with other nuclear receptors, but it also exhibits
several unique features (Fig. 2A and Refs. 5 and 9). The
hypervariable amino-terminal A/B domain of VDR is
unusually short and, in contrast to that of most othernuclear receptors, is generally thought to lack potent
transactivation domains. However, as discussed later
(see VDR Isoforms), there is increasing evidence that
the VDR A/B domain helps determine the overall trans-
activation capacity of the VDR (12). The DNA-binding
domain (DBD, or region C) of VDR is similar to that of
other nuclear receptors and is characterized by two
zinc-binding modules that direct sequence-specific
binding of receptors to DNA (13). The ligand-binding
domain (LBD, or region E) is a multifunctional globular
domain that mediates selective interactions of the re-
ceptor with its cognate hormone (13), with other nu-
clear receptor partners (14), and with comodulatory or
adapter proteins (15). The LBD contains the ligand-dependent activation function-2 (AF-2), which is cru-
cial to ligand-activated transcription. Mutation of the
AF-2 renders the nuclear receptor transcriptionally in-
active despite retaining the ability to bind ligand (1416). The DBD and LBD are bridged by the hinge region
(domain D), which is thought to confer rotational flex-
ibility between the DBD and LBD and allow for recep-
tor dimerization and interaction with the DNA (17).
Although detailed crystal structures for several nu-
clear receptors have been available for nearly a de-
cade (1822), the structure of the VDR LBD was notsolved until recently (23). Numerous attempts to crys-
tallize VDR failed, likely due to the presence of a
unique insertion sequence in the LBD (see Fig. 2A) that
is largely unordered, leading to decreased protein sol-
ubility. Removal of this insertion domain allowed for
efficient crystallization and structure determination of
the VDR LBD complexed to 1,25-(OH)2D3 (23). Al-though the lack of this domain may compromise the
interpretation of the VDR structure, the mutant VDR
displays normal ligand binding and similar transacti-
vation properties in vitro (24). Thus, the absence of the
insertion sequence does not alter the conformation
significantly so as to compromise VDR function.
The structure of the VDR LBD is similar to that of
other nuclear receptors, being most closely related to
that of the retinoic acid receptor (RAR)LBD (23). The
VDR LBD is organized into 13 -helices and 3
-sheets, which together form a hydrophobic ligand-
binding pocket. This pocket is larger than that of RAR
Fig. 2. Domain Structure of VDR and Two-Step Model of
VDR-Mediated Transcription
A, Functional domains of VDR. A/B, Amino-terminal region;
DBD, DBD showing two zinc finger modules (Zn); LBD, LBDincluding the long insertion domain and helix 12 encompass-
ing AF-2. B, Temporal association of coactivators during
VDR-mediated transcription. The liganded (D) VDR-RXR het-
erodimer recruits SRCs and CBP/p300, resulting in acetyla-
tion (Ac) of histones. The open chromatin template allows for
binding of the DRIP complex and entry of the core transcrip-
tional machinery.
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due to variations in the positions of helices H2 and H3n
and the H6H7 loop. Helix 12, containing the ligand-dependent AF-2, of ligand-bound VDR is positioned
similarly to other nuclear receptors (20), highlighting its
central importance in creating a coactivator interaction
surface (see VDR Coactivators). In fact, several resi-
dues of H12 directly contact the ligand, indicating thatthe ligand conformation may modulate H12 conforma-
tion and, therefore, coactivator binding and transcrip-
tional activity. When additional structures of liganded
VDR complexed with various coactivators are solved,
they will likely provide a molecular framework on which
to develop new compounds to modulate the vitamin D
endocrine system.
In this regard, numerous synthetic analogs have
already been developed that mimic the advantageous
effects of 1,25-(OH)2
D3
without the hypercalcemic
side effects (see Novel Vitamin D Ligands). Specula-
tion about the mechanisms behind the selective, pleio-
tropic effects of 1,25-(OH)2
D3
analogs centers on the
concept that these analogs induce distinct conforma-
tions in VDR compared with that of the natural ligand,
ultimately resulting in analog-selective gene regulation
(25, 26). Protease digestions and coactivator binding
studies provide experimental support for this model
(25, 27). However, the VDR ligand-binding cavity is
larger than that of many other nuclear receptors, and
the ligand occupies less than half of this volume. Con-
sequently, the VDR ligand-binding pocket can accom-
modate rather significant structural changes in the
ligand including 1,25-(OH)2
D3
analogs with bulky side
chains (23, 28). Indeed, the crystal structures of VDR
complexed with the MC1288 and KH1060 analogs
show that these low calcemic analogs do not inducedifferent conformations in the VDR compared with the
natural ligand (29). Thus, other mechanisms must be
considered to explain the different potencies and cal-
cemic profiles of the analogs. One potential answer
resides in the observation that the VDR-analog com-
plexes are more energetically stable than the VDR-
1,25-(OH)2D3 complex (29). The increased half-life of
the activated VDR may result in altered transcriptional
activity, which may explain the differences both in
potencies and in target gene selectivity between the
natural and synthetic ligands. Alternatively, the solid-
state crystal structure may not reveal subtle dynamic
conformational changes in solubilized VDR evoked by
various analogs.
VDR COACTIVATORS
The existence of limiting accessory factors or adapter
proteins in steroid hormone receptor action was pro-
posed in the late 1980s based on the squelchingphenomenon, in which the LBD of one receptor inter-
feres with ligand-activated transcription mediated by a
second receptor (30). A decade later, these comodu-
latory proteins were identified as specific molecules
that interact with nuclear receptors and influence their
transactivation potential (3133). The emergence ofcoactivators, and their inhibitory counterparts core-
pressors, provides new insight into the molecular
mechanism of nuclear receptor-mediated transcrip-
tion. Upon association with its cognate hormone, the
receptor LBD undergoes a subtle conformationalchange (34). The critical change occurs in helix 12, the
carboxy-terminal-helix containing the ligand-depen-
dent AF-2. In response to ligand binding, helix 12 folds
over top of the globular LBD and caps the ligand-
binding cavity (20). This ligand-dependent conforma-
tional shift creates a hydrophobic cleft composed of
helices 3, 4, 5, and 12 (3537). The hydrophobic cleftserves as a docking surface for many nuclear receptor
coactivators by interacting with a complementary hy-
drophobic domain in the coactivator containing the
consensus LXXLL motif, also referred to as the nuclear
receptor box (38). Although these studies provide an
elegant structural model for ligand-activated tran-scription by nuclear receptors and LXXLL-containing
coactivators, the precise mechanisms governing nu-
clear receptor-mediated transactivation are less clear.
The ability of coactivators to interact with components
of the preinitiation complex, with other transcription
factors, and with histone-modifying proteins implies
that a complex integration of transactivator cues oc-
curs at the promoter of nuclear receptor target genes.
The growing number of coactivators identified in the
last decade adds yet another level of complexity to the
paradigm of nuclear receptor-mediated transcription.
Extensive reviews on comodulatory proteins can be
found elsewhere (3941). Here, we will highlight a few
significant developments in the VDR coactivator field.
Steroid receptor coactivator (SRC)-1 [or nuclear re-
ceptor coactivator (NCoA)1] is the founding member of
the LXXLL motif-containing SRC family of coactivators
(33). This family also includes transcriptional interme-
diary factor-2 (TIF2; Refs. 42 and 43) and receptor-
associated coactivator-3 (4448). The SRCs interactwith VDR and potentiate its transcriptional activity (15,
49). Each of the SRCs possess an autonomous tran-
scriptional activation domain, as evidenced by their
ability to enhance transcription when fused to a het-
erologous DNA-binding sequence such as GAL4.
SRCs stimulate transcription possibly by recruiting
other transcription factors to the promoter. For exam-ple, SRCs interact with cAMP response element bind-
ing protein (CBP)/p300, a histone acetyltransferase
(HAT) that remodels chromatin structure at the pro-
moter (46, 47, 50). SRCs also possess intrinsic HAT
activity (51). CBP/p300 directly associates with nu-
clear receptors and, together with SRCs, synergisti-
cally stimulates transcription (52, 53). Thus, SRCs di-
rectly alter chromatin structure and recruit other
factors that modify histones, potentially providing
more accessible promoter templates on which the
transcriptional machinery assembles and initiates
transcription of target genes.
