Vitamin D receptor mutations in patients with hereditary 1,25-dihydroxyvitamin D-resistant rickets.

CONTEXT: Hereditary vitamin D resistant rickets (HVDRR), also known as vitamin D-dependent rickets type II, is an autosomal recessive disorder characterized by the early onset of rickets with hypocalcemia, secondary hyperparathyroidism and hypophosphatemia and is caused by mutations in the vitamin D receptor (VDR) gene. The human gene encoding the VDR is located on chromosome 12 and comprises eight coding exons and seven introns. OBJECTIVES, PATIENTS, AND METHODS: We analyzed the VDR gene of 5 previously unreported patients, two from Singapore and one each from Macedonia (former Yugoslav Republic), Saudi Arabia and Turkey. Each patient had clinical and radiographic features of rickets, hypocalcemia, and the 4 cases that had the measurement showed elevated serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)(2)D). Mutations were re-created in the WT VDR cDNA and examined for 1,25(OH)(2)D(3)-mediated transactivation in COS-7 monkey kidney cells. RESULTS: Direct sequencing identified four novel mutations and two previously described mutations in the VDR gene. The novel mutations included a missense mutation in exon 3 causing the amino acid change C60W; a missense mutation in exon 4 causing the amino acid change D144N; a missense mutation in exon 7 causing the amino acid change N276Y; and a 2bp deletion in exon 3 5'-splice site (IVS3∆+4-5) leading to a premature stop. CONCLUSIONS: These 4 unique mutations add to the previous 45 mutations identified in the VDR gene in patients with HVDRR.

Cell-autonomous regulation of brown fat identity gene UCP1 by unliganded vitamin D receptor.

White adipose tissue stores energy in the form of lipids, and brown adipose tissue expends energy via uncoupled fatty acid oxidation, which leads to the generation of heat. Obesity reflects an imbalance between energy storage and energy expenditure and is strongly associated with metabolic and cardiovascular disease. Therefore, there are important medical and biological implications for elucidating the mechanisms that promote energy expenditure in humans. Animal models with altered vitamin D receptor (VDR) expression have changes in energy expenditure. However, the specific mechanism for this effect has not been elucidated and the relevance for humans is unclear. Here we show, using human patient samples from individuals with hereditary vitamin D resistant rickets, that the VDR directly inhibits the expression of uncoupling protein-1 (UCP1), the critical protein for uncoupling fatty acid oxidation in brown fat and burning energy. The inhibition is enforced by VDR occupancy of a negative response element in the promoter proximal region of the UCP1 gene. Deletion of VDR increases UCP1 expression and results in a "browning" of adipocytes. Importantly, we found that this process occurs cell autonomously and is independent of the physiologic VDR hormone ligand, 1,25-dihydroxyvitamin D. These results identify a mechanism for modulating energy balance in humans.

Genetic disorders and defects in vitamin D action.

The biochemical and genetic analysis of the VDR in patients with HVDRR has yielded important insights into the structure and function of the receptor in mediating 1,25(OH)2D3 action. Similarly, study of children affected by HVDRR continues to provide a more complete understanding of the biologic role of 1,25(OH)2D3 in vivo. A concerted investigative approach to HVDRR at the clinical, cellular, and molecular levels has proved valuable in gaining knowledge of the functions of the domains of the VDR and elucidating the detailed mechanism of action of 1,25(OH)2D3. These studies have been essential to promote the well-being of the families with HVDRR and in improving the diagnostic and clinical management of this rare genetic disease.

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3.

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

Report of two unrelated patients with hereditary vitamin D resistant rickets due to the same novel mutation in the vitamin D receptor.

BACKGROUND/AIMS: Two unrelated patients found to have hereditary vitamin D resistant rickets (HVDRR) were admitted to our hospital. METHODS: This article describes the diagnosis, management and molecular basis for their disease. RESULTS: Both patients had severe growth and motor developmental retardation, rickets with chest deformities and pulmonary abnormalities, but no alopecia. Both had hypocalcemia, secondary hyperparathyroidism and susceptibility to pulmonary infections. In both cases, good response with normalization of abnormal biochemistries and healing of rickets was achieved with IV calcium infusion. Subsequently, improvement was maintained with oral calcium. Both children harbored the same unique missense mutation in the vitamin D receptor (VDR) gene that substituted arginine with histidine at amino acid 274 (R274H) in the VDR ligand-binding domain (LBD). R274 is a contact point for the 1alpha-hydroxyl group of 1,25(OH)2D3, the active ligand for the VDR. Functional analyses of the R274H mutation revealed a 100-fold decrease in activity compared to wild-type VDR. CONCLUSION: We describe a novel missense mutation at R274H in the VDR gene that resulted in the HVDRR syndrome in two unrelated children. Vigorous treatment using IV calcium to normalize their hypocalcemia achieved dramatic improvement in these complex and severely ill patients.

