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Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106
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: pnm2{at}po.cwru.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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). A major function of 1,25-(OH)2D3 is to promote intestinal absorption of calcium and phosphate. However, it also may have direct effects 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 terminally 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).
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-hydroxylase (1OHase), resulting in the production of the most bioactive metabolite, 1,25-(OH)2D3. A classic endocrine feedback system operates to tightly control serum levels of 1,25-(OH)2D3 (5, 6). For example, renal 1OHase activity is stimulated by low serum calcium and phosphorus levels and by PTH. The expression of 1OHase is negatively regulated by high levels of 1,25-(OH)2D3. Inactivation, or catabolism, of vitamin D metabolites is initiated by the ubiquitous enzyme 25-hydroxyvitamin D3-24-hydroxylase (24OHase) to generate either 24,25(OH)2D3 or 1,24,25(OH)3D3. The 24-hydroxylated metabolites are further degraded and eventually excreted as either calcitroic acid or 23-carboxyl derivatives. This catabolic process is also carefully regulated as 1,25-(OH)2D3 stimulates 24OHase expression to prevent excessive synthesis of the hormone. The biological effects of 1,25-(OH)2D3 are mediated through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of ligand-activated transcription factors (7, 8). Binding of 1,25-(OH)2D3 to VDR initiates a cascade of macromolecular interactions ultimately leading to transcription of select target genes (9). 1,25-(OH)2D3 associates with the VDR and promotes its heterodimerization with retinoid X receptor (RXR), a common heterodimeric partner for other class II nuclear receptors (10). The liganded VDR-RXR heterodimer is the functionally active transcription factor in 1,25-(OH)2D3-mediated transcription. The heterodimer binds with high affinity to vitamin D response elements (VDREs) in the promoters of target genes. VDREs are characterized by two direct hexameric repeats with an intervening spacer of three nucleotides (DR-3 elements). Thus, 1,25-(OH)2D3 target gene selectivity is conferred, in part, through ligand binding, VDR-RXR heterodimerization, and high-affinity binding 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 interact directly with VDR and other nuclear receptors in a ligand-dependent manner (11). These coactivators participate in an intricate multiprotein complex together with the basal transcriptional machinery and histone modifiers 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 insight into the ligand-binding pocket of VDR complexed 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 unraveled. 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, investigators have begun to identify novel ligands and alternative 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.
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VDR CRYSTAL STRUCTURE |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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and Refs. 5 and 9). The hypervariable amino-terminal A/B domain of VDR is unusually short and, in contrast to that of most other nuclear 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 transactivation 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 receptor with its cognate hormone (13), with other nuclear receptor partners (14), and with comodulatory or adapter proteins (15). The LBD contains the ligand-dependent activation function-2 (AF-2), which is crucial to ligand-activated transcription. Mutation of the AF-2 renders the nuclear receptor transcriptionally inactive despite retaining the ability to bind ligand (14, 15, 16). The DBD and LBD are bridged by the hinge region (domain D), which is thought to confer rotational flexibility between the DBD and LBD and allow for receptor dimerization and interaction with the DNA (17).
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) that is largely unordered, leading to decreased protein solubility. Removal of this insertion domain allowed for efficient crystallization and structure determination of the VDR LBD complexed to 1,25-(OH)2D3 (23). Although the lack of this domain may compromise the interpretation of the VDR structure, the mutant VDR displays normal ligand binding and similar transactivation 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 ß-sheets, which together form a hydrophobic ligand-binding pocket. This pocket is larger than that of RAR due to variations in the positions of helices H2 and H3n and the H6–H7 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 residues of H12 directly contact the ligand, indicating that the ligand conformation may modulate H12 conformation and, therefore, coactivator binding and transcriptional 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)2D3 without the hypercalcemic side effects (see Novel Vitamin D Ligands). Speculation about the mechanisms behind the selective, pleiotropic effects of 1,25-(OH)2D3 analogs centers on the concept that these analogs induce distinct conformations 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. Consequently, the VDR ligand-binding pocket can accommodate rather significant structural changes in the ligand including 1,25-(OH)2D3 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 induce different conformations in the VDR compared with the natural ligand (29). Thus, other mechanisms must be considered to explain the different potencies and calcemic profiles of the analogs. One potential answer resides in the observation that the VDR-analog complexes 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.