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A large multiprotein complex called DRIP (VDR-
interacting proteins) was identified as a coactivator for
VDR and other nuclear receptors (54, 55). Many com-
ponents of this complex were discovered separately
as thyroid receptor activating protein (TRAP) and the
mammalian Mediator complex (56, 57). The diversity
of transactivator interactions with the DRIP/TRAP/Mediator complex clearly suggests a more fundamen-
tal role for this complex in stimulus-activated tran-
scriptional processes. In VDR-mediated transcription,
DRIP205/TRAP220 acts as an anchoring subunit of
the complex by interacting directly with VDR/RXR het-
erodimers through one of two LXXLL motifs (58). Bio-
chemical depletion of DRIP in cell-free transcription
assays shows that DRIP is essential for VDR-activated
transcription in vitro (54). Because the DRIP complex
does not contain SRCs and is not associated with HAT
activity (58), it is likely that DRIP and SRCs potentiate
the transcriptional activation of VDR through distinct
mechanisms. Chromatin immunoprecipitations stud-
ies indicate that a coactivator exchange occurs in the
transcriptional complex on native nuclear receptor-
responsive promoters (5961). Specifically, SRCs ap-pear to enter the transcriptional complex first and dis-
sociate followed by binding of the DRIP multimeric
complex (60, 61). DRIP is also known to recruit the
RNA polymerase II holoenzyme to VDR upon ligand
binding (62). Although these data conflict, to some
extent, with previous studies that show simultaneous
association of SRCs and DRIP with activated nuclear
receptor complexes (59), they do suggest a temporal
model in which SRCs enter the complex first to re-
model the chromatin, followed by DRIP complex entry
and subsequent recruitment of RNA polymerase II(Fig. 2B).
In addition to DRIP and SRCs, several other proteins
that potentiate VDR-mediated transcription have been
described. One example is NCoA-62/ski-interacting
protein (SKIP), which is a coactivator unrelated to
DRIPs, SRCs, and other LXXLL-containing coactiva-
tors (63). It interacts with VDR and other nuclear re-
ceptors and augments their transcriptional activity.
Bx42, the Drosophila melanogasterortholog of NCoA-
62/SKIP, is also implicated in transcriptional pro-
cesses activated by the insect steroid ecdysone (64).
NCoA-62/SKIP was identified independently through
its interaction with ski, placing it as part of the TGF-
-dependent Smad transcriptional complex (65). It isalso implicated in a number of other transcriptional
pathways. NCoA-62/SKIP lacks LXXLL motifs and se-
lectively associates with the VDR-RXR heterodimer
through the LBD, but through a domain that is distinct
from the H3-H5/H12 interactions surface (66). NCoA-
62/SKIP binds VDR simultaneously with SRC-1 to
form a ternary complex that synergistically enhances
VDR-stimulated transcription (66), suggesting a poten-
tial interplay between different coactivator classes for
maximal activity. Recently, NCoA-62/SKIP was iden-
tified in subcomplexes of the spliceosome (67, 68).
This, combined with NCoA62/SKIPs ability to contact
varied transcription factors including the VDR, sug-
gests a potentially important role for this and other
coactivators in coupling nuclear receptor-mediated
transcription with mRNA splicing (69).
Although a variety of coactivator proteins have been
identified for VDR and other nuclear receptors, their
physiological significance and their discrete and/orredundant functions in different signal-activated tran-
scriptional systems remain unclear. Mice with individ-
ual targeted deletions of the three SRCs have been
developed (7072). These models show that the SRCsshare several similar functions, especially in the devel-
opment and maintenance of the female reproductive
system. However, the individual SRCs also serve dis-
tinct physiological roles. For example, ablation of TIF2/
glucocorticoid receptor-interacting protein 1 results in
testicular defects, whereas deletion of either SRC-1 or
receptor-associated coactivator 3 does not affect
male reproduction (71). As the effect of these deletions
on 1,25-(OH)2
D3
-mediated transcription has not been
reported in any of these three knockout models, the in
vivo relevance of SRCs in VDR-activated transcription
remains to be determined. Deletion of the receptor-
interacting subunit of the DRIP complex, DRIP205/
TRAP220, results in attenuated thyroid hormone-stim-
ulated transcription but does not affect retinoic acid
responses. Again, 1,25-(OH)2
D3
responses have not
been examined in this model, so it is unknown whether
DRIP is required for VDR-activated transcription in
vivo. Silencing of the Caenorhabditis elegans ortholog
of NCoA-62/SKIP by RNA interference results in early
embryonic lethality due to a potential general tran-
scription defect (73). Although delineation of the phys-
iological function of NCoA-62/SKIP awaits develop-ment of a mammalian knockout model, these data
suggest that NCoA-62/SKIP plays a fundamental role
in RNA polymerase II-mediated transcription. More
studies are needed to build an integrative vision of
how the entire ensemble of coactivator proteins asso-
ciates and stimulates the transcriptional activity of
VDR and other nuclear receptors. Initial forays into
deciphering this complex process have begun with the
application of chromatin immunoprecipitation assays
and in vivo imaging of fluorescently tagged nuclear
receptors and coactivators to assess the temporal
assembly of transcription factors and nuclear recep-
tors on native promoters (5961, 74, 75). These strat-
egies, combined with in vitro systems composed ofpurified components and chromatin-packaged tem-
plates, will be required for a more complete under-
standing of the molecular details of VDR-activated
transcription.
KNOCKOUT MODELS
Much of our understanding of the physiology of the
vitamin D endocrine system has stemmed from classic
dietary manipulations and from the analysis of inher-
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ited disorders in humans (Ref. 76 and reviewed in Ref.
77). The recent development of murine genetic models
in which key genes in this endocrine system have been
systematically eliminated highlights the essential role
of 1,25-(OH)2
D3
in maintaining mineral homeostasis as
well as reveals more subtle actions of this hormone in
other physiological processes.
VDR Knockout (VDRKO) Mice
Two groups independently created mouse strains with
targeted deletions in the VDR gene by disrupting either
exon 2 (78) or exon 3 (79). Not surprisingly, the VDRKO
mice displayed all of the features of the human disease
hereditary vitamin D-resistant rickets (HVDRR), a rare
genetic disorder caused by mutations in the VDR gene
(Ref. 76 and reviewed in Ref. 77). The VDRKO mice are
viable and develop normally until the weaning period.
However, shortly after weaning, VDR-null mice exhibit
alopecia and growth retardation accompanied by pro-
gressive hypocalcemia, hypophosphatemia, and com-
pensatory hyperparathyroidism. These metabolic im-
balances result in severe skeletal defects, including
decreased bone mineral density, thinned bone cortex,
and widened undermineralized growth plates. How-
ever, when VDRKO mice are fed a rescue diet rich in
calcium and phosphorus to normalize serum calcium
and PTH levels, the mice develop normally without
bone abnormalities (80). The skeletons of these mice
appear grossly, histologically, and biometrically nor-
mal (81). This indicates that the bone defect in VDR-
null mice is secondary to the malabsorption of calcium
in the intestine and is not due to the lack of a direct
effect of 1,25-(OH)2D3 on the bone. The impaired in-testinal absorption of calcium in the VDRKO mice is
linked to diminished intestinal expression of several
1,25-(OH)2
D3
-regulated genes putatively involved in
calcium transport, including calbindin D9K, calcium
transport protein-1, and epithelial calcium channel (82,
83). In addition to the intestinal defect in calcium ab-
sorption, mice that express a mutant VDR that lacks
the DBD have decreased renal reabsorption of calcium
(84). These studies reinforce the concept that both the
intestine and the kidney are essential VDR target or-
gans in maintaining calcium homeostasis.
The lack of a skeletal phenotype in the VDRKO mice
weaned onto the rescue diet is somewhat surprising in
light of the vast literature supporting direct, primaryroles of VDR in both osteoblast and osteoclast biology
(85). On the surface, the VDRKO studies indicate that
1,25-(OH)2
D3
is not essential for normal bone devel-
opment and for maintaining skeletal integrity beyond
its classic role in calcium and phosphate absorption in
the intestine. However, a standard caveat with gene
disruption is that the developing animal may acquire
adaptive mechanisms or utilize redundant compensa-
tory pathways to bypass the effects of the gene dele-
tion. Conditional knockout approaches that ablate
VDR in a temporally controlled or tissue-specific man-
ner may be more informative. Such strategies could be
designed to explore the role of VDR at stage-selective
checkpoints in skeletal maturation, at which the pos-
sibility of developing compensatory mechanisms has
been minimized. Finally, the skeletal phenotypes of
normocalcemic VDRKO mice may be more pro-
nounced at different life stages or under different
physiological stresses. Thus, it will be important todetermine whether normocalcemic VDRKO mice are
more susceptible to age-related or ovariectomy-
induced loss of bone mineral density and whether they
are compromised in their ability to repair skeletal
fractures.
