Title: The Nuclear Vitamin D Receptor: Biological and Molecular Regulatory Properties Revealed
Abstract: In the decade since the vitamin D receptor (VDR) was cloned1 and recognized as a member of the superfamily of nuclear receptors that regulate gene expression in a ligand-dependent manner,2, 3 the central role of VDR in the biology of vitamin D action has been illuminated and is being defined at the molecular level. Following renal production as the hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone–receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling. VDR not only mediates the action of 1,25(OH)2D3 in calcium/phosphate translocating tissues, primarily intestine, but also elicits a myriad of apparent bioactivities in other major cell systems in the organism, including immune, neural, epithelial, and endocrine. The scope of this review will be limited to highlighting the actions of 1,25(OH)2D3 mediated by nuclear VDR and discussing new developments in the structure/function analysis of the receptor, including the phenotype of VDR knockout mice and the biochemical classification of patients with point mutations in the receptor. These new advances, along with other recent research, will be interpreted to update our understanding of the molecular role of VDR, ranging from characterization of its natural gene and clinically significant polymorphisms, through its DNA contact sites and protein partners, to novel ligand analogs that hold the promise of influencing VDR conformation in a therapeutically beneficial fashion. The traditional action of vitamin D, via its 1,25(OH)2D3 hormonal metabolite, is to effect calcium and phosphate homeostasis to ensure the deposition of bone mineral (summarized in Fig. 1A). 1,25(OH)2D3 stimulates intestinal calcium and phosphate absorption, bone calcium and phosphate resorption, and renal calcium and phosphate reabsorption, thus increasing the blood Ca•PO4 ion product. Failure to achieve normal bone mineral accretion by these mechanisms leads to rachitic syndromes. Nutritional rickets, caused by the simultaneous deprivation of sunlight exposure4 and dietary vitamin D, was ameliorated in classic experiments by administration of this fat soluble vitamin (reviewed in Ref. 5). Subsequently, it was recognized that the pathways comprising the metabolic activation of the vitamin to its hormonal form and consequent functions of the hormone in target tissues (Fig. 1) present additional steps where defects directly elicit vitamin D–resistant rachitic syndromes. Two such disorders involve the inadequate bioactivation of 25-hydroxyvitamin D3 (25(OH)D3), a constitutively produced intermediary metabolite, to 1,25- (OH)2D3. This step is catalyzed by the 1α-OHase enzyme in kidney (Fig. 1A). Chronic renal failure results in renal rickets and secondary hyperparathyroidism when compromised renal mass reduces 1α-OHase activity,6 whereas pseudo–vitamin D–deficiency rickets (PDDR) involves a specific hereditary defect in the gene coding for the 1α-OHase enzyme.7 The latter conclusion has been verified by the recent cloning of cDNAs for the rat8 and mouse9 1α-OHase P450 and the pinpointing of the human 1α-OHase gene to a chromosomal locus coincident with PDDR.8 Interestingly, the 1α-OHase/PDDR locus maps rather closely to the VDR gene on chromosome 12 in the 12q13–14 region,7 a proximity that may be relevant to the evolution and control of the vitamin D ligand–receptor system. Calcemic and phosphatemic biological actions of vitamin D in mammals. (A) Effects of vitamin D and its metabolites to ensure skeletal integrity, especially when calcium is limiting. (Central open box) Vitamin D3, obtained from diet or derived from sunlight-initiated photobiogenesis in skin, is converted via two hydroxylation reactions to the 1,25(OH)2D3 hormonal form that circulates in blood. The final step in bioactivation of vitamin to hormone is catalyzed by the renal 1α-OHase when stimulated by PTH under conditions of low calcium. (Lower portion) Integrated actions of the 1,25(OH)2D3 metabolite, via binding to the intracellular VDR, to control calcium homeostasis in bone, intestine, kidney, and parathyroid as explained in the text. (Top left) Action of 1,25(OH)2D3–VDR in skin cell differentiation. (Top