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VDR COACTIVATORS |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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-helix containing the ligand-dependent 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 conformational shift creates a hydrophobic cleft composed of helices 3, 4, 5, and 12 (35, 36, 37). The hydrophobic cleft serves as a docking surface for many nuclear receptor coactivators by interacting with a complementary hydrophobic 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 transcription by nuclear receptors and LXXLL-containing coactivators, the precise mechanisms governing nuclear 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 occurs 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 (39, 40, 41). Here, we will highlight a few significant developments in the VDR coactivator field. Steroid receptor coactivator (SRC)-1 [or nuclear receptor coactivator (NCoA)1] is the founding member of the LXXLL motif-containing SRC family of coactivators (33). This family also includes transcriptional intermediary factor-2 (TIF2; Refs. 42 and 43) and receptor-associated coactivator-3 (44, 45, 46, 47, 48). The SRCs interact with VDR and potentiate its transcriptional activity (15, 49). Each of the SRCs possess an autonomous transcriptional activation domain, as evidenced by their ability to enhance transcription when fused to a heterologous DNA-binding sequence such as GAL4. SRCs stimulate transcription possibly by recruiting other transcription factors to the promoter. For example, SRCs interact with cAMP response element binding protein (CBP)/p300, a histone acetyltransferase (HAT) that remodels chromatin structure at the promoter (46, 47, 50). SRCs also possess intrinsic HAT activity (51). CBP/p300 directly associates with nuclear receptors and, together with SRCs, synergistically stimulates transcription (52, 53). Thus, SRCs directly 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.
A large multiprotein complex called DRIP (VDR-interacting proteins) was identified as a coactivator for VDR and other nuclear receptors (54, 55). Many components 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 fundamental role for this complex in stimulus-activated transcriptional processes. In VDR-mediated transcription, DRIP205/TRAP220 acts as an anchoring subunit of the complex by interacting directly with VDR/RXR heterodimers through one of two LXXLL motifs (58). Biochemical 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 studies indicate that a coactivator exchange occurs in the transcriptional complex on native nuclear receptor-responsive promoters (59, 60, 61). Specifically, SRCs appear to enter the transcriptional complex first and dissociate 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 remodel 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 coactivators (63). It interacts with VDR and other nuclear receptors and augments their transcriptional activity. Bx42, the Drosophila melanogaster ortholog of NCoA-62/SKIP, is also implicated in transcriptional processes 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-ß-dependent Smad transcriptional complex (65). It is also implicated in a number of other transcriptional pathways. NCoA-62/SKIP lacks LXXLL motifs and selectively 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 potential interplay between different coactivator classes for maximal activity. Recently, NCoA-62/SKIP was identified in subcomplexes of the spliceosome (67, 68). This, combined with NCoA62/SKIP’s ability to contact varied transcription factors including the VDR, suggests 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/or redundant functions in different signal-activated transcriptional systems remain unclear. Mice with individual targeted deletions of the three SRCs have been developed (70, 71, 72). These models show that the SRCs share several similar functions, especially in the development and maintenance of the female reproductive system. However, the individual SRCs also serve distinct 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)2D3-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-stimulated transcription but does not affect retinoic acid responses. Again, 1,25-(OH)2D3 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 transcription defect (73). Although delineation of the physiological function of NCoA-62/SKIP awaits development 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 associates 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 receptors on native promoters (59, 60, 61, 74, 75). These strategies, combined with in vitro systems composed of purified components and chromatin-packaged templates, will be required for a more complete understanding of the molecular details of VDR-activated transcription.
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KNOCKOUT MODELS |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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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 progressive hypocalcemia, hypophosphatemia, and compensatory hyperparathyroidism. These metabolic imbalances result in severe skeletal defects, including decreased bone mineral density, thinned bone cortex, and widened undermineralized growth plates. However, 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 normal (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 intestinal absorption of calcium in the VDRKO mice is linked to diminished intestinal expression of several 1,25-(OH)2D3-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 absorption, 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 organs 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, primary roles of VDR in both osteoblast and osteoclast biology (85). On the surface, the VDRKO studies indicate that 1,25-(OH)2D3 is not essential for normal bone development 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 compensatory pathways to bypass the effects of the gene deletion. Conditional knockout approaches that ablate VDR in a temporally controlled or tissue-specific manner 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 possibility of developing compensatory mechanisms has been minimized. Finally, the skeletal phenotypes of normocalcemic VDRKO mice may be more pronounced at different life stages or under different physiological stresses. Thus, it will be important to determine 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 enzyme and the catabolic 24OHase, the VDRKO animals have abnormally high levels of 1,25-(OH)2D3. 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 undetectable levels of 1,25-(OH)2D3, fifth-generation vitamin D-deficient VDRKO mice still have alopecia. Thus, 1,25-(OH)2D3 toxicity does not cause alopecia in VDRKO mice. Because wild-type littermates of VDRKO mice raised under the same vitamin D-deficient conditions do not display alopecia, Demay and colleagues (87) proposed that VDR may regulate hair follicle cycling in a ligand-independent fashion. Further support for this hypothesis comes from the observation 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 various transcription factors and induces select genes in the absence of ligand (63, 89, 90, 91). Alternatively, unliganded VDR may repress a subset of target genes in a manner analogous to other nuclear receptors through corepressor interactions (92, 93, 94). 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 establishing molecular 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)2D3 plays important roles in physiological processes beyond mineral homeostasis and keratinocyte function. For example, VDRKO mice have impaired reproductive function (78). Both male and female VDR-null mice exhibit diminished estradiol levels and elevated gonadotropins, indicating a gonadal dysfunction in the secretion of sex hormones (95). Histological analysis of reproductive glands shows abnormal ovarian follicle development in the females and dilated seminiferous tubules with diminished spermatogenesis in the males. However, serum calcium normalization with the rescue diet mostly corrects the hormonal imbalances and histological abnormalities (95) and completely restores fertility (95, 96), suggesting that the gonadal dysfunction in VDRKO mice primarily results from hypocalcemia.