In contrast to the skeletal phenotype, the mineral-
rich diet does not correct the alopecia (i.e. the absence
of functional hair follicles) observed in VDRKO mice
(80). Although the VDRKO keratinocytes proliferate
and differentiate normally, they fail to properly initiate
hair regrowth after depilation (86, 87). Due to the lack
of feedback regulation of the anabolic 1OHase en-
zyme and the catabolic 24OHase, the VDRKO animals
have abnormally high levels of 1,25-(OH)2
D3
. Thus,
one potential cause of alopecia in VDRKO animals is
1,25-(OH)2D3 toxicity. To address this possibility,
VDRKO mice were raised and bred for five generations
in a UV light-free environment and on a diet lacking
vitamin D derivatives (87). Despite having undetect-
able levels of 1,25-(OH)2
D3
, fifth-generation vitamin
D-deficient VDRKO mice still have alopecia. Thus,
1,25-(OH)2
D3
toxicity does not cause alopecia in
VDRKO mice. Because wild-type littermates of
VDRKO mice raised under the same vitamin D-defi-
cient conditions do not display alopecia, Demay and
colleagues (87) proposed that VDR may regulate hair
follicle cycling in a ligand-independent fashion. Furthersupport for this hypothesis comes from the observa-
tion that mice with a targeted deletion in 25(OH)D3
-
1-OHase (see below), the biosynthetic enzyme that
produces 1,25-(OH)2D3, do not display alopecia (88).
In vitro studies indicate that VDR associates with var-
ious transcription factors and induces select genes in
the absence of ligand (63, 8991). Alternatively, unli-ganded VDR may repress a subset of target genes in
a manner analogous to other nuclear receptors
through corepressor interactions (9294). Such VDR-RXR-repressed genes could be involved in negatively
regulating hair follicle cycling. Although these studies
introduce a novel and potentially significant concept in
VDR biology, identifying target genes and establishingmolecular mechanisms that govern the function of
unliganded VDR in keratinocytes are important goals
for future research.
The global tissue distribution of VDR suggests that
1,25-(OH)2
D3
plays important roles in physiological
processes beyond mineral homeostasis and keratino-
cyte function. For example, VDRKO mice have im-
paired reproductive function (78). Both male and fe-
male VDR-null mice exhibit diminished estradiol levels
and elevated gonadotropins, indicating a gonadal dys-
function in the secretion of sex hormones (95). Histo-
logical analysis of reproductive glands shows abnor-
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mal ovarian follicle development in the females and
dilated seminiferous tubules with diminished spermat-
ogenesis in the males. However, serum calcium nor-
malization with the rescue diet mostly corrects the
hormonal imbalances and histological abnormalities
(95) and completely restores fertility (95, 96), suggest-
ing that the gonadal dysfunction in VDRKO mice pri-marily results from hypocalcemia.
Mammary gland development is also emerging as a
biological process that is impacted by 1,25-(OH)2
D3
.
The mammary glands of VDRKO mice demonstrate a
hyperproliferative phenotype as evidenced by in-
creased numbers of terminal end buds and enhanced
ductal branching compared with wild-type littermates
(97). VDRKO mammary glands also show accelerated
ductal development and increased proliferation in re-
sponse to exogenously administered estrogen and
progesterone (97). Although dietary calcium supple-
mentation normalizes estrogen levels in VDRKO mice,
the abnormal mammary phenotype is retained. These
data indicate a significant developmental role for VDRin the mammary gland potentially in restricting ductal
growth. Combined with the observations that 1,25-
(OH)2
D3
and its analogs inhibit the growth and induce
the differentiation of breast cancer cell lines (98, 99),
the mammary phenotype in the VDRKO model indi-
cates that 1,25-(OH)2
D3
and its derivatives may be
useful therapies for breast cancer.
An important immunomodulatory function for 1,25-
(OH)2D3 and VDR has been indicated by decades of in
vitro studies. For example, 1,25-(OH)2D3 potently in-
hibits proliferation and drives the differentiation of leu-
kemic cells along the monocyte/macrophage lineage
(100). However, VDRKO mice lack a striking immune
system phenotype. Although OKelly et al. (101) ob-served that VDRKO mice have abnormal T cell re-
sponses due to diminished cytokine production by
macrophages, the calcium-rich rescue diet was not
tested in this study. Thus, it is not known whether
these abnormalities can be attributed to a lack VDR, to
1,25-(OH)2
D3
toxicity, or to hypocalcemia. Mathieu
et al. (102) found that VDRKO mice had a defect in
calcium-dependent T cell proliferation resulting in pro-
tection from experimentally induced autoimmune dia-
betes and that these abnormalities could be corrected
by restoring serum calcium to normal levels. The nor-
mal development of the immune system in the VDRKO
mice suggests that VDR is not essential for immune
function or that other compensatory pathways exist.Several clinical studies have proposed that 1,25-
(OH)2
D3
may also be beneficial to the cardiovascular
system by decreasing blood pressure (103, 104). Con-
sistent with these observations in humans, VDRKO
mice have increased renin expression, resulting in
higher levels of angiotensin II, increased water intake,
electrolyte disturbances, elevated blood pressure, and
cardiac hypertrophy (105). Furthermore, high levels of
renin and angiotensin II persist despite normalization
of mineral ion levels with the rescue diet. This re-
sponse appears to be at the transcriptional level, as
1,25-(OH)2D3 suppresses renin promoter activity. This
study suggests that VDR negatively regulates the ex-
pression of renin, allowing for decreased angiotensin
production and lower blood pressure. The relevance of
this study to human hypertension is not entirely clear
because there are no reports of hypertensive HVDRR
patients. Regardless, these are provocative observa-
tions in the VDRKO model that may stimulate a moreextensive examination of 1,25-(OH)
2D3
and its syn-
thetic analogs as potential therapies for some forms of
hypertension.
VDR/RXR Double Knockout
VDR heterodimerizes with RXR to modulate transcrip-
tion of target genes in response to 1,25-(OH)2D3. To
examine the effect of abolishing both of the active
partners in 1,25-(OH)2D3 signaling, Yagishita and col-
leagues (106) crossed VDRKO mice with RXR-null
mice to generate VDR/RXR-double-knockout mice.
The phenotype of the double-knockout mice is nearly
identical with that of the single-VDR-knockout mouse,including growth retardation, hypocalcemia, hy-
pophosphatemia, hyperparathyroidism, rickets, and
alopecia. Upon closer inspection, however, a unique
abnormality was noted in the growth plates of long
bones of VDR/RXR-knockout mice that is not present
in either of the single-knockout mice. Specifically,
VDR/RXR-null mice have a defect in the development
of hypertrophic chondrocytes, the most mature type of
chondrocyte in the growth plate. Normalization of se-
rum calcium and phosphorus rescues all of the skel-
etal anomalies except for the disordered growth
plates. Because this chondrocyte defect is not present
in either of the single-knockout strains, the authors
suggested that a functionally redundant VDR-relatedreceptor exists that selectively heterodimerizes with
RXR. Such a receptor would likely share a consider-
able amount of sequence similarity with the classical
VDR because it must 1) bind to and be transcriptionally
activated by 1,25-(OH)2
D3
or its metabolites; 2) het-
erodimerize with RXR; and 3) recognize and stimulate
transcription of the same target genes as classical
VDR in chondrocytes. Although the public genome
databases do not indicate that highly related se-
quences exist, potential candidates might include the
most closely related nuclear receptors, such as farne-
soid X receptor, steroid xenobiotic receptor/pregnane
X receptor, and liver X receptor (107). Alternatively, any
nuclear receptor unrelated to VDR that selectively het-
erodimerizes with RXRand binds 1,25-(OH)2
D3
or its
metabolites might fulfill this role.
25(OH)D3-1-Hydroxylase Knockout
Arguably, one of the most significant advances in the
vitamin D field over the past five years has been the
identification and cloning of the renal 1OHase,
the enzyme responsible for the regulated synthesis of
the active hormone (108, 109). To study the effects
of the absence of 1,25-(OH)2
D3
, two groups indepen-
dently disrupted the 1OHase gene in mice (88, 110).
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The phenotype of the 1OHase-null mice is strikingly
similar to that of the VDR-knockout mouse, including
hypocalcemia, hyperparathyroidism, growth retarda-
tion, and osteomalacia, consistent with rickets (88,
110). The 1OHase-mutant female mice are anovula-
tory and, therefore, infertile (88). Another key differ-
ence between VDRKO mice and the 1OHase-ablatedmice is that the 1OHase-knockout animals do not
display alopecia (88, 110). The role of hypocalcemia/
hypophosphatemia in any aspect of the abnormal
phenotype of the 1OHase-ablated mice has yet to be
addressed using the rescue diet. Importantly, the de-
velopment of knockout models of both the receptor
and ligand of 1,25-(OH)2
D3
will provide powerful tools
to delineate the overlapping and distinct roles of VDR
and its ligand in diverse processes such as mineral
homeostasis, hair follicle cycling, mammary gland de-
velopment, and blood pressure regulation.