center) Conversion of 1,25(OH)2D3 or the preceding 25(OH)D3 metabolite to 24-hydroxylated forms in response to 1,25(OH)2D3–VDR induction of the 24-OHase gene. This conversion serves to initiate catabolism of the vitamin D molecule, but may also produce 24-hydroxylated metabolites with novel hormonal activity with respect to chondrocyte differentiation and bone mineralization (see text). (B) The vitamin D bioactivation-phosphate homeostatic loop: proposed novel roles for phosphatonin, the PEX gene product, and NPT2. (Left and lower portion) Under normal physiologic conditions, low PO4 enhances the synthesis of 1,25(OH)2D3, which then acts through VDR to effect phosphate reclamation by suppressing PTH as well as inducing NPT2 and PEX gene expression. NPT2 acts directly to reabsorb PO4, while the PEX enzyme eliminates phosphatonin. (Top right) Tumor-induced osteomalacia and XLH each elicit increased phosphatonin, an uncharacterized phosphaturic hormone that is postulated to inhibit both NPT2 and the 1α-OHase, to cause severe phosphate wasting. Clinical and molecular genetic data from the last decade have provided unequivocal evidence for the obligatory role of VDR in mediating the action of vitamin D. Familial target tissue insensitivity to 1,25(OH)2D3, known as hereditary hypocalcemic vitamin D–resistant rickets (HVDRR), is an autosomal recessive disorder resulting in a phenotype characterized by severe bowing of the lower extremities, short stature, and often alopecia.10 In virtually all cases, the cause of this syndrome has been shown to be a defect in the gene encoding human VDR (hVDR) (reviewed in Refs. 10-13), although potential exceptions have been described.14, 15 The fact that the phenotype of HVDRR patients, excluding alopecia, mimics classic nutritional rickets indicates that 1,25(OH)2D3-liganded VDR not only executes all of the bone mineral homeostatic actions of 1,25(OH)2D3 but suggests that VDR itself also participates in the normal hair growth cycle in skin. Recently, VDR knockout mice have been created by two groups,16, 17 revealing apparently normal heterozygotes but homozygotes that display a phenotype very similar to HVDRR, including the progressive development of alopecia over 4–7 weeks of age. At various intervals after birth (differing somewhat between the two studies), VDR null mice acquired low bone mass, hypocalcemia, hypophosphatemia, hyperparathyroidism, and 10-fold elevated 1,25(OH)2D3, coincident with extremely low 24,25(OH)2D3. Affected homozygotes died within 15 weeks16 or exhibited near normal survival rates for up to 6 months.17 The differing survival times may be related to diet or environmental variations, since ablation of exon II/first zinc finger16 or exon III/second zinc finger17 should produce equivalent functional consequences of VDR gene knockout. Despite a lack of VDR throughout early development, VDR null mice are born phenotypically normal, exhibiting symptoms of rickets/osteomalacia and secondary hyperparathyroidism primarily after weaning.16 This observation suggests that the vitamin D endocrine system is principally required for maintaining bone mineral homeostasis when the organism is deprived of a consistent and plentiful supply of calcium, such as occurs after weaning in mammals, after hatching in birds, or after leaving the aqueous environment in the case of amphibians.18 In addition to the data reported in their abstract, Demay and coworkers19 also presented preliminary results describing the prevention of many, but not all, of the phenotypic effects of VDR knockout by means of a “rescue diet,” consisting of high levels of lactose, calcium, and phosphate. By manipulating blood calcium and phosphate levels in this manner, parathyroid hormone (PTH) was normalized and bone mineralization was greatly improved in the VDR knockout animals, to a degree that the histology of the growth plate was indistinguishable from that of normal littermates. However, alopecia and skin abnormalities, such as dermal cysts, persisted in the VDR null mice on the rescue diet, intimating that VDR plays an indispensable role in hair and skin development independent of bone mineral homeostasis. That normalizing circulating mineral concentrations via dietary intervention in VDR