Hereditary vitamin D-resistant rickets (HVDRR) owing to a heterozygous mutation in the vitamin D receptor.

Hereditary vitamin D-resistant rickets (HVDRR) is a rare autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). Patients exhibit severe rickets and hypocalcemia. Heterozygous parents and siblings appear normal and exhibit no symptoms of the disease. We analyzed the VDR gene of a young girl who exhibited the clinical features of HVDRR without alopecia. The patient had clinical and radiographic features of rickets, hypocalcemia, and elevated serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. A single heterozygous missense mutation was found in the VDR gene that substituted glutamic acid with alanine at amino acid 420 (E420A). Sequencing of the girl's VDR cDNAs showed that the f/M1 allele contained the E420A mutation, whereas the F/M4 allele was completely normal. The girl's father, who was also heterozygous for the E420A mutation on the f/M1 allele, exhibited minor symptoms of vitamin D resistance. In contrast, the mother had no signs of the disease and had no mutations in her VDR gene. Both the girl and the father's skin fibroblasts showed resistance to 1,25(OH)(2)D(3) by their severely reduced induction of CYP24A1 gene expression. In transactivation assays, the E420A mutant VDR showed dominant-negative activity towards the wild-type VDR. This is the first report that we are aware of describing a patient with HVDRR caused by a single heterozygous missense mutation in the VDR gene. The E420A mutant appears to act in a dominant-negative fashion, silencing the wild-type VDR and resulting in an attenuated response to 1,25(OH)(2)D(3).

The role of vitamin D receptor mutations in the development of alopecia.

Hereditary Vitamin D Resistant Rickets (HVDRR) is a rare disease caused by mutations in the vitamin D receptor (VDR). The consequence of defective VDR is the inability to absorb calcium normally in the intestine. This leads to a constellation of metabolic abnormalities including hypocalcemia, secondary hyperparathyroidism and hypophosphatemia that cause the development of rickets at an early age in affected children. An interesting additional abnormality is the presence of alopecia in some children depending on the nature of the VDR mutation. The data indicate that VDR mutations that cause defects in DNA binding, RXR heterodimerization or absence of the VDR cause alopecia while mutations that alter VDR affinity for 1,25(OH)(2)D(3) or disrupt coactivator interactions do not cause alopecia. The cumulative findings indicate that hair follicle cycling is dependent on unliganded actions of the VDR. Further research is ongoing to elucidate the role of the VDR in hair growth and differentiation.

Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D(3) analogues hydroxymethylated at C-26.

We designed by docking and synthesized two novel analogues of 1α,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.

Two new unrelated cases of hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from the same novel nonsense mutation in the vitamin D receptor gene.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) an important regulator of bone homeostasis, mediates its actions by binding to the vitamin D receptor (VDR), a nuclear transcription factor. Mutations in the VDR cause the rare genetic disease hereditary vitamin D resistant rickets (HVDRR). In this study, we examined two unrelated young female patients who exhibited severe early onset rickets, hypocalcemia, and hypophosphatemia. Both patients had partial alopecia but with different unusual patterns of scant hair. Sequencing of the VDR gene showed that both patients harbored the same unique nonsense mutation that resulted in a premature stop codon (R50X). Skin fibroblasts from patient #1 were devoid of VDR protein and 1,25(OH)2D3 treatment of these cells failed to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 action. In conclusion, we identified a novel nonsense mutation in the VDR gene in two patients with HVDRR and alopecia. The mutation truncates the VDR protein and causes 1,25(OH)2D3 resistance.

Genetic disorders and defects in vitamin d action.

Two rare genetic diseases can cause rickets in children. The critical enzyme to synthesize calcitriol from 25-hydroxyvitamin D, the circulating hormone precursor, is 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). When this enzyme is defective and calcitriol can no longer be synthesized, the disease 1alpha-hydroxylase deficiency develops. The disease is also known as vitamin D-dependent rickets type 1 or pseudovitamin D deficiency rickets. When the VDR is defective, the disease hereditary vitamin D-resistant rickets, also known as vitamin D-dependent rickets type 2, develops. Both diseases are rare autosomal recessive disorders characterized by hypocalcemia, secondary hyperparathyroidism, and early onset severe rickets. In this article, these 2 genetic childhood diseases, which present similarly with hypocalcemia and rickets in infancy, are discussed and compared.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from a novel missense mutation in the DNA-binding domain of the vitamin D receptor.