Mammary gland development is also emerging as a biological process that is impacted by 1,25-(OH)2D3. The mammary glands of VDRKO mice demonstrate a hyperproliferative phenotype as evidenced by increased 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 response to exogenously administered estrogen and progesterone (97). Although dietary calcium supplementation normalizes estrogen levels in VDRKO mice, the abnormal mammary phenotype is retained. These data indicate a significant developmental role for VDR in the mammary gland potentially in restricting ductal growth. Combined with the observations that 1,25-(OH)2D3 and its analogs inhibit the growth and induce the differentiation of breast cancer cell lines (98, 99), the mammary phenotype in the VDRKO model indicates that 1,25-(OH)2D3 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 inhibits proliferation and drives the differentiation of leukemic cells along the monocyte/macrophage lineage (100). However, VDRKO mice lack a striking immune system phenotype. Although O’Kelly et al. (101) observed that VDRKO mice have abnormal T cell responses 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)2D3 toxicity, or to hypocalcemia. Mathieu et al. (102) found that VDRKO mice had a defect in calcium-dependent T cell proliferation resulting in protection from experimentally induced autoimmune diabetes and that these abnormalities could be corrected by restoring serum calcium to normal levels. The normal 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)2D3 may also be beneficial to the cardiovascular system by decreasing blood pressure (103, 104). Consistent 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 response appears to be at the transcriptional level, as 1,25-(OH)2D3 suppresses renin promoter activity. This study suggests that VDR negatively regulates the expression 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 observations in the VDRKO model that may stimulate a more extensive examination of 1,25-(OH)2D3 and its synthetic analogs as potential therapies for some forms of hypertension.
VDR/RXR Double Knockout
VDR heterodimerizes with RXR to modulate transcription 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 colleagues (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, hypophosphatemia, 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 serum calcium and phosphorus rescues all of the skeletal 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-related receptor exists that selectively heterodimerizes with RXR. Such a receptor would likely share a considerable amount of sequence similarity with the classical VDR because it must 1) bind to and be transcriptionally activated by 1,25-(OH)2D3 or its metabolites; 2) heterodimerize 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 sequences exist, potential candidates might include the most closely related nuclear receptors, such as farnesoid X receptor, steroid xenobiotic receptor/pregnane X receptor, and liver X receptor (107). Alternatively, any nuclear receptor unrelated to VDR that selectively heterodimerizes with RXR and binds 1,25-(OH)2D3 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)2D3, two groups independently disrupted the 1OHase gene in mice (88, 110). The phenotype of the 1OHase-null mice is strikingly similar to that of the VDR-knockout mouse, including hypocalcemia, hyperparathyroidism, growth retardation, and osteomalacia, consistent with rickets (88, 110). The 1OHase-mutant female mice are anovulatory and, therefore, infertile (88). Another key difference between VDRKO mice and the 1OHase-ablated mice 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 development of knockout models of both the receptor and ligand of 1,25-(OH)2D3 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 development, and blood pressure regulation.