knockout mice prevents the rachitic phenotype is consistent with results in HVDRR patients whose bone abnormalities are resolved by frequent therapy with overnight intravenous calcium infusions.20, 21 Thus, the generation of VDR null mice and the reversal of their bone abnormalities by diet dramatizes the concept that the major physiologic effect of 1,25(OH)2D3 is on intestinal absorption of calcium and phosphate, although certain calcium regulating end-points such as depressed renal calbindin-D9k mRNA expression in kidney are not corrected when VDR null mice are maintained on the rescue diet (M. Demay et al., unpublished results). Moreover, in another preliminary study, Kato and colleagues22 found that, when utilizing the coculture system of Suda and coworkers,23 osteoblasts/stromal cells from VDR knockout mice will not support 1,25(OH)2D3-induced osteoclastogenesis of normal spleen cells, whereas the reverse experiment (normal osteoblasts/stromal cells and VDR null mouse spleen cells) results in the production of osteoclasts upon exposure of the coculture to 1,25(OH)2D3. Therefore, as depicted in Fig. 1(A), VDR appears to be essential for 1,25(OH)2D3 to elicit a paracrine signal from osteoblasts, which in turn facilitates osteoclast differentiation, at least in vitro. However, the fact that VDR null mice curiously possess normal, or even increased numbers of osteoclasts16 suggests that other osteoclast-activating factors, such as PTH or interleukin-1 (IL-1), can still support osteoclastogenesis in the absence of functional VDR. Superimposed upon its pivotal role in controlling bone mineral transport and differentiation in hair follicles, another function of the 1,25(OH)2D3–VDR complex is to govern the level of the renal 1,25(OH)2D3 hormone by feedback regulation of its biosynthesis and by induction of a key catabolic enzyme. 1,25(OH)2D3 appears to effect a short feedback loop (Fig. 1A) to repress the 1α-OHase enzyme,24 and is also a potent suppressor of the synthesis and secretion of PTH,25 the primary tropic hormone stimulating the 1α-OHase (Fig. 1).26 The mechanism whereby 1,25(OH)2D3 curtails PTH production involves a VDR-mediated silencing of PTH gene transcription.27-29 Based upon preliminary data from dietarily rescued VDR null mice, excess calcium is able to adequately control PTH secretion and parathyroid cell growth, suggesting that the role of 1,25(OH)2D3 in these processes is cooperative with physiologic calcium levels but can be overridden in situations of calcium abundance. Turnover of 1,25(OH)2D3 is accomplished via several catabolic routes,30, 31 with 24-hydroxylation initiating the apparent primary pathway for elimination of vitamin D metabolites. The 24-OHase enzyme is markedly enhanced by 1,25(OH)2D3 through a VDR-dependent mechanism (Fig. 1A), a phenomenon predominating in kidney but also occurring in all 1,25(OH)2D3 target cells.32, 33 The dependency of 24-OHase activity on VDR action is emphasized by the extremely low levels of 24-hydroxylated metabolites found in VDR null mice, as discussed above.16 Both the 25(OH)D3 precursor and the 1,25(OH)2D3 hormone serve as effective substrates for the 24-OHase enzyme, rendering the latter capable of catalyzing a potent attenuation of active vitamin D metabolite concentrations. In fact, homozygous 24-OHase null mice34 display reduced 1,25(OH)2D3 clearance, with the F1 progeny exhibiting signs of vitamin D intoxication, such as calcified kidneys. Interestingly, the F1 progeny also have defective intramembranous ossification. The failure of bones such as the calvaria and mandible to calcify in this situation could be a developmental consequence of excess 1,25(OH)2D3. Another possibility is that a 24-hydroxylated D-metabolite(s) is required for some aspect of cartilage or bone formation (Fig. 1A),35-38 perhaps via pharmacokinetic interactions with 1,25(OH)2D3 or through an uncharacterized novel receptor for the 24-hydroxylated metabolite. Other than negative feedback on PTH synthesis and secretion27, 39 to suppress this phosphaturic hormone, how does the 1,25(OH)2D3–VDR complex influence phosphate metabolism to maintain the Ca•PO4 ion product? One mechanism seems to be the primary induction of phosphate translocating proteins in kidney and