RESUMEN
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.
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Raquitismo Hipofosfatémico Familiar/genética , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados , Animales , Sitios de Unión , Células COS , Células Cultivadas , Chlorocebus aethiops , Codón sin Sentido , Exones , Raquitismo Hipofosfatémico Familiar/patología , Femenino , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Mutación Missense , Vitamina D/metabolismoRESUMEN
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.
Asunto(s)
Resistencia a Medicamentos/genética , Mutación Missense/genética , Receptores de Calcitriol/genética , Raquitismo/genética , Árabes/genética , Sitios de Unión/genética , Femenino , Humanos , Lactante , Masculino , Radiografía , Receptores de Calcitriol/química , Raquitismo/diagnóstico por imagen , Raquitismo/tratamiento farmacológico , Vitamina D/uso terapéuticoRESUMEN
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.
Asunto(s)
Alopecia/tratamiento farmacológico , Mutación Missense , Receptores de Calcitriol/genética , Raquitismo/tratamiento farmacológico , Vitamina D/análogos & derivados , Alopecia/genética , Alopecia/patología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Calcio/uso terapéutico , Células Cultivadas , Chlorocebus aethiops , Análisis Mutacional de ADN , Resistencia a Medicamentos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Raquitismo/genética , Raquitismo/patología , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina D/metabolismo , Vitamina D/uso terapéutico , Vitamina D3 24-HidroxilasaRESUMEN
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.
Asunto(s)
Alopecia/genética , Codón sin Sentido , Receptores de Calcitriol/genética , Raquitismo/genética , Biomarcadores/metabolismo , Preescolar , Análisis Mutacional de ADN , Resistencia a Medicamentos , Inducción Enzimática/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Humanos , Receptores de Calcitriol/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Esteroide Hidroxilasas/biosíntesis , Vitamina D/análogos & derivados , Vitamina D/farmacología , Vitamina D/uso terapéutico , Vitamina D3 24-HidroxilasaRESUMEN
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.
Asunto(s)
Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Metribolona/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Western Blotting , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Masculino , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa.
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Calcitriol/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata/metabolismoRESUMEN
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.
Asunto(s)
Andrógenos/farmacología , Andrógenos/fisiología , Crecimiento/efectos de los fármacos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Neoplasias de la Próstata/metabolismo , Elementos de Respuesta/fisiología , Western Blotting , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Línea Celular Tumoral , Cromatina/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Humanos , Inmunoprecipitación , Masculino , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
UNLABELLED: In this study, we report that the function of certain mutant VDRs from patients with hereditary HVDRR can at least be partially restored by phosphorylation and hexafluoro 1,25(OH)2D3 analogs. Our study provides new insights into mechanisms involved in enhancement of mutant VDR function. INTRODUCTION: 1,25-Dihydroxyvitamin D-resistant rickets (HVDRR) is a rare genetic disorder caused by inactivating mutations in the vitamin D receptor (VDR). In this study, we examined VDR from patients with HVDRR having mutations in the ligand-binding domain (F251C, I268T, H305Q, E420K). We examined methods of restoring transcriptional activity of these mutants and the mechanisms involved. MATERIALS AND METHODS: Reporter gene transcriptional assays were used to examine the activation of mutant VDRs. Western-blot analysis, glutathione S-transferase (GST) pull-down assays, and chromatin immunoprecipitation (ChIP) assays were also used in this study. RESULTS: Using mutant VDRs, H305Q, F251C, I268T, and 10(-8) M 1,25(OH)2D3, only 10-30% of the activity of wildtype (WT) VDR in activating 24(OH)ase transcription was observed. The transcriptional response of mutant VDR mutants was significantly enhanced 2- to 3-fold by co-treatment of VDR mutant transfected COS-7 cells with 1,25(OH)2D3 and okadaic acid (OA; inhibitor of phosphatase; 50 nM). The H305Q mutant was the most responsive (90% of the response exhibited by WT VDR was restored). The E420K mutant was unresponsive to 1,25(OH)2D3 in the presence or absence of OA. The increased transcriptional response correlated with an increase in the interaction between DRIP205 and the mutant VDR. We further provide evidence that OA induces the phosphorylation of CREB-binding protein (CBP), indicating for the first time a correlation between phosphorylation of CBP and enhanced VDR function. Hexafluoro 1,25(OH)2D3 analogs (RO-26-2198 and RO-4383561) also resulted in at least a partial restoration of the transcriptional responsiveness of mutant VDRs I268T, F251C, and H305Q. Our data indicate that the enhanced potency of the hexafluoro analogs may be caused by increased DRIP205 and glucocorticoid receptor interacting protein 1 (GRIP-1) binding to VDRs and enhanced association of VDRs with DNA, as suggested by results of ChIP assays. CONCLUSION: Our study provides new insights into the mechanisms involved in the enhancement of VDR function by both phosphorylation and hexafluoro analogs and forms a basis for future study of vitamin D analogs or specifically designed kinase activity mediators as potential therapy for the treatment of selected patients with HVDRR.