24OHase Knockout
24OHase metabolizes both the bioactive 1,25-(OH)2D3 and its precursor, 25(OH)D3. 24OHase gene transcription is positively regulated by 1,25-(OH)2D3, thereby completing a negative feedback loop to prevent excessive hormone synthesis. One of the major products of 24OHase, namely 24,25(OH)2D3, is considered to be an inactive metabolite, an initial product destined for further degradation and eventual excretion as calcitroic acid (5). However, there is substantial evidence that 24,25(OH)2D3 has biological activity of its own, most strikingly in the function of chondrocytes, or cartilage-forming cells (111, 112, 113). To determine the consequences of abolishing this enzyme and, therefore, its 24-hydroxylated products, a knockout model of 24OHase was created (114). This mutation results in reduced embryonic viability and aberrant intramembranous ossification. The obvious possibility is that the diminished levels of 24,25(OH)2D3 causes these abnormalities. However, the phenotype is not rescued in 24OHase-knockout progeny by feeding pregnant mice exogenous 24,25(OH)2D3. Alternatively, the phenotype could be caused by the abnormally high levels of 1,25-(OH)2D3 resulting from the deletion of this catabolic enzyme. Indeed, crossing the 24OHase-null mice with VDR-null mice completely rescues the decreased embryonic viability and ossification defects, thus supporting the concept that 1,25-(OH)2D3 toxicity leads to the abnormal phenotype in the 24OHase-knockout mice. Moreover, this model also suggests that 24-hydroxylated metabolites are not required for normal intramembranous ossification (114).
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BEYOND 1,25-(OH)2D3 AND VDR: NOVEL LIGANDS AND ALTERNATIVE RECEPTORS |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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Calcipotriol (MC 903) is an analog that has been used to treat psoriasis for nearly 15 yr, and it is currently considered a first-line therapy for the disease (116). Calcipotriol improves psoriasis by inhibiting proliferation and promoting differentiation of keratinocytes, but it does not cause hypercalcemia or decreased bone mass. This selectivity can be attributed to calcipotriol’s low affinity for vitamin D binding protein, the major vitamin D transport protein in the circulation (117), and the fact that it is applied topically, thus restricting its actions to the skin. 1,25-(OH)2D3 analogs are also valuable in treating renal osteodystrophy, a devastating consequence of chronic renal failure. Kidney disease, due to a variety of causes, often leads to reduced 1,25-(OH)2D3 production and impaired phosphate excretion resulting in abnormally high PTH levels. The decreased calcitriol levels combined with secondary hyperparathyroidism, in turn, result in increased bone turnover (118). Treatment with 1,25-(OH)2D3 is effective in suppressing PTH levels and improving the initial skeletal abnormalities. However, due to the narrow therapeutic window, 1,25-(OH)2D3 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 mechanisms of the selectivity of these two analogs remain unclear, they represent significant improvements in treatment regimens for renal osteodystrophy.
Although neither vitamin D3 nor 1,25-(OH)2D3 analogs 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 recommended as a dietary supplement in addition to any pharmacological treatment for all patients with decreased bone mass or osteoporosis (123). Still, the higher doses of 1,25-(OH)2D3 required for maximal improvement in bone density cause significant hypercalcemia. Peleg and colleagues (124) tested 1,25-(OH)2D3 analogs for their ability to improve bone mineral 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 observations are potentially explained by the tissue-selective action 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)2D3 (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)2D3 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)2D3 may serve as tissue-selective activators of VDR-mediated responses. An unexpected ligand for VDR was discovered from studies with bile acid compounds (126). Metabolic lipophilic molecules such as bile acids activate many of the orphan nuclear receptors, including farnesoid X receptor and steroid xenobiotic receptor/pregnane X receptor (127, 128, 129). Recently, Makishima et 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 micromolar concentrations of LCA, whereas VDR is activated by nanomolar amounts of 1,25-(OH)2D3. Nonetheless, LCA- or 1,25-(OH)2D3-liganded VDR also stimulates the expression of endogenous CYP3A, the P450 enzyme 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)2D3 is protective against colon cancer (132). Thus, induction of CYP3A by 1,25-(OH)2D3 and by LCA itself may represent a detoxification pathway for LCA, as well as explain the potential preventative effects of 1,25-(OH)2D3 in colon cancer. Although this study awaits further in vivo confirmation, it clearly raises the possibility that VDR may be activated by other naturally occurring ligands.
VDR Isoforms
Many nuclear receptors, such as RAR, RXR, and thyroid receptor, have multiple isoforms that are encoded by separate genes (133). Unlike these nuclear receptors, 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 transcripts 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 427 amino acids in length. However, two transcripts have upstream in-frame methionines that potentially generate 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 detected in osteoblast, colon cancer, and kidney cell lines (136). Interestingly, VDRB1 has reduced transcriptional activity compared with classical VDR. Whether the levels of expression of these isoforms are substantial and whether these isoforms result in altered 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 endonuclease site. In some humans, there is an ATG 224 ACG transition at the +1 position, eliminating the translational initiation site and Fok I recognition sequence. This transition results in the use of an in-frame methionine as the initiator codon at the +4 position. Thus, either a 427 (Met-1) or a 424 (Met-4) amino acid protein is expressed. Numerous epidemiological studies suggest an association between the shorter form of VDR and increased bone mineral density in humans (139, 140, 141, 142, 143). The molecular mechanism of this association remains unclear, but there is suggestive evidence that the Met-4 VDR displays enhanced transcriptional activity due to increased interaction with basal transcription factor II B (12). Although these findings remain 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, suggests that there may be inhibitory domains at the extreme N terminus of VDR that decrease its transactivation potential.