24OHase Knockout
24OHase metabolizes both the bioactive 1,25-(OH)2
D3
and its precursor, 25(OH)D3
. 24OHase gene transcrip-
tion is positively regulated by 1,25-(OH)2
D3
, thereby
completing a negative feedback loop to prevent ex-
cessive hormone synthesis. One of the major products
of 24OHase, namely 24,25(OH)2
D3
, is considered to
be an inactive metabolite, an initial product destined
for further degradation and eventual excretion as cal-
citroic acid (5). However, there is substantial evidence
that 24,25(OH)2
D3
has biological activity of its own,
most strikingly in the function of chondrocytes, or
cartilage-forming cells (111113). To determine theconsequences of abolishing this enzyme and, there-
fore, its 24-hydroxylated products, a knockout modelof 24OHase was created (114). This mutation results in
reduced embryonic viability and aberrant intramem-
branous ossification. The obvious possibility is that the
diminished levels of 24,25(OH)2
D3
causes these ab-
normalities. However, the phenotype is not rescued in
24OHase-knockout progeny by feeding pregnant mice
exogenous 24,25(OH)2
D3
. Alternatively, the phenotype
could be caused by the abnormally high levels of
1,25-(OH)2
D3
resulting from the deletion of this cata-
bolic enzyme. Indeed, crossing the 24OHase-null mice
with VDR-null mice completely rescues the decreased
embryonic viability and ossification defects, thus sup-
porting the concept that 1,25-(OH)2D3 toxicity leads to
the abnormal phenotype in the 24OHase-knockoutmice. Moreover, this model also suggests that 24-
hydroxylated metabolites are not required for normal
intramembranous ossification (114).
BEYOND 1,25-(OH)2D3 AND VDR: NOVEL
LIGANDS AND ALTERNATIVE RECEPTORS
Novel Vitamin D Ligands
The therapeutic potential of 1,25-(OH)2
D3
continues to
expand. In addition to treating disorders of mineral
metabolism and diseases of the skeleton, such as
rickets, osteoporosis, and renal osteodystrophy, 1,25-
(OH)2
D3
has significant therapeutic potential for pa-
thologies such as cancer, autoimmune syndromes,
and psoriasis. However, 1,25-(OH)2
D3
itself has a nar-
row therapeutic window limited by the development of
toxic hypercalcemia. The increase in calcium isachieved both by enhanced intestinal absorption and
by liberation of calcium from the skeleton, eventually
leading to decreased bone mass at higher doses. This,
of course, counteracts the beneficial effects of 1,25-
(OH)2D3 for the treatment of bone diseases. Conse-
quently, more than 800 synthetic 1,25-(OH)2
D3
ana-
logs have been developed in attempt to preserve the
favorable activities of 1,25-(OH)2
D3
while avoiding the
side effects (115).
Calcipotriol (MC 903) is an analog that has been
used to treat psoriasis for nearly 15 yr, and it is cur-
rently considered a first-line therapy for the disease
(116). Calcipotriol improves psoriasis by inhibiting pro-
liferation and promoting differentiation of keratino-
cytes, but it does not cause hypercalcemia or de-
creased bone mass. This selectivity can be attributed
to calcipotriols low affinity for vitamin D binding pro-tein, the major vitamin D transport protein in the cir-
culation (117), and the fact that it is applied topically,
thus restricting its actions to the skin. 1,25-(OH)2
D3
analogs are also valuable in treating renal osteodys-
trophy, a devastating consequence of chronic renal
failure. Kidney disease, due to a variety of causes,
often leads to reduced 1,25-(OH)2
D3
production and
impaired phosphate excretion resulting in abnormally
high PTH levels. The decreased calcitriol levels com-
bined with secondary hyperparathyroidism, in turn,result in increased bone turnover (118). Treatment with
1,25-(OH)2
D3
is effective in suppressing PTH levels
and improving the initial skeletal abnormalities. How-
ever, due to the narrow therapeutic window, 1,25-
(OH)2
D3
often causes hypercalcemia and additional
bone disease from inappropriately low bone turnover.
Two 1,25-(OH)2D3 analogs are currently approved for
treatment of secondary hyperparathyroidism in the
United States. 19-Nor-D2
(paricalcitol; Ref. 119) and
1(OH)D2
(doxercalciferol; Ref. 120) are as effective as
1,25-(OH)2D3 in reducing PTH levels but do not result
in significant hypercalcemia. Although the mecha-
nisms of the selectivity of these two analogs remain
unclear, they represent significant improvements intreatment regimens for renal osteodystrophy.
Although neither vitamin D3
nor 1,25-(OH)2
D3
ana-
logs are currently Food and Drug Administration
(FDA)-approved for treating osteoporosis in the United
States, these compounds are widely used to prevent
and treat osteoporosis throughout the world (121,
122). Furthermore, vitamin D3
is currently recom-
mended as a dietary supplement in addition to any
pharmacological treatment for all patients with de-
creased bone mass or osteoporosis (123). Still, the
higher doses of 1,25-(OH)2
D3
required for maximal
improvement in bone density cause significant hyper-
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calcemia. Peleg and colleagues (124) tested 1,25-
(OH)2
D3
analogs for their ability to improve bone min-
eral density in the ovariectomized rat model of
osteoporosis. One novel analog, Ro-26-9228, protects
against osteopenia, but it does not increase serum
calcium except at very high doses. These observa-
tions are potentially explained by the tissue-selectiveaction of Ro-26-9228, which stimulates osteocalcin
and osteopontin expression in osteoblasts but does
not affect calbindin D9K
or plasma membrane calcium
pump expression in the intestine (124). Shevde et al.
(125) found that another analog, 2-methylene-19-nor-
(20S)-1,25(OH)2
D3
(2MD), potently stimulates bone
formation in vitro and markedly improves bone mass in
ovariectomized rats without dramatically increasing
serum calcium. Such studies support the concept that
more selective 1,25-(OH)2
D3
analogs will be useful
therapies for osteoporosis by enhancing bone mineral
density without causing toxic hypercalcemia.
In addition to these and numerous other designer
analogs, a recent study suggests the possibility that
natural compounds other than 1,25-(OH)2
D3
may
serve as tissue-selective activators of VDR-mediated
responses. An unexpected ligand for VDR was discov-
ered from studies with bile acid compounds (126).
Metabolic lipophilic molecules such as bile acids ac-
tivate many of the orphan nuclear receptors, including
farnesoid X receptor and steroid xenobiotic receptor/
pregnane X receptor (127129). Recently, Makishimaet al. (126) screened classical nuclear receptors to
identify those that were activated by the bile acid
lithocholic acid (LCA). Surprisingly, they found that
LCA and its metabolites directly bind and activate
VDR. However, this activation requires micromolarconcentrations of LCA, whereas VDR is activated by
nanomolar amounts of 1,25-(OH)2
D3
. Nonetheless,
LCA- or 1,25-(OH)2
D3
-liganded VDR also stimulates
the expression of endogenous CYP3A, the P450 en-
zyme responsible for degradation of LCA in the liver
and the intestine. LCA is implicated as a toxin that
promotes colorectal carcinogenesis (130, 131),
whereas 1,25-(OH)2
D3
is protective against colon can-
cer (132). Thus, induction of CYP3A by 1,25-(OH)2
D3
and by LCA itself may represent a detoxification path-
way for LCA, as well as explain the potential preven-
tative effects of 1,25-(OH)2
D3
in colon cancer. Al-
though this study awaits further in vivo confirmation, it
clearly raises the possibility that VDR may be activatedby other naturally occurring ligands.
VDR Isoforms
Many nuclear receptors, such as RAR, RXR, and thy-
roid receptor, have multiple isoforms that are encoded
by separate genes (133). Unlike these nuclear recep-
tors, only one human VDR genetic locus has been
identified (134), and the genomic database does not
indicate additional highly related sequences. Although
the cDNA encoding the human VDR was cloned nearly
15 yr ago (8), only recently have several significant
variations in the VDR gene, transcript, and protein
sequences been discovered. At least 14 distinct tran-
scripts of human VDR have been identified that differ
in their 5 ends (135). These transcripts arise from
alternative mRNA splicing and differential promoter
usage. Most of these variant transcripts utilize the
same initiator codon, producing a VDR that is 427amino acids in length. However, two transcripts have
upstream in-frame methionines that potentially gener-
ate N-terminal extensions in VDR of 50 or 23 amino
acids (135). Low levels of endogenous VDRB1 protein,
the 50-amino-acid-extended variant, have been de-
tected in osteoblast, colon cancer, and kidney cell
lines (136). Interestingly, VDRB1 has reduced tran-
scriptional activity compared with classical VDR.
Whether the levels of expression of these isoforms are
substantial and whether these isoforms result in al-
tered biological activity in vivo remains unresolved.
Multiple polymorphic variations also exist in VDR in
the human population (137). The vast majority of these
polymorphisms do not result in a structural alteration
in the VDR protein, with the exception of the Fok I
variant (138). The Fok I polymorphism is located at the
original initiator ATG, which is part of a Fok I endonu-
clease site. In some humans, there is an ATG 224 ACG
transition at the 1 position, eliminating the transla-
tional initiation site and Fok I recognition sequence.