perhaps intestine (Fig. 1). An example is the renal sodium–phosphate cotransporter-2 (NPT2) (Fig. 1B),40, 41 a likely 1,25(OH)2D3-induced protein because its gene contains a vitamin D responsive element (VDRE) in the promoter region (see below). It is also well established that hypophosphatemia stimulates the 1α-OHase (Fig. 1B) to elevate circulating 1,25(OH)2D3 levels.26 However, circulating 1,25(OH)2D3 is inappropriately low for the prevailing phosphate concentrations in patients with X-linked hypophosphatemic rickets (XLH),42 a dominant familial disorder of renal phosphate wasting. Importantly, such patients can be cured with a therapeutic combination of oral phosphate and 1,25(OH)2D3.43 The defective gene responsible for XLH has been identified as PEX, or phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome.44 Further, there exists a significant number of cases of tumor-induced osteomalacia, an acquired disorder that closely resembles the phosphate wasting of XLH and is characterized by low circulating 1,25(OH)2D3.45 Finally, renal cross-transplantation46 and parabiosis47 studies in normal and genetically hypophosphatemic mice demonstrate that a novel uncharacterized phosphaturic hormone is present in the circulation. Taken together, these observations argue strongly that the XLH and tumor-induced osteomalacia syndromes are both caused by excess amounts of this novel humoral factor, which is distinct from PTH and has been named phosphatonin (Fig. 1B).48, 49 Like PTH, phosphatonin is presumably a potent inactivator of NPT2 (Fig. 1B), but in contrast to PTH, this newly recognized factor apparently also inhibits the 1α-OHase, thus suppressing 1,25(OH)2D3. As depicted in Fig. 1(B), the normal role of the PEX gene product is postulated to be the proteolytic inactivation of this phosphaturic principle, such that inactivating PEX mutations in XLH elicit the appearance of abnormally high circulating levels of phosphatonin.44 In the case of tumor-induced osteomalacia, the tumor appears to produce phosphatonin ectopically. Thus, as illustrated in Fig. 1(B), the PEX/phosphatonin system could participate in a novel regulatory loop for maintaining normal phosphate homeostasis, which becomes deranged in XLH, or when tumors directly secrete phosphatonin. Another role of 1,25(OH)2D3/VDR in phosphate control is postulated to be the induction of PEX gene expression (Fig. 1B), thus creating an additional strategy for protection by 1,25(OH)2D3 against hypophosphatemia. Indeed, the PEX gene appears to possess a VDRE based upon Southwestern analysis of the relevant yeast artificial chromosome.50 The present hypothesis is that a low phosphate level triggers an increase in circulating 1,25(OH)2D3, which in turn augments the PEX gene product to destroy phosphatonin, constituting a novel phosphate homeostatic loop that is overwhelmed when high levels of phosphatonin diminish 1,25(OH)2D3 synthesis and inhibit NPT2. This concept is consistent with the relatively depressed 1,25(OH)2D3 levels observed either in patients with tumor-induced osteomalacia or in kindreds with XLH caused by inactivating PEX mutations. Given the present indications that phosphatonin represents a pathophysiologically relevant phosphaturic hormone that regulates vitamin D bioactivation, its characterization at the chemical level is eagerly anticipated. As summarized in Table 1, the potential actions of 1,25(OH)2D3 via its nuclear VDR extend far beyond the bone mineral homeostasis realm pictured in Fig. 1. The following three independent methodologies have been employed to provide evidence that 1,25(OH)2D3 functions in a diverse array of cells: autoradiographic localization of the ligand following administration to vitamin D–deficient animals, immunohistochemical detection of VDR in the nucleus of target cells, and responsiveness of specific cell types in culture to 1,25(OH)2D3 and its active analogs. In many cases, these data are coupled to biological responses or to loss of function in VDR null mice, in vivo, for instance in the maintenance of insulin secretion by 1,25(OH)2D3,51 the uterine hypoplasia reported in VDR knockout female mice apparently caused by suboptimal ovarian estrogen production,16 and the exploitation of the prodifferentiation/antiproliferative actions of 1,25(OH)2D3 in the treatment of psoriasis.52 One major neoclassical target for 1,25(OH)2D3 is the immune system (reviewed in Ref. 53), with the suppression of IL-1 to IL-6 and interferon-γ constituting prominent in vitro 1,25(OH)2D3 effects mediated by VDR (Table 1). Moreover, in vivo immunomodulatory actions of the hormone also have been documented (reviewed in Ref. 54), such as reduced macrophage and lymphocyte function in vitamin D–deficient rats.55 1,25(OH)2D3 functions as a general suppressor of the immune system, especially of T-helper cells (subset type 1), suggesting that analogs of vitamin D might be useful therapeutic agents in procedures such as organ transplants56 or in the treatment of autoimmune disorders.57 In addition, 1,25(OH)2D3 is thought to play an important role in the differentiation of cells in the hematopoietic lineage. Several illustrations of this action have been reported, including differentiation of a human promyelocytic leukemia cell line (HL-60) into macrophage-like cells,58, 59 and the development of osteoclasts in bone from colony forming unit–granulocyte/macrophage precursors (see Ref. 53 and references therein). Many of these effects of 1,25(OH)2D3–VDR, although of potential therapeutic significance, may be biologically redundant with other immune modulators, perhaps offering survival advantages. The tentative conclusion of redundant immunoregulation is based upon the normal immune profile of VDR null mice at 7 weeks of age,16 but it is reasonable to hypothesize that the potential immunomodulatory power of 1,25(OH)2D3–VDR could become more significant during pathophysiological stress situations or under conditions of senescence. Other apparent sites of action for 1,25(OH)2D3 and VDR include the central nervous system (CNS) (Table 1), where one of the outcomes is immunosuppression. For example, 1,25(OH)2D3 treatment elicits a partial improvement of symptoms in rodents with developing experimental allergic encephalomyelitis.60, 61 Moreover, 1,25(OH)2D3 also has been shown to induce expression of the following neurotrophic hormones or their mRNAs: glial cell-derived neurotrophic factor (a protein that may be important in protecting certain types of neural tissue from degenerative processes),62 leukemia inhibitory factor (a widely distributed protein in the brain with neurotrophic activity),63 neurotrophin-3 mRNA (in primary astrocytes),64 and nerve growth factor (both in primary newborn astrocytes65 and in the hippocampus and cortex of the adult rat66). Furthermore, the activity of cholinergic acetyltransferase is elevated in specific brain regions in response to 1,25(OH)2D3 administration.67 In some neurons, analogous to the antiapoptotic effect of calbindin-D28k in lymphocytes,68 the induction of calbindins by 1,25(OH)2D3 may protect against cell death in the face of repetitive calcium transients,69 and in fact the expression of calbindin-D28k mRNA is decreased in the hippocampus of Alzheimer's patients as assessed by in situ hybridization.70, 71 Taken together, these observations not only imply a modulatory role for VDR in neural cell growth and differentiation but also intimate a possible role for 1,25(OH)2D3 in therapeutic intervention for neurodegenerative disorders. Similarly, 1,25(OH)2D3 appears to affect dramatically the maturation and functions of certain normal and neoplastic epithelial cells (Table 1). As discussed above, VDR plays a key role in the hair growth cycle, an action related to the ability of 1,25(OH)2D3 to stimulate the differentiation of keratinocytes (Table 1). Also, the proliferation of a number of epithelially derived cancer cells (e.g., mammary, prostate, and colon) is inhibited in culture by 1,25(OH)2D3, with some cells being directed toward a more differentiated phenotype. This effect in neoplastic cells may be related to the reported ability of liganded VDR to arrest cells at the G1 stage by influencing cell cycle regulatory proteins, such as p21 and p27, to control cell growth transcription factors such as c-myc and c-fos, or to elicit apoptosis by down-regulating Bcl-2 (Table 1 and references therein). Therefore, the 1,25(OH)2D3 hormone seems to resemble its nutritionally derived, lipophilic ligand cousins, vitamin A and thyroid hormone, in possessing the ability to influence the program of cell development as well as to evoke classic metabolic and growth regulatory effects. 1,25(OH)2D3 also reportedly affects several major endocrine processes, such as TRH/TSH action and pancreatic insulin secretion (Table 1). However, only further investigations of VDR null mice and other transgenic strategies, such as tissue-specific expression of a dominant negative VDR, will allow us to sort out which of the putative neoclassical effects of 1,25(OH)2D3 mediated by VDR are biologically relevant. At this early juncture, of those sites enumerated in Table 1, only the skin/hair growth cycle and ovarian/uterine systems appear to be markedly affected in the VDR null mouse. However, the limited phenotype of VDR knockout mice does not preclude 1,25(OH)2D3 and its analogs from being valuable as biological response modifiers useful in the treatment of hyperproliferative disorders, autoimmunity, and CNS deterioration, as well as traditional maladies of calcium and phosphorus metabolism such as renal osteodystrophy, hypoparathyroidism, and osteoporosis. The recently characterized gene encoding hVDR (Fig. 2A),72 previously localized to chromosome 12,73 is similar to other nuclear receptor genes74 in that each of the two zinc fingers is encoded by separate exons (II and III), and the 5′ end of the gene exhibits some complexity in the form of alternate splice and/or translation start sites. For hVDR, alternate splicing of three exons (IA–IC) encoding portions of the 5′ untranslated region generates at least three mRNA variants,72 while the presence of a polymorphic sequence in exon II determines the presence or absence of an alternative translation start site (see discussion of FokI polymorphism below).72 A unique feature of the hVDR gene is the presence of an additional exon (V) that is not found in other nuclear receptor genes (Fig. 2A)72; it resides near the center of the gene and encodes residues 155–194 in hVDR. This region of the VDR protein is more expansive than the corresponding segment in other nuclear receptors, suggesting that the VDR may have acquired a novel exon of unknown function as it diverged evolutionarily from other nuclear receptor genes.72 Schematic view of genomic and deduced amino acid sequences for hVDR, displaying known natural variations. (A) The hVDR chromosomal gene, containing a total of 11 exons, three of which (IA, IB, and IC) encode 5′ UTR region and are variably present in VDR transcripts.72 Several polymorphic variants, including a FokI site in exon II, and a cluster of linked sites near or in exon IX, are discussed in the text. (B) Schematized linear amino acid sequence of hVDR, highlighting functional domains as currently understood from mutagenesis analysis. ⊕ signifies a cluster of five basic amino acids in the intervening sequence between the two zinc fingers. Two reported sites of modulatory serine phosphorylation at serine-51 and serine-208 are indicated by S51 and S208, respectively. Known natural point mutations in human patients with the HVDRR syndrome are indicated by single-letter abbreviations (e.g., G33D is a glycine to aspartate substitution at position 33). X refers to a premature stop codon, while f indicates a frame-shift mutation, leading to premature termination. Point mutants from positions 33–80 are defective in DNA binding, and all X and f mutants can neither bind hormone nor heterodimerize with RXR. Other point mutations in the C-terminal half of the receptor (from positions 259–391) display defects in either hormone-binding or heterodimer formation with RXR (or both) as discussed in the text. One of the most intriguing, yet controversial, areas of bone-related genetic research in the past few years has been the discovery of common polymorphisms in the hVDR gene and their potential relationship to bone mineral density (BMD) and the pathophysiology of osteoporosis, hyperparathyroidism, and cancers of the breast and prostate. Morrison, Eisman, and colleagues75 first reported that VDR alleles could predict BMD, contending that the occurrence of a BsmI restriction site (Fig. 2A, denoted b) in the intron separating exons VIII and IX of the gene (Fig. 2A) is associated with enhanced lumbar spine BMD. Conversely, the absence of the BsmI site (denoted B) in VDR was correlated with low BMD. In a population of Australian twins of Irish ancestry, Morrison et al.75 concluded that the VDR genotype (b vs. B) accounted for up to 75% of the genetic component of BMD, although a correction/partial retraction of this report appeared recently.76 Numerous subsequent studies with other population samples have found more modest,77, 78 little if any,79-82 and even conflicting associations83 of the B versus b alleles with BMD. A meta-analytic approach84 that incorporated the results of 16 VDR polymorphic studies revealed a 1.5–2.5% decrease in BMD associated with BB (versus bb) homozygotes, far less dramatic than the 12% effect originally proposed.75 Additional studies have also suggested a trend toward lower bone mass with the B allele,85-88 but it has become clear that other confounding factors, such as age, estrogen status, ethnicity, and calcium intake, must be accounted for to reveal the true impact, if any, of this VDR polymorphism on BMD. Further evaluation of the VDR gene has revealed a cluster of linked polymorphisms in the 3′ portion of the VDR gene (Fig. 2A),89 including the aforementioned BsmI site, a nearby ApaI site (in the same intron), and a silent mutation within codon 352 of the ninth exon that alters a TaqI site. These VDR polymorphisms have been linked not only to variations in bone-specific parameters but also to a higher occurrence of sporadic primary hyperparathyroidism in patients with the b allele,90 as well as to prostate cancer, which possesses a particularly strong association with the T allele (lack of the TaqI site).91 However, given that none of these polymorphisms change the encoded VDR protein in any way, the explanation for these findings has been unclear. Recently, an additional genetic variation in the hVDR gene was found in the form of a microsatellite poly(A) repeat in the 3′ UTR, approximately 1 kb upstream of the poly(A) tail (Fig. 2A). Multiple (≥12) allelic variants of this microsatellite were detected and classified into two groupings, long (L) and short (S), based upon the length of the repeat.92, 93 The L grouping (linked to T; see Fig. 2A) exhibits a strong association with prostate cancer incidence93 but displays a contrasting protective effect against breast cancer.94 It is still not established whether this poly(A) microsatellite is the functionally relevant locus, although by analogy to short tandem repeats in other genes95 the length of the repeat may affect a crucial parameter, such as mRNA stability. Alternatively, the poly(A) microsatellite may be tightly linked to yet another site which is the true functional locus. Regardless of which scenario is correct, a complicating factor in interpreting earlier findings using the BsmI, TaqI, or ApaI polymorphic restriction sites is that the linkage is imperfect between these restriction sites and the L versus S groupings, such that some ethnic groups exhibit a very tight linkage (thus displaying a clear correlation between BMD and the BsmI site), while in others (e.g., African-Americans) the presence of the BsmI or TaqI site is not a good predictor of the L versus S genotype,92 leading to a loss of functional correlation between BsmI or TaqI and BMD. Nonetheless, the study of the poly(A) microsatellite sequence may have brought us closer to a functional understanding of VDR genetic diversity, while also providing a partial explanation for the variability in association of BsmI or TaqI genotypes with BMD and hyperproliferative disorders. Near the 5′ end of the hVDR gene, a FokI restriction endonuclease site has been identified,96, 97 the presence of which (denoted f) dictates that the 427-residue, M1 isoform of VDR is expressed (so named because it contains an ATG methionine translational start site corresponding to codon #1). An evolutionarily more recent polymorphism (Fig. 3),98 or neomorph, has been reported in which both the FokI restriction site and the ATG codon #1 are changed, causing an alternative 424-residue isoform of the receptor to be translated. The F neomorph, encoding M4 hVDR (for initiation of translation at the fourth codon) already constitutes approximately 65% of VDR allel