A Membrane Receptor? Rapid, Nongenomic Effects of Vitamin D and Its Metabolites
According to the classical paradigm of nuclear receptor 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 transcription and translation of target genes, they are typically 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 transcriptional and translational inhibitors, such as actinomycin D and cycloheximide, precludes the possibility that the traditional "genomic" model is operating. Recent attention to these rapid, "nongenomic" hormone effects 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 movement of calcium across chick enterocytes within several 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 translation of calcium-binding proteins or calcium transporters (147). In addition to the enterocyte, the osteoblast is a target for 1,25-(OH)2D3-induced rapid calcium mobilization from internal stores, a process that involves a membrane-initiated signaling cascade including phospholipase C activation and inositol triphosphate formation (148). This process also occurs in skeletal muscle cells, in which 1,25-(OH)2D3 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)2D3 have been studied.
Although the rapid effects of 1,25-(OH)2D3 and numerous other steroids are well documented, the field is hindered by the inability to identify the putative membrane receptors that trigger these nongenomic 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. A promising new approach in the nongenomic field is the use of 1,25-(OH)2D3 analogs to discriminate between receptors that mediate membrane-initiated events and those that mediate the classical nuclear effects of 1,25-(OH)2D3. Song et al. (152) showed that 6-s-cis-locked analogs of 1,25-(OH)2D3 stimulate 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)2D3. In fact, two preliminary reports indicate that annexin II, a membrane-associated calcium-binding protein, may bind 1,25-(OH)2D3 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). Osteoblast cultures derived from VDR-mutant mice are unable to initiate a rapid calcium flux in response to 1,25-(OH)2D3, suggesting that some nongenomic responses require a functional nuclear VDR. Although a full array of other nongenomic responses needs to be tested, the VDRKO strains will provide important tools to decipher the molecular requirements of classical VDR that mediate the genomic and nongenomic effects of 1,25-(OH)2D3 in vivo.
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SUMMARY: FRONTIERS IN VITAMIN D |
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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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)2D3 and bone biology. In particular, specific cells of the osteoblast and osteoclast lineage have garnered considerable research attention over the past several decades as important direct targets of 1,25-(OH)2D3 in preserving skeletal integrity. However, the striking skeletal defects observed in the VDRKO are corrected by simply providing the animals with supplemental dietary calcium. Does this mean that 1,25-(OH)2D3 does not act as a direct "bone-a-fide" hormone 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)2D3 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 follicle cycling, 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 stimulate 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 view of the VDR protein, highlighting the expansive binding pocket associated with both natural and synthetic 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-liganded 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)2D3 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 analogs and natural compounds such as bile acids, are broadening our understanding of the therapeutic potential and physiological intricacies of vitamin D. Likewise, alternative receptors, both related to and distinct from the classical nuclear VDR, are emerging as significant 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF-2, Activation function-2; CBP, cAMP response element binding protein-binding protein; DBD, DNA-binding domain; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; 1OHase, 25(OH)D3-1-hydroxylase; 25-(OH)D3, 25hydroxyvitamin D3; 24-OHase, 25-(OH)D3-24-hydroxylase; DRIP, VDR-interacting protein; HAT, histone acetyltransferase; HVDRR, hereditary vitamin D-resistant rickets; LBD, ligand-binding domain; LCA, lithocholic acid; NCoA, nuclear receptor coactivator; RAR, retinoic acid receptor; RXR, retinoid X receptor; SKIP, ski-interacting protein; SRC, steroid receptor coactivator; TIF2, transcription intermediary factor-2; TRAP, thyroid receptor-activating protein; VDR, vitamin D receptor; VDRE, vitamin D response element; VDRKO, VDR knockout.
Received for publication November 1, 2002. Accepted for publication March 5, 2003.
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TOPABSTRACTINTRODUCTIONVDR CRYSTAL STRUCTUREVDR COACTIVATORSKNOCKOUT MODELSBEYOND 1,25-(OH)2D3 AND VDR:...SUMMARY: FRONTIERS IN VITAMIN...REFERENCES |
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