This transition results in the use of an in-frame methi-
onine as the initiator codon at the 4 position. Thus,
either a 427 (Met-1) or a 424 (Met-4) amino acid pro-
tein is expressed. Numerous epidemiological studies
suggest an association between the shorter form of
VDR and increased bone mineral density in humans
(139143). The molecular mechanism of this associa-tion remains unclear, but there is suggestive evidencethat the Met-4 VDR displays enhanced transcriptional
activity due to increased interaction with basal tran-
scription factor II B (12). Although these findings re-
main controversial (144), they raise the possibility that
the N terminus possesses some type of structure that
influences transcriptional activity. This, combined with
the observations that other N-terminal extensions of
VDR may have reduced transcriptional activity, sug-
gests that there may be inhibitory domains at the
extreme N terminus of VDR that decrease its transac-
tivation potential.
A Membrane Receptor? Rapid, NongenomicEffects of Vitamin D and Its Metabolites
According to the classical paradigm of nuclear recep-
tor action, ligand-activated nuclear receptors recruit
the basal transcriptional machinery and other activator
complexes to the promoters of target genes to induce
transcription. Because these responses require tran-
scription and translation of target genes, they are typ-
ically delayed by at least 30 min. However, more rapid
(within seconds to minutes) effects in response to
steroid hormones are also apparent. The rapid nature
of these effects and their relative insensitivity to tran-
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scriptional and translational inhibitors, such as actino-
mycin D and cycloheximide, precludes the possibility
that the traditional genomic model is operating. Re-cent attention to these rapid, nongenomic hormoneeffects has spawned renewed interest in this long-
standing area of membrane-initiated signaling in the
steroid hormone field (145). Over two decades ago,1,25-(OH)
2D3 was shown to evoke transcellular move-
ment of calcium across chick enterocytes within sev-
eral minutes (146, 147). This phenomenon is theorized
to be adaptively beneficial for a hypocalcemic animal
in that rapid absorption of calcium occurs without a
delayed response involving transcription and transla-
tion of calcium-binding proteins or calcium transport-
ers (147). In addition to the enterocyte, the osteoblast
is a target for 1,25-(OH)2
D3
-induced rapid calcium
mobilization from internal stores, a process that in-
volves a membrane-initiated signaling cascade includ-
ing phospholipase C activation and inositol triphos-
phate formation (148). This process also occurs in
skeletal muscle cells, in which 1,25-(OH)2
D3
induces
calcium release from the sarcoplasmic reticulum (149),
potentially through MAPK activation (150). These are
just a few of the many examples of in vitro systems in
which these nongenomic actions of 1,25-(OH)2
D3
have been studied.
Although the rapid effects of 1,25-(OH)2
D3
and
numerous other steroids are well documented, the
field is hindered by the inability to identify the puta-
tive membrane receptors that trigger these non-
genomic effects. Although suggestive biochemical
and immunological data (151) indicate that the
membrane VDR is a distinct gene product, a true
protein representing this receptor remains elusive. Apromising new approach in the nongenomic field is
the use of 1,25-(OH)2
D3
analogs to discriminate be-
tween receptors that mediate membrane-initiated
events and those that mediate the classical nuclear
effects of 1,25-(OH)2
D3
. Song et al. (152) showed
that 6-s-cis-locked analogs of 1,25-(OH)2
D3
stimu-
late rapid phosphorylation of MAPK in leukemic
cells, yet these analogs bind poorly to VDR and are
weak activators of VDR-mediated transcription.
These studies also support the possibility of two
separate gene products involved in either rapid,
nongenomic signaling or in classical transcriptional
activation by 1,25-(OH)2
D3
. In fact, two preliminary
reports indicate that annexin II, a membrane-asso-ciated calcium-binding protein, may bind 1,25-
(OH)2
D3
and function as its membrane receptor
(153, 154). In contrast to these studies, analysis of
mice carrying a mutated VDR lacking a DBD implies
that the classical nuclear VDR mediates both
genomic and nongenomic responses (84). Osteo-
blast cultures derived from VDR-mutant mice are
unable to initiate a rapid calcium flux in response to
1,25-(OH)2
D3
, suggesting that some nongenomic
responses require a functional nuclear VDR. Al-
though a full array of other nongenomic responses
needs to be tested, the VDRKO strains will provide
important tools to decipher the molecular require-
ments of classical VDR that mediate the genomic
and nongenomic effects of 1,25-(OH)2
D3
in vivo.
SUMMARY: FRONTIERS IN VITAMIN D
The molecular, cellular, structural, and genetic studies
of the past decade have impacted our understanding
of the vitamin D endocrine system in a number of
significant ways. First and foremost, the genetic mod-
els solidify the fundamental roles that both 1,25-
(OH)2
D3
and VDR play in ensuring that an organism
obtains sufficient calcium and phosphate from the
environment to sustain life and normal development.
Lack of either functional VDR or active 1,25-(OH)2
D3
leads to profound, life-threatening hypocalcemia and
undermineralized skeletal tissue. Previous classical
nutritional manipulations and identification of mutated
VDR as the causative defect in HVDRR have estab-
lished this central function of 1,25-(OH)2
D3
long before
the creation of genetic mouse models. However, the
development and analysis of such models are central
in formulating a detailed physiological picture of 1,25-
(OH)2
D3
signaling and in reinforcing a direct link be-
tween the various genes within the endocrine system
and mineral ion dysregulation.
On the other hand, the knockout studies provide
somewhat of a surprise and, to a certain extent, a bit
of a disappointment for those interested in 1,25-
(OH)2
D3
and bone biology. In particular, specific cells
of the osteoblast and osteoclast lineage have gar-
nered considerable research attention over the pastseveral decades as important direct targets of 1,25-
(OH)2
D3
in preserving skeletal integrity. However, the
striking skeletal defects observed in the VDRKO are
corrected by simply providing the animals with sup-
plemental dietary calcium. Does this mean that 1,25-
(OH)2
D3
does not act as a direct bone-a-fide hor-mone in the skeleton? Clearly, more cellular and
genetic approaches are needed to fully answer this
question and to test whether VDR and 1,25-(OH)2
D3
play more limited or specialized roles in the developing
or aging skeleton. Although the jury is still out on the
bone, striking new biologies are emerging from the
VDRKO studies, indicating potential functions for
1,25-(OH)2D3 in diverse processes such as hair folliclecycling, blood pressure regulation, and mammary
gland development that are independent of mineral ion
homeostasis. Moreover, studies on alopecia in
VDRKO mice raise the exciting possibility that VDR
acts in a ligand-independent fashion and may stimu-
late further exploration into the molecular and cellular
functions of unliganded VDR.
Beyond the physiological information gleaned from
the genetic mouse models, recent progress in other
areas of the vitamin D field is revealing additional
molecular details of the endocrine system. Description
of the crystal structure of VDR has supplied an atomic
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view of the VDR protein, highlighting the expansive
binding pocket associated with both natural and syn-
thetic ligands. Further refinement of this structure
complexed with coactivators undoubtedly will allow
for the future rational design of selective VDR-targeted
drugs. Some of the additional molecular requirements
for the transcriptional activity of 1,25-(OH)2D3-ligan-ded VDR have been elucidated and are beginning to
be assembled into an integrated model of coactivator
cooperativity. Furthermore, it is becoming increasingly
clear that some actions of 1,25-(OH)2
D3
cannot be
explained by the traditional model of 1,25-(OH)2D3-
bound VDR acting solely as a transcription factor in the
nucleus. Novel VDR ligands, including synthetic ana-
logs and natural compounds such as bile acids, are
broadening our understanding of the therapeutic po-
tential and physiological intricacies of vitamin D. Like-
wise, alternative receptors, both related to and distinct
from the classical nuclear VDR, are emerging as sig-
nificant participants mediating the biological effects of
vitamin D compounds. The four areas of progress
covered in this minireview have filled in many gaps in
our knowledge of vitamin D, but they also have raised
a multitude of new questions that await answering with
the new molecular, pharmacological, and genetic tools
developed in recent years.
Acknowledgments
We apologize to many colleagues whose excellent primarypublications may not have been cited in this minireview dueto space limitations.
Received November 1, 2002. Accepted March 5, 2003.Address all correspondence and requests for reprints to:
Paul N. MacDonald, Ph.D., Department of Pharmacology,Case Western Reserve University, 10900 Euclid Avenue,Cleveland, Ohio 44106. E-mail: [email protected].
This work was supported by NIH Grants R01-DK-50348and R01-DK-53980 (to P.N.M.), by an awardfrom theMedicalScientist Training Program NIH Grant T32-GM-007250 (toA.L.M.S.), and by a Pharmaceutical Manufacturers Associa-tion Foundation Pre-Doctoral Fellowship (to A.L.M.S.).
REFERENCES
1. Brown AJ, Dusso A, Slatopolsky E 1999 Vitamin D. Am JPhysiol 277:F157F1752. van Leeuwen JP, van Driel M, van den Bemd GJ, Pols
HA 2001 Vitamin D control of osteoblast function andbone extracellular matrix mineralization. Crit Rev Eu-karyot Gene Expr 11:199226
3. Owen TA, Aronow MS, Barone LM, Bettencourt B, SteinGS, Lian JB 1991 Pleiotropic effects of vitamin D onosteoblast gene expression are related to the prolifera-tive and differentiated state of the bone cell phenotype:dependency upon basal levels of gene expression, du-ration of exposure, and bone matrix competency innormal rat osteoblast cultures. Endocrinology 128:14961504
4. Bar-Shavit Z, Teitelbaum SL, Reitsma P, Hall A, PeggLE, Trial J, Kahn AJ 1983 Induction of monocytic differ-
entiation and bone resorption by 1,25-dihydroxyvitaminD3. Proc Natl Acad Sci USA 80:59075911
5. Jones G, Strugnell SA, DeLuca HF 1998 Current under-standing of the molecular actions of vitamin D. PhysiolRev 78:11931231
6. Horst RL, Reinhardt TA 1997 Vitamin D metabolism. In:Feldman D, Glorieux, FH, Pike JW, eds. Vitamin D. San
Diego: Academic Press; 13327. McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR,
OMalley BW 1987 Molecular cloning of complementaryDNA encoding the avian receptor for vitamin D. Science235:12141217
8. Baker AR, McDonnell DP, Hughes M, Crisp TM, Man-gelsdorf DJ, Haussler MR, Pike JW, Shine J, OMalleyBW 1988 Cloning and expression of full-length cDNAencoding human vitamin D receptor. Proc Natl Acad SciUSA 85:32943298
9. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC,Thompson PD, Selznick SH, Dominguez CE, JurutkaPW 1998 The nuclear vitamin D receptor: biological andmolecular regulatory properties revealed. J Bone MinerRes 13:325349
10. Mangelsdorf DJ, Evans RM 1995 TheRXR heterodimers
and orphan receptors. Cell 83:84185011. McKenna NJ, Lanz RB, OMalley BW 1999 Nuclearreceptor coregulators: cellular and molecular biology.Endocr Rev 20:321344
12. Jurutka PW, Remus LS, Whitfield GK, Thompson PD,Hsieh J-C, Zitzer H, Tavakkoli P, Galligan MA, DangHTL, Haussler CA, Haussler MR 2000 The polymorphicN terminus in human vitamin D receptor isoforms influ-ences transcriptional activity by modulating interactionwith transcription factor IIB. Mol Endocrinol 14:401420
13. McDonnell DP, Scott RA, Kerner SA, OMalley BW, PikeJW 1989 Functional domains of the human vitamin D3receptor regulate osteocalcin gene expression. Mol En-docrinol 3:635644
14. Nakajima S, Hsieh JC, MacDonald PN, Galligan MA,Haussler CA, Whitfield GK, Haussler MR 1994 The C-terminal region of the vitamin D receptor is essential toform a complex with a receptor auxiliary factor requiredfor high affinity binding to the vitamin D-responsiveelement. Mol Endocrinol 8:159172
15. Masuyama H, Brownfield CM, St-Arnaud R, MacDonaldPN 1997 Evidence for ligand-dependent intramolecularfolding of the AF-2 domain in vitamin D receptor-acti-vated transcription and coactivator interaction. Mol En-docrinol 11:15071517
16. Danielian PS, White R, Lees JA, Parker MG 1992 Iden-tification of a conserved region required for hormonedependent transcriptional activation by steroid hor-mone receptors. EMBO J 11:10251033
17. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P,Schutz G, Umesono K, Blumberg B, Kastner P, Mark M,Chambon P, Evans RM 1995 The nuclear receptorsuperfamily: the second decade. Cell 83:835839
18. Schwabe JW, Chapman L, Finch JT, Rhodes D 1993The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discrim-inatebetween their response elements. Cell 75:567578
19. Wagner RL, Apriletti JW, McGrath ME, West BL, BaxterJD, Fletterick RJ 1995 A structural role for hormone inthe thyroid hormone receptor. Nature 378:690697
20. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P,Gronemeyer H, Moras D 1995 Crystal structure of theRAR- ligand-binding domain bound to all-trans reti-noic acid. Nature 378:681689
21. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Mo-ras D 1995 Crystal structure of the ligand-binding do-main of the human nuclear receptor RXR-. Nature375:377382
Minireview Sutton and MacDonald Mol Endocrinol, May 2003, 17(5):777791 787
by on November 16, 2006mend.endojournals.orgDownloaded from
http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/ -
8/7/2019 D Vitamin D- More Than a Bone-a-Fide Hormone
12/15
22. WeathermanRV, Fletterick RJ,ScanlanTS1999 Nuclear-receptor ligands and ligand-binding domains. Annu RevBiochem 68:559581
23. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D2000 The crystal structure of the nuclear receptor forvitamin D bound to its natural ligand. Mol Cell5:173179
24. Rochel N, Tocchini-Valentini G, Egea PF, Juntunen K,Garnier JM, Vihko P, Moras D 2001 Functional andstructural characterization of the insertion region in theligand binding domain of the vitamin D nuclear receptor.Eur J Biochem 268:971979
25. Peleg S, Sastry M, Collins ED, Bishop JE, Norman AW1995 Distinct conformational changes induced by 20-epi analogues of 1,25-dihydroxyvitamin D3 are asso-ciated with enhanced activation of the vitamin D recep-tor. J Biol Chem 270:1055110558
26. Carlberg C, Quack M, Herdick M, Bury Y, Polly P, ToellA 2001 Central role of VDR conformations for under-standing selective actions of vitamin D(3) analogues.Steroids 66:213221
27. Yang W, Freedman LP 1999 20-Epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers of DRIPcoactivator complex binding to the vitamin D3 receptor.J Biol Chem 274:1683816845
28. Norman AW, Manchand PS, Uskokovic MR, OkamuraWH, Takeuchi JA, Bishop JE, Hisatake JI, Koeffler HP,Peleg S 2000 Characterization of a novel analogue of1,25(OH)
2-vitamin D
3with two side chains: interaction
with its nuclear receptor and cellular actions. J MedChem 43:27192730
29. Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A,Moras D 2001 Crystal structures of the vitamin D re-ceptor complexed to superagonist 20-epi ligands. ProcNatl Acad Sci USA 98:54915496
30. Tasset D, Tora L, Fromental C, Scheer E, Chambon P1990 Distinct classes of transcriptional activating do-mains function by different mechanisms. Cell 62:11771187
31. Cavailles V, Dauvois S, Danielian PS, Parker MG 1994
Interaction of proteins with transcriptionally active es-trogen receptors. Proc Natl Acad Sci USA 91:1000910013
32. Halachmi S, Marden E, Martin G, MacKay H, Abbon-danza C, Brown M 1994 Estrogen receptor-associatedproteins: possible mediators of hormone-induced tran-scription. Science 264:14551458
33. Onate S, Tsai, SY, Tsai, M-J, OMalley, BW 1995 Se-quence and characterization of a coactivator for thesteroid hormone receptor superfamily. Science 270:13541357
34. Leid M 1994 Ligand-induced alteration of the proteasesensitivity of retinoid X receptor . J Biol Chem 269:1417514181
35. Feng W, Ribeiro RCnJ, Wagner RL, Nguyen H, AprilettiJW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998Hormone-dependent coactivator binding to a hydro-phobic cleft on nuclear receptors. Science 280:17471749
36. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH,Kurokawa R, Rosenfeld MG, Willson TM, Glass CK,Milburn MV 1998 Ligand binding and co-activatorassembly of the peroxisome proliferator-activatedreceptor-. Nature 395:137143
37. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ,Agard DA, Greene GL 1998 The structural basis ofestrogen receptor/coactivator recognition and the an-tagonism of this interaction by tamoxifen. Cell 95:927937
38. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 Asignature motif in transcriptional co-activators mediatesbinding to nuclear receptors. Nature 387:733736
39. McKenna NJ, OMalley BW 2002 Combinatorial controlof gene expression by nuclear receptors and coregula-tors. Cell 108:465474
40. McKenna NJ, OMalley BW 2002 Minireview: nuclearreceptor coactivatorsan update. Endocrinology 143:24612465
41. Rosenfeld MG, Glass CK 2001 Coregulator codes of
transcriptional regulation by nuclear receptors. J BiolChem 276:3686536868
42. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR1996 GRIP1, a novel mouse protein that serves as atranscriptional coactivator in yeast for the hormonebinding domains of steroid receptors. Proc Natl AcadSci USA 93:49484952
43. Voegel JJ, Heine MJ, Zechel C, Chambon P, Grone-meyer H 1996 TIF2, a 160 kDa transcriptional mediatorfor the ligand-dependent activation function AF-2 ofnuclear receptors. EMBO J 15:36673675
44. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclearreceptor-associated coactivator that is related toSRC-1 andTIF2. Proc Natl Acad Sci USA94:84798484
45. Anzick SL, Kononen J, Walker RL, Azorsa DO, TannerMM, Guan XY, Sauter G, Kallioniemi OP, Trent JM,Meltzer PS 1997 AIB1, a steroid receptor coactivatoramplified in breast and ovarian cancer. Science 277:965968
46. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, NagyL, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclearreceptor coactivator ACTR is a novel histone acetyl-transferase and forms a multimeric activation complexwith P/CAF and CBP/p300. Cell 90:569580
47. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S,Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-recep-tor function. Nature 387:677684
48. Takeshita A, Cardona GR, Koibuchi N, Suen CS, ChinWW 1997 TRAM-1, a novel 160-kDa thyroid hormonereceptor activator molecule, exhibits distinct propertiesfrom steroid receptor coactivator-1. J Biol Chem 272:2762927634
49. Kraichely DM, Collins 3rd JJ, DeLisle RK, MacDonaldPN 1999 The autonomous transactivation domain inhelix H3 of the vitamin D receptor is required for trans-activation and coactivator interaction. J Biol Chem 274:1435214358
50. Ikeda M, Kawaguchi A, Takeshita A, Chin WW, Endo T,Onaya T 1999 CBP-dependent and independent en-hancing activity of steroid receptor coactivator-1 in thy-roid hormone receptor-mediated transactivation. MolCell Endocrinol 147:103112
51. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J,Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ,OMalley BW 1997 Steroid receptor coactivator-1 is ahistone acetyltransferase. Nature 389:194198
52. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T,Schulman IG, Juguilon H, Montminy M, Evans RM 1996Role of CBP/P300 in nuclear receptor signalling. Nature383:99103
53. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, GlossB, Lin SC, Heyman RA, Rose DW, Glass CK, RosenfeldMG 1996 A CBP integrator complex mediates transcrip-tional activation and AP-1 inhibition by nuclear recep-tors. Cell 85:403414
54. Rachez C, Suldan Z, Ward J, Chang CP, Burakov D,Erdjument-Bromage H, Tempst P, Freedman LP 1998 Anovel protein complex that interacts with the vitamin D3receptor in a ligand-dependent manner and enhancesVDR transactivation in a cell-free system. Genes Dev12:17871800
55. Rachez C, Lemon BD, Suldan Z, Bromleigh V, GambleM, Naar AM, Erdjument-Bromage H, Tempst P, Freed-man LP 1999 Ligand-dependent transcription activation
788 Mol Endocrinol, May 2003, 17(5):777791 Minireview Sutton and MacDonald
by on November 16, 2006mend.endojournals.orgDownloaded from
http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/ -
8/7/2019 D Vitamin D- More Than a Bone-a-Fide Hormone
13/15
by nuclear receptors requires the DRIP complex. Nature398:824828
56. Fondell JD, Ge H, Roeder RG 1996 Ligand induction ofa transcriptionally active thyroid hormone receptor co-activator complex. Proc Natl Acad Sci USA 93:83298333
57. Jiang YW, Veschambre P, Erdjument-Bromage H,
Tempst P, Conaway JW, Conaway RC, Kornberg RD1998 Mammalian mediator of transcriptional regulationand its possible role as an end-point of signal transduc-tion pathways. Proc Natl Acad Sci USA 95:85388543
58. Rachez C, Gamble M, Chang CP, Atkins GB, Lazar MA,Freedman LP 2000 The DRIP complex and SRC-1/p160coactivators share similar nuclear receptor binding de-terminants but constitute functionally distinct com-plexes. Mol Cell Biol 20:27182726
59. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000Cofactor dynamics and sufficiency in estrogen recep-tor-regulated transcription. Cell 103:843852
60. Burakov D, Crofts LA, Chang CP, Freedman LP 2002Reciprocal recruitment of DRIP/mediator and p160 co-activator complexes in vivo by estrogen receptor. J BiolChem 277:1435914362
61. Sharma D, Fondell JD 2002 Ordered recruitment of
histone acetyltransferases and the TRAP/Mediatorcomplex to thyroid hormone-responsive promoters invivo. Proc Natl Acad Sci USA 99:79347939
62. Chiba N, Suldan Z, Freedman LP, Parvin JD 2000 Bind-ing of liganded vitamin D receptor to the vitamin Dreceptor interacting protein coactivator complex in-duces interaction with RNA polymerase II holoenzyme.J Biol Chem 275:1071910722
63. Baudino TA, Kraichely DM, Jefcoat Jr SC, WinchesterSK, Partridge NC, MacDonald PN 1998 Isolation andcharacterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription. J BiolChem 273:1643416441
64. Wieland C, Mann S, von Besser H, Saumweber H 1992The Drosophila nuclear protein Bx42, which is found inmany puffs on polytene chromosomes, is highlycharged. Chromosoma 101:517525
65. Dahl R, Wani B, Hayman MJ 1998 The Ski oncoproteininteracts with Skip, the human homolog of DrosophilaBx42. Oncogene 16:15791586
66. Zhang C, Baudino TA, Dowd DR,Tokumaru H, Wang W,MacDonald PN 2001 Ternary complexes and coopera-tive interplay between NCoA-62/Ski-interacting proteinand steroid receptor coactivators in vitamin D receptor-mediated transcription. J Biol Chem 276:4061440620
67. Zhou Z, Licklider LJ, Gygi SP, Reed R 2002 Compre-hensive proteomic analysis of the human spliceosome.Nature 419:182185
68. Makarov EM, Makarova OV, Urlaub H, Gentzel M, WillCL, Wilm M, Luhrmann R 2002 Small nuclear ribonu-cleoprotein remodeling during catalytic activation of thespliceosome. Science 298:22052208
69. Auboeuf D, Honig A, Berget SM, OMalley BW 2002
Coordinate regulation of transcription and splicing bysteroid receptor coregulators. Science 298:41641970. Xu J, Qiu Y, DeMayo FJ, Tsai SY, Tsai M-J, OMalley
BW 1998 Partial hormone resistance in mice with dis-ruption of the steroid receptor coactivator-1 (SRC-1)gene. Science 279:19221925
71. Gehin M, Mark M, Dennefeld C, Dierich A, GronemeyerH, Chambon P 2002 The function of TIF2/GRIP1 inmouse reproduction is distinct from those of SRC-1 andp/CIP. Mol Cell Biol 22:59235937
72. Xu J, Liao L, Ning G, Yoshida-Komiya H, Deng C,OMalley BW 2000 The steroid receptor coactivatorSRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is requiredfor normal growth, puberty, female reproductive func-tion, and mammary gland development. Proc Natl AcadSci USA 97:63796384
73. Kostrouchova M, Housa D, Kostrouch Z, Saudek V, RallJE 2002 SKIP is an indispensable factor for Caenorhab-ditis elegans development. Proc Natl Acad Sci USA99:92549259
74. Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M,OMalley BW, Smith CL, Belmont AS, Mancini MA 2001Ligand-mediated assembly and real-time cellular dy-
namics of estrogen receptor -coactivator complexesin living cells. Mol Cell Biol 21:4404441275. Becker M, Baumann C, John S, Walker DA, Vigneron M,
McNally JG, Hager GL 194 2002 Dynamic behavior oftranscription factors on a natural promoter in living cells.EMBO Rep 3:11881181
76. Hughes MR, Malloy PJ, Kieback DG, Kesterson RA,Pike JW, Feldman D, OMalley BW 1988 Point muta-tions in the human vitamin D receptor gene associatedwith hypocalcemic rickets. Science 242:17021705
77. Malloy PJ, Pike JW, Feldman D 1999 The vitamin Dreceptor and the syndrome of hereditary 1,25-dihy-droxyvitamin D-resistant rickets. Endocr Rev 20:156188
78. Yoshizawa T, HandaY, Uematsu Y, Takeda S, Sekine K,Yoshihara Y, Kawakami T, Arioka K, Sato H, UchiyamaY, Masushige S, Fukamizu A, Matsumoto T, Kato S