The rare genetic recessive disease, hereditary vitamin D resistant rickets (HVDRR), is caused by mutations in the vitamin D receptor (VDR) that result in resistance to the active hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) or calcitriol). In this study, we examined the VDR from a young boy with clinical features of HVDRR including severe rickets, hypocalcemia, hypophosphatemia and partial alopecia. The pattern of alopecia was very unusual with areas of total baldness, adjacent to normal hair and regions of scant hair. The child failed to improve on oral calcium and vitamin D therapy but his abnormal chemistries and his bone X-rays normalized with intravenous calcium therapy. We found that the child was homozygous for a unique missense mutation in the VDR gene that converted valine to methionine at amino acid 26 (V26M) in the VDR DNA-binding domain (DBD). The mutant VDR was studied in the patient's cultured skin fibroblasts and found to exhibit normal [(3)H]1,25(OH)(2)D(3) binding and protein expression. However, the fibroblasts were unresponsive to treatment with high concentrations of 1,25(OH)(2)D(3) as demonstrated by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)(2)D(3) responsiveness. We recreated the V26M mutation in the WT VDR and showed that in transfected COS-7 cells the mutation abolished 1,25(OH)(2)D(3)-mediated transactivation. The mutant VDR exhibited normal ligand-induced binding to RXRalpha and to the coactivator DRIP205. However, the V26M mutation inhibited VDR binding to a consensus vitamin D response element (VDRE). In summary, we have identified a novel V26M mutation in the VDR DBD as the molecular defect in a patient with HVDRR and an unusual pattern of alopecia.

Modulation of vitamin d receptor activity by the corepressor hairless: differential effects of hairless isoforms.

The vitamin D receptor (VDR) and its corepressor Hairless (HR) are thought to regulate key steps in the hair cycle because mutations in VDR or HR cause alopecia in humans and mice. Many mammalian cells express two major HR isoforms due to alternative splicing of exon 17. HR isoform-a encodes an 1189-amino acid protein (full-length HR), and isoform-b encodes an 1134-amino acid protein (HRDelta1072-1126). We demonstrated that both HR isoforms are expressed in primary human keratinocytes and in the human keratinocyte cell line HaCaT. In transfected COS-7 cells, the full-length HR repressed VDR-mediated transactivation. In contrast, HRDelta1072-1126 failed to suppress and even stimulated VDR-mediated transactivation. In coimmunoprecipitation, both HR isoforms interacted with the VDR, but only the full-length HR interacted with histone deacetylase 1 (HDAC1). Alanine mutagenesis of two conserved glutamic acids residues (E1100A/E1101A) encoded by exon 17 completely eliminated HR corepressor activity and interactions with HDAC1. When the two HR isoforms were coexpressed in COS-7 cells, the corepressor activity of the full-length HR was not antagonized by the HRDelta1072-1126 isoform. When transfected into HaCaT cells, the full-length HR inhibited endogenous CYP24A1 basal gene expression as well as 1,25-dihydroxyvitamin D3-stimulated CYP24A1 expression. HRDelta1072-1126 failed to suppress basal or 1,25-dihydroxyvitamin D3-stimulated CYP24A1 gene expression. In conclusion, we have demonstrated that both HR isoforms are expressed in keratinocytes and that the HRDelta1072-1126 isoform lacks corepressor activity and is unable to bind HDACs. HRDelta1072-1126 may function as a coactivator in some settings by inhibiting HDAC recruitment to the VDR transcriptional complex.

Hereditary vitamin D resistant rickets: identification of a novel splice site mutation in the vitamin D receptor gene and successful treatment with oral calcium therapy.

OBJECTIVE: To study the vitamin D receptor (VDR) gene in a young girl with severe rickets and clinical features of hereditary vitamin D resistant rickets, including hypocalcemia, hypophosphatemia, partial alopecia, and elevated serum levels of 1,25-dihydroxyvitamin D. STUDY DESIGN: We amplified and sequenced DNA samples from blood from the patient, her mother, and the patient's two siblings. We also amplified and sequenced the VDR cDNA from RNA isolated from the patient's blood. RESULTS: DNA sequence analyses of the VDR gene showed that the patient was homozygous for a novel guanine to thymine substitution in the 5'-splice site in the exon 8-intron J junction. Analysis of the VDR cDNA using reverse transcriptase-polymerase chain reaction showed that exons 7 and 9 were fused, and that exon 8 was skipped. The mother was heterozygous for the mutation and the two siblings were unaffected. CONCLUSIONS: A novel splice site mutation was identified in the VDR gene that caused exon 8 to be skipped. The mutation deleted amino acids 303-341 in the VDR ligand-binding domain, which is expected to render the VDR non-functional. Nevertheless, successful outpatient treatment was achieved with frequent high doses of oral calcium.

