d vitamin d- more than a bone-a-fide hormone

8/7/2019 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.

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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.

Minireview Sutton and MacDonald Mol Endocrinol, May 2003, 17(5):777791 779



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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).




8/15

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-




9/15

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-




10/15

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




11/15

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.).

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