1997 Mice lacking the vitamin D receptor exhibit im-paired bone formation, uterine hypoplasia and growthretardation after weaning. Nat Genet 16:391396
79. Li YC, Pirro AE, Amling M, Delling G, Baron R, BronsonR, Demay MB 1997 Targeted ablation of the vitamin Dreceptor: an animal model of vitamin D-dependent rick-ets type II with alopecia. Proc Natl Acad Sci USA 94:98319835
80. Li YC, Amling M,Pirro AE, Priemel M,Meuse J, Baron R,Delling G, Demay MB 1998 Normalization of mineral ionhomeostasis by dietary means prevents hyperparathy-roidism, rickets, and osteomalacia, but not alopecia invitamin D receptor-ablated mice. Endocrinology 139:43914396
81. Amling M, Priemel M, Holzmann T, Chapin K, RuegerJM, Baron R, Demay MB 1999 Rescue of the skeletalphenotype of vitamin D receptor-ablated mice in thesetting of normal mineral ion homeostasis: formal his-tomorphometric and biomechanical analyses. Endocri-nology 140:49824987
82. Van Cromphaut SJ, Dewerchin M, Hoenderop JG,Stockmans I, Van Herck E, Kato S, Bindels RJ, Collen D,Carmeliet P, Bouillon R, Carmeliet G 2001 Duodenalcalcium absorption in vitamin D receptor-knockoutmice: functional and molecular aspects. Proc Natl AcadSci USA 98:1332413329
83. Li YC, Bolt MJG, Cao L-P, Sitrin MD 2001 Effects ofvitamin D receptor inactivation on the expression ofcalbindins and calcium metabolism. Am J Physiol En-docrinol Metab 281:E558E564
84. Erben RG, Soegiarto DW, Weber K, Zeitz U, LieberherrM, Gniadecki R, Moller G, Adamski J, Balling R 2002Deletion of deoxyribonucleic acid binding domain of the
vitamin D receptor abrogates genomic and nongenomicfunctions of vitamin D. Mol Endocrinol 16:1524153785. Haussler MR, Haussler CA, Jurutka PW, Thompson PD,
Hsieh JC, Remus LS, Selznick SH, Whitfield GK 1997The vitamin D hormone and its nuclear receptor: mo-lecular actions and disease states. J Endocrinol 154:S57S73
86. Sakai Y, Demay MB 2000 Evaluation of keratinocyteproliferation and differentiation in vitamin D receptorknockout mice. Endocrinology 141:20432049
87. Sakai Y, Kishimoto J, Demay MB 2001 Metabolic andcellular analysis of alopecia in vitamin D receptor knock-out mice. J Clin Invest 107:961966
88. Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R,Hendy GN, Goltzman D 2001 Targeted ablation of the25-hydroxyvitamin D 1-hydroxylase enzyme: evidence
Minireview Sutton and MacDonald Mol Endocrinol, May 2003, 17(5):777791 789
by on November 16, 2006mend.endojournals.orgDownloaded from
http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/http://mend.endojournals.org/ -
8/7/2019 D Vitamin D- More Than a Bone-a-Fide Hormone
14/15
for skeletal, reproductive, and immune dysfunction.Proc Natl Acad Sci USA 98:74987503
89. Masuyama H, Jefcoat Jr SC, MacDonald PN 1997 TheN-terminal domain of transcription factor IIB is requiredfor direct interaction with the vitamin D receptor andparticipates in vitamin D-mediated transcription. MolEndocrinol 11:218228
90. Lavigne AC, Mengus G, Gangloff YG, Wurtz JM, David-son I 1999 Human TAF(II)55 interacts with the vitaminD(3) and thyroid hormone receptors and with derivativesof the retinoid X receptor that have altered transactiva-tion properties. Mol Cell Biol 19:54865494
91. Tolon RM, Castillo AI, Jimenez-Lara AM, Aranda A 2000Association with Ets-1 causes ligand- and AF2-inde-pendent activation of nuclear receptors. Mol Cell Biol20:87938802
92. Chen JD,Evans RM 1995 A transcriptional co-repressorthat interacts with nuclear hormone receptors. Nature377:454457
93. Kurokawa R, Soderstrom M, Horlein A, HalachmiS, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determinedby a co-repressor. Nature 377:451454
94. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B,Kurokawa R, Ryan A, Kamei Y, Soderstrom M, GlassCK, Rosenfeld MG 1995 Ligand-independent repres-sion by the thyroid hormone receptor mediated by anuclear receptor co-repressor. Nature 377:397404
95. Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, SeinoY 2000 Vitamin D is an important factor in estrogenbiosynthesis of both female and male gonads. Endocri-nology 141:13171324
96. Johnson LE, DeLuca HF 2001 Vitamin D receptor nullmutant mice fed high levels of calcium are fertile. J Nutr131:17871791
97. Zinser G, Packman K, Welsh J 2002 Vitamin D(3) recep-tor ablation alters mammary gland morphogenesis. De-velopment 129:30673076
98. Elstner E, Linker-Israeli M, Said J, Umiel T, de Vos S,Shintaku IP, Heber D, Binderup L, Uskokovic M, Koef-
fler HP 1995 20-Epi-vitamin D3 analogues: a novel classof potent inhibitors of proliferation and inducers of dif-ferentiation of human breast cancer cell lines. CancerRes 55:28222830
99. Campbell MJ, Gombart AF, Kwok SH, Park S, KoefflerHP 2000 The anti-proliferative effects of 1,25(OH)2D3on breast and prostate cancer cells are associated withinduction of BRCA1 gene expression. Oncogene 19:50915097
100. Abe E, Miyaura C, Sakagami H, Takeda M, Konno K,Yamazaki T, Yoshiki S, Suda T 1981 Differentiation ofmouse myeloid leukemia cells induced by 1,25-dihy-droxyvitamin D3. Proc Natl Acad Sci USA 78:49904994
101. OKelly J, Hisatake J, Hisatake Y, Bishop J, Norman A,Koeffler HP 2002 Normal myelopoiesis but abnormal Tlymphocyte responses in vitamin D receptor knockoutmice. J Clin Invest 109:10911099
102. Mathieu C, Van Etten E, Gysemans C, Decallonne B,Kato S, Laureys J, Depovere J, Valckx D, Verstuyf A,Bouillon R 2001 In vitro and in vivo analysis of theimmune system of vitamin D receptor knockout mice.J Bone Miner Res 16:20572065
103. Kristal-Boneh E, Froom P, Harari G, Ribak J 1997 As-sociation of calcitriol and blood pressure in normoten-sive men. Hypertension 30:12891294
104. Lind L, Hanni A, Lithell H, Hvarfner A, Sorensen OH,Ljunghall S 1995 Vitamin D is related to blood pressureand other cardiovascular risk factors in middle-agedmen. Am J Hypertens 8:894901
105. Li YC, Kong J, Wei M, Chen Z-F, Liu SQ, Cao L-P 20021,25-Dihydroxyvitamin D3 is a negative endocrine reg-
ulator of the renin-angiotensin system. J Clin Invest110:229238
106. Yagishita N, Yamamoto Y, Yoshizawa T, Sekine K, Ue-matsu Y, Murayama H, Nagai Y, Krezel W, Chambon P,Matsumoto T, Kato S 2001 Aberrant growth plate de-velopment in VDR/RXR double null mutant mice. En-docrinology 142:53325341
107. Whitfield GK, Jurutka PW, Haussler CA, Haussler MR1999 Steroid hormone receptors: evolution, ligands,and molecular basis of biologic function.J Cell Biochem(Suppl)3233:110122
108. Takeyama K, Kitanaka S, Sato T, Kobori M, YanagisawaJ, Kato S 1997 25-Hydroxyvitamin D3 1-hydroxylaseand vitamin D synthesis. Science 277:18271830
109. St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glo-rieux FH 1997 The 25-hydroxyvitamin D 1--hydroxy-lase gene maps to the pseudovitamin D-deficiency rick-ets (PDDR) disease locus. J Bone Miner Res 12:15521559
110. Dardenne O, Prudhomme J, Arabian A, Glorieux FH,St-Arnaud R 2001 Targeted inactivation of the 25-hydroxyvitamin D
3-1-hydroxylase gene (CYP27B1)
creates an animal model of pseudovitamin D-deficiencyrickets. Endocrinology 142:31353141
111. Seo E-G, Einhorn TA, Norman AW 1997 24R,25-Dihy-droxyvitamin D3: an essential vitamin D3 metabolite forboth normal bone integrity and healing of tibial fracturein chicks. Endocrinology 138:38643872
112. Seo EG, Norman AW 1997 Three-fold induction of renal25-hydroxyvitamin D324-hydroxylase activity and in-creased serum 24,25-dihydroxyvitamin D3 levels arecorrelated with the healing process after chick tibialfracture. J Bone Miner Res 12:598606
113. Boyan BD, Sylvia VL, Dean DD, Schwartz Z 2001 24,25-(OH)
2D3
regulates cartilage and bone via autocrine andendocrine mechanisms. Steroids 66:363374
114. St-Arnaud R, Arabian A, Travers R, Barletta F, Raval-Pandya M, Chapin K, Depovere J, Mathieu C, Christa-kos S, Demay MB, Glorieux FH 2000 Deficient