Asunto(s)
Resistencia a Medicamentos , Mutación , Receptores de Calcitriol/genética , Raquitismo/tratamiento farmacológico , Raquitismo/genética , Vitamina D/análogos & derivados , Animales , Western Blotting , Células COS , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Genes Reporteros , Glutatión Transferasa/metabolismo , Ligandos , Subunidad 1 del Complejo Mediador , Ratones , Proteínas del Tejido Nervioso/metabolismo , Ácido Ocadaico/farmacología , Osteoblastos/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/farmacología , Transcripción Genética , Transfección , Vitamina D/farmacologíaRESUMEN
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.
Asunto(s)
Calcitriol/farmacología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genética , Elemento de Respuesta a la Vitamina D/genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Hipofosfatemia Familiar/genética , Mutación , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Alopecia/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Calcitriol/metabolismo , Calcitriol/farmacología , Cesárea , Sistema Enzimático del Citocromo P-450/genética , Genotipo , Humanos , Recién Nacido , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/genética , Receptores de Calcitriol/química , Esteroide Hidroxilasas/genética , Activación Transcripcional , Estados Unidos , Vitamina D3 24-Hidroxilasa , Población BlancaRESUMEN
INTRODUCTION: Hereditary vitamin D--resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young boy who exhibited the typical clinical features of HVDRR but without alopecia. MATERIALS AND METHODS: The patient's VDR was studied using cultured dermal fibroblasts, and the recreated mutant VDR was analyzed in transfected cells. RESULTS: The patient's fibroblasts were resistant to 1,25-dihydroxyvitamin D [1,25(OH)2D3], exhibiting only a slight induction of 24-hydroxylase gene expression when treated with 1 microM 1,25(OH)2D3 x [3H]1,25(OH)2D3 binding was absent in cell extracts from the patient's fibroblasts. Sequence analysis of the VDR gene uncovered a unique 5-bp deletion/8-bp insertion in exon 4. The mutation in helix HI of the ligand-binding domain deletes two amino acids (H141 and T142) and inserts three amino acids (L141, W142, and A143). In transactivation assays, the recreated mutant VDR was 1000-fold less active than the wildtype (WT) VDR. In glutathione S-transferase (GST) pull-down assays, the mutant VDR bound GST-retinoid X receptor (RXR) weakly in the absence of 1,25(OH)2D3; however, the binding did not increase with increasing concentrations of ligand. The mutant VDR did not bind to GST-vitamin D receptor interacting protein (DRIP) 205 at concentrations up to 1 microM 1,25(OH)2D3. We also examined effects of the three individual mutations on VDR transactivation. Only the insertion of A143 into the WT VDR disrupted VDR transactivation to the same extent observed with the natural mutation. CONCLUSION: We describe a novel insertion/substitution mutation in helix Hl of the VDR ligand-binding domain (LBD) that abolishes ligand binding and result in the syndrome of HVDRR. This is the first time an insertion/substitution has been found as the defect-causing HVDRR.
Asunto(s)
Receptores de Calcitriol/metabolismo , Raquitismo/genética , Vitamina D/análogos & derivados , Vitamina D/uso terapéutico , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Preescolar , Cartilla de ADN , Humanos , Ligandos , Masculino , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Receptores de Calcitriol/genética , Raquitismo/tratamiento farmacológicoRESUMEN
Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.
Asunto(s)
Calcitriol/uso terapéutico , Agonistas de los Canales de Calcio/uso terapéutico , Receptores de Calcitriol/genética , Raquitismo/tratamiento farmacológico , Raquitismo/genética , Alopecia , Animales , Sitios de Unión/genética , Células COS , Dimerización , Resistencia a Medicamentos/genética , Femenino , Genotipo , Humanos , Lactante , Péptido Hidrolasas/farmacología , Mutación Puntual , Polimorfismo de Longitud del Fragmento de Restricción , Estructura Terciaria de Proteína , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores X Retinoide/química , Receptores X Retinoide/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Heterogeneous loss of function mutations in the vitamin D receptor (VDR) interfere with vitamin D signaling and cause hereditary vitamin D-resistant rickets (HVDRR). HVDRR is characterized by hypocalcemia, secondary hyperparathyroidism and severe early-onset rickets in infancy and is often associated with consanguinity. Affected children may also exhibit alopecia of the scalp and total body. The children usually fail to respond to treatment with calcitriol; in fact, their endogenous levels are often very elevated. Successful treatment requires reversal of hypocalcemia and secondary hyperparathyroidism and is usually accomplished by administration of high doses of calcium given either intravenously or sometimes orally to bypass the intestinal defect in VDR signaling.
RESUMEN
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.
Asunto(s)
Fibroblastos/fisiología , Silenciador del Gen , Canales Iónicos/genética , Proteínas Mitocondriales/genética , Receptores de Calcitriol/fisiología , Tejido Adiposo Pardo/metabolismo , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Raquitismo Hipofosfatémico Familiar/metabolismo , Raquitismo Hipofosfatémico Familiar/patología , Humanos , Canales Iónicos/metabolismo , Ligandos , Proteínas Mitocondriales/metabolismo , Unión Proteica , Proteína Desacopladora 1 , Elemento de Respuesta a la Vitamina DRESUMEN
BACKGROUND: We report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube. CLINICAL CASE: A 22 month old male (length -3.4 SDS; weight -2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1mg/dL, (8.8-10.8); phosphorus 4.1mg/dL, (4.5-5.5); alkaline phosphatase 1481 U/L (80-220); intact PTH 537.1 pg/mL (10-71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34 ng/mL (20-100); 1,25-dihydroