A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.     

Calcium and vitamin D metabolism in hypocalcemic vitamin D-resistant rickets carriers

BACKGROUND/AIMS: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare monogenic autosomal recessive disorder associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D3 action. Although many investigations have discussed the clinical manifestations and molecular etiology of this disease, only a few have investigated the biochemical and hormonal status of heterozygous HVDRR. The aim of the current work was to investigate the profile of selected biochemical and hormonal parameters related to the vitamin D endocrine system in a large number of HVDRR heterozygotes. METHODS: 67 relatives of 2 HVDRR patients, all members of an extended Greek kindred of five generations with a common ancestor, were included in the study. Direct sequencing was used to identify VDR gene mutations. Serum Ca, P, 25(OH)D, iPTH, and 1,25(OH)2D levels were determined in all members of the kindred. RESULTS: DNA analysis of the participants led to the design of two study groups: the HVDRR carriers (24) and the control subjects (43). Our results showed elevated circulating serum levels of 1,25(OH)2D3 and lower levels of PTH than their age- and sex-matched controls. No hypocalcemia or hypophosphatemia were detected in HVDRR carriers. CONCLUSIONS: Our findings suggest that HVDRR carriers may have compensatory elevated serum levels of 1,25(OH)2D3 through which they restrain PTH secretion. The study of HVDRR carriers could be a useful tool for the investigation of the vitamin D endocrine system.     

Vitamin D receptor polymorphisms in hypocalcemic vitamin D-resistant rickets carriers

BACKGROUND/AIMS: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare autosomal recessive disorder characterized by severe rickets, hypocalcemia, secondary hyperparathyroidism, elevated levels of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], and occasionally, alopecia. In most cases, the disease is associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)(2)D(3) action. The apparently healthy HVDRR heterozygotes express both normal and mutant VDR alleles, and they present higher levels of 1,25(OH)(2)D(3) than their respective controls. Because VDR function, except for the disease-causative mutations, might be influenced by the presence of certain polymorphisms, we investigated the distribution of four common VDR polymorphisms--BsmI, ApaI, TaqI and FokI--in HVDRR carriers compared with their respective controls. METHODS: Sixty-seven relatives of 2 HVDRR patients, all members of an extended Greek kindred, were included in the study. VDR allelic polymorphisms were assessed by restriction fragment length polymorphisms after specific polymerase chain reaction amplification. RESULTS: The distribution of genotypic and allelic frequencies differed between HVDRR carriers and their respective controls regarding BsmI and TaqI polymorphisms. The bb genotype and the T allele (presence of BsmI and absence of TaqI polymorphisms) were less frequent in the HVDRR carrier group than in the control group in a statistically significant manner (p = 0.029 and p = 0.025, respectively). CONCLUSIONS: Our findings showed that the apparently healthy HVDRR carriers present a different distribution of BsmI and TaqI VDR polymorphisms than their controls, suggesting that further investigation of the HVDRR carrier population may elucidate the implication of VDR alleles in VDR function and the vitamin D endocrine system.     

A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR). Here we describe a patient with HVDRR who also exhibited some hypotrichosis of the scalp but otherwise had normal hair and skin. A 102 bp insertion/duplication was found in the VDR gene that introduced a premature stop (Y401X). The patient's fibroblasts expressed the truncated VDR, but were resistant to 1,25(OH)2D3. The truncated VDR weakly bound [3H]-1,25(OH)2D3 but was able to heterodimerize with RXR, bind to DNA and interact with the corepressor hairless (HR). However, the truncated VDR failed to bind coactivators and was transactivation defective. Since the patient did not have alopecia or papular lesions of the skin generally found in patients with premature stop mutations this suggests that this distally truncated VDR can still regulate the hair cycle and epidermal differentiation possibly through its interactions with RXR and HR to suppress gene transactivation.     

Ligand recognition by the vitamin D receptor.

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on, is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1,25-(OH)(2)D(3) (OCT), 20-epi-1,25-(OH)(2)D(3) and 20-epi-22-oxa-24,26,27-trihomo-1,25-(OH)(2)D(3). In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1,25-(OH)(2)D(3) and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands.     

Vitamin D and cancer

Vitamin D, a steroid hormone and exerts its biological effects through its active metabolite 1alpha, 25 dihydroxyvitamin D3 [1,25(OH)2D3]. Like steroid hormones, 1,25(OH)2D3 is efficacious at very low concentrations and serves as a ligand for vitamin D receptors (VDR), associating with VDR very high affinity. Despite its potent property as a differentiating agent, its use in the clinical practice is hampered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. Therefore nearly 400 structural analogs of vitamin D3 have been synthesized and evaluated for their efficacy and toxicity. Among these analogs, relatively less toxic but highly efficacious analogs, EB1089, RO24-5531, 1alpha-hydroxyvitamin D5 and a few others have been evaluated in a preclinical toxicity and in Phase I clinical trials for dose tolerance in advanced cancer patients. Clinical trials using vitamin D analogs for prevention or therapy of cancer patients are still in their infancy. Vitamin D mediates its action by two independent pathways. Genomic pathway involves nuclear VDR and induces biological effects by interactions with hormone response elements and modulation of differential gene expressions. Evidence also suggests that vitamin D analogs also interact with steroid hormone(s) inducible genes. The non-genomic pathway is characterized by rapid actions of vitamin D. It involves interactions with membrane-VDR interactions and its interactions with protein kinase C and by altering intracellular calcium channels. Thus, the development of nontoxic analogs of vitamin D analogs and understanding of their molecular mechanism(s) of action are of significant importance in the prevention and treatment of cancer by vitamin D.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Convergent synthesis, chiral HPLC, and vitamin D receptor affinity of analogs of 1,25-dihydroxycholecalciferol

A series of analogs of 1,25-dihydroxycholecalciferol was obtained with an additional chiral center at the terminus of the aliphatic side chain (C-25). The analogs were obtained from (+)-(R)- and (-)-(S)-2-methylglycidols, by opening of the oxirane ring with the carbanions derived from vitamin D C23a,24- or C22-sulfones. The diastereomeric purity of the analogs was determined by high-performance liquid chromatography on a chiral stationary phase. The binding affinity of analogs for the calf thymus intracellular vitamin D receptor (VDR) was two orders of magnitude lower than that of the lead compound of this group, 24a,24b-dihomo-1,25-dihydroxycholecalciferol, and it was comparable to the affinity of analogs of 24-nor-1,25-dihydroxycholecalciferol. However, a twofold difference was observed for analogs diastereomeric at C-25 in their affinity for VDR. The diastereodifferentiation of the binding affinity was found to be specific for vitamin D vicinal 25,26-diols as it disappears for analogs where 26-hydroxyl, neighboring the C-25 chiral center, is replaced with methyl.     

Highly potent cell differentiation-inducing analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1alpha,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-22-oxa-1alpha,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D3 (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.     

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

Synthesis and biological evaluation of a 3-positon epimer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)(2)D(3). ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)(2)D(3). Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)(2)D(3), an epimer of 1,25(OH)(2)D(3) at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)(2)D(3) at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D(3) analogs were found to be 1,25(OH)(2)D(3)>ED-71> or =3-epi-1,25(OH)(2)D(3)>3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Nucleo-cytoplasmic cycling of the vitamin D receptor in the enterocyte-like cell line, Caco-2

We examined the effects of 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the distribution and mobility of the vitamin D receptor (VDR) in the enterocyte-like Caco-2 cell. Confocal microscopy showed that a green fluorescent protein-vitamin D receptor (GFP-VDR) fusion protein is predominantly nuclear (58%) and it does not associate with the apical or basolateral membrane of proliferating or polarized, differentiated cells. In contrast to the previously studied cell types, neither endogenous VDR nor GFP-VDR levels accumulate in the nucleus following 1,25(OH)(2)D(3) treatment (100 nM, 30 min). However, in nuclear photobleaching experiments nuclear GFP-VDR import was significantly increased by 1,25(OH)(2)D(3) during both an early (0-5 min) and later (30-35 min) period (20% per 5 min). Compared to the natural ligand, nuclear import of GFP-VDR was 60% lower in cells treated with the 1,25(OH)(2)D(3) analog, 1-alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228, 5 min, 100 nM). Downstream events like ligand-induced association of VDR with chromatin at 1 h and the accumulation of CYP24 mRNA were significantly lower in Ro-26-9228 treated cells compared to 1,25(OH)(2)D(3) (60 and 95% lower, respectively). Collectively our data are consistent with a role for ligand-induced nuclear VDR import in receptor activation. In addition, ligand-dependent VDR nuclear import appears to be balanced by export, thus accounting for the lack of nuclear VDR accumulation even when VDR import is significantly elevated.