Inactivation of the human vitamin D receptor by caspase-3.

Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [(3)H]1,25-dihydroxy vitamin D(3) binding and VDR protein, suggesting that the VDR may be targeted for inactivation by caspases during apoptosis. A potential caspase-3 site (D(195)MMD(198)S) was identified in the human VDR ligand-binding domain. Mutations D195A, D198A, and S199A were generated in the putative capase-3 cleavage site. In transfected COS-7 cells, STS treatment resulted in the cleavage of the wild-type (WT) VDR and S199A mutant VDR but not the D195A or D198A mutants. In in vitro assays, the WT VDR and S199A mutant VDR were cleaved by caspase-3, although the D195A and D198A mutants were resistant to caspase-3. In vitro, the WT VDR was also cleaved by caspase-6 and caspase-7 and in extracts of STS-treated LNCaP cells. In STS-treated LNCaP cells and human skin fibroblasts, the proteasome inhibitor MG-132 protected the VDR caspase cleavage fragment from further degradation by the 26S proteasome. The rat VDR that does not contain the caspase-3 cleavage site was not cleaved in STS-treated COS-7 cells. In conclusion, our results demonstrate that the human VDR is a target of caspase-3 and suggest that activation of caspase-3 may limit VDR activity.

Compound heterozygous mutations in the vitamin D receptor in a patient with hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia.

Hereditary vitamin D-resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)(2)D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA-binding domain and a unique 3-bp in-frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (DeltaK246). The child and her mother were both heterozygous for the 3-bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)(2)D(3) as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)(2)D(3) responsiveness. [(3)H]1,25(OH)(2)D(3) binding and immunoblot analysis showed that the patient's cells expressed the VDRDeltaK246 mutant protein; however, the amount of VDRDeltaK246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDRDeltaK246 mutant was unresponsive to 1,25(OH)(2)D(3). The DeltaK246 mutation abolished heterodimerization of the mutant VDR with RXRalpha and binding to the coactivators DRIP205 and SRC-1. However, the DeltaK246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the DeltaK246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions.

Interaction of the vitamin D receptor with a vitamin D response element in the Mullerian-inhibiting substance (MIS) promoter: regulation of MIS expression by calcitriol in prostate cancer cells.

Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth of a variety of cancer cells including human prostate cancer. Müllerian-inhibiting substance (MIS) also exhibits antiproliferative and proapoptotic actions on multiple cancer cells including human prostate cancer. In this study, we investigated whether calcitriol regulated MIS expression in prostate cancer, an action that might contribute to its antiproliferative activity. We identified a 15-bp sequence, GGGTGAgcaGGGACA, in the MIS promoter that was highly similar to direct repeat 3-type vitamin D response elements (VDREs). The human MIS promoter containing the putative VDRE was cloned into a luciferase reporter vector. In HeLa cells transfected with the vitamin D receptor (VDR), MIS promoter activity was stimulated by calcitriol. Coexpression of steroidogenic factor 1, a key regulator of MIS, increased basal MIS promoter activity that was further stimulated by calcitriol. Mutation or deletion of the VDRE reduced calcitriol-induced transactivation. In addition, the MIS VDRE conferred calcitriol responsiveness to a heterologous promoter. In gel shift assays, VDR and retinoid X receptor bound to the MIS VDRE and the binding was increased by calcitriol. Chromatin immunoprecipitation assays showed that VDR and retinoid X receptor were present on the MIS promoter in prostate cancer cells. In conclusion, we demonstrated that MIS is a target of calcitriol action. MIS is up-regulated by calcitriol via a functional VDRE that binds the VDR. Up-regulation of MIS by calcitriol may be an important component of the antiproliferative actions of calcitriol in some cancers.

The role of insulin-like growth factor binding protein-3 in the growth inhibitory actions of androgens in LNCaP human prostate cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3), an antiproliferative and proapoptotic protein, has been shown to be upregulated by growth inhibitory concentrations of androgens in LNCaP human prostate cancer (PCa) cells, but the mechanism of regulation and the role of IGFBP-3 in the modulation of PCa cell proliferation are unknown. In this study, we have examined the effects of a range of concentrations of the synthetic androgen R1881 on IGFBP-3 expression and cell growth in LNCaP cells. We have also investigated the role of androgen-stimulated IGFBP-3 in androgen-induced growth inhibition. We show that low doses of R1881 stimulate LNCaP cell proliferation, but do not induce IGFBP-3 expression, whereas high doses of R1881 that inhibit cell growth, significantly increase expression of IGFBP-3. Importantly, we demonstrate that the combination of calcitriol and androgens not only synergistically upregulates IGFBP-3 expression but also inhibits cell growth better than either hormone alone. siRNA knockdown of IGFBP-3 expression partially reverses the growth inhibition by calcitriol and by androgens. Furthermore, we find that the growth inhibitory dose of R1881 leads to increases in the cyclin dependent kinase inhibitors (CDKIs), p21 and p27 as well as to G1 arrest. These changes can be blocked or partially reversed by IGFBP-3 siRNA, indicating that the induction of CDKIs is downstream of IGFBP-3. Our data suggest, for the first time, that IGFBP-3 is involved in the antiproliferative action of high doses of androgens partly through p21 and p27 pathways and that IGFBP-3 may contribute significantly to androgen-induced changes in LNCaP cell growth.

Interactions of the vitamin D receptor with the corepressor hairless: analysis of hairless mutants in atrichia with papular lesions.

Atrichia with papular lesions (APL) and hereditary vitamin D-resistant rickets have a similar congenital hair loss disorder caused by mutations in hairless (HR) and vitamin D receptor (VDR) genes, respectively. HR is a VDR corepressor, and it has been hypothesized that VDR.HR suppress gene expression during specific phases of the hair cycle. In this study, we examined the corepressor activity of HR mutants (E583V, C622G, N970S, V1056M, D1012N, V1136D, and Q1176X) previously described as the molecular cause of APL as well as HR variants (P69S, C397Y, A576V, E591G, R620Q, T1022A) due to non-synonymous polymorphisms in the HR gene. We found that the corepressor activities of all but one of the pathogenic HR mutants were completely abolished. HR mutant E583V exhibited normal corepressor activity, suggesting that it may not be pathogenic. In co-immunoprecipitation assays, all of the pathogenic HR mutants bound VDR but exhibited reduced binding to histone deacetylase 1 (HDAC1), suggesting that the impaired corepressor activity is due in part to defective interactions with HDACs. The HR variants exhibited two classes of corepressor activity, those with normal activity (C397Y, E591G, R620Q) and those with partially reduced activity (P69S, A576V, T1022A). All of the variants interacted with VDR and HDAC1 with the exception of P69S, which was degraded. When coexpressed with VDR, all of the HR pathogenic mutants and variants increased the level of VDR protein, demonstrating that this function of HR was not impaired by these mutations. This study of HR mutations provides evidence for the molecular basis of APL.

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.

Growth inhibitory concentrations of androgens up-regulate insulin-like growth factor binding protein-3 expression via an androgen response element in LNCaP human prostate cancer cells.

IGF binding protein-3 (IGFBP-3), the most abundant circulating IGF binding protein, inhibits cell growth and induces apoptosis by both IGF-I-dependent and -independent pathways. The ability of IGFBP-3 to inhibit tumor growth has been demonstrated in many cancers including prostate cancer (PCa). High concentrations of androgens, which inhibit the growth of the LNCaP human PCa cell line, have been shown to have both positive and negative effects on IGFBP-3 expression by different laboratories. To further explore the relationship between IGFBP-3 and androgens, we examined IGFBP-3 expression in LNCaP cells. We demonstrate that IGFBP-3 expression can be induced by 10 nm of the synthetic androgen R1881 or dihydrotestosterone. Transactivation assays show that the 6-kb IGFBP-3 promoter sequence directly responds to androgen treatment. In silico analysis identified a putative androgen response element (ARE) at -2,879/-2,865 in the IGFBP-3 promoter. A single point mutation in this ARE disrupted transactivation by R1881. Combining the data obtained from EMSA, chromatin immunoprecipitation and mutational analysis, we conclude that a novel functional ARE is present in the IGFBP-3 promoter that directly mediates androgen induction of IGFBP-3 expression. Furthermore, we found that the combination of androgens and calcitriol significantly potentiated the IGFBP-3 promoter activity, suggesting that enhanced induction of the expression of the endogenous IGFBP-3 gene may contribute to the greater inhibition of LNCaP cell growth by combined calcitriol and androgens. Because androgens are well known to stimulate PCa growth and androgen deprivation therapy causes PCa to regress, the stimulation by androgens of this antiproliferative and proapoptotic protein is paradoxical and raises interesting questions about the role of androgen-stimulated IGFBP-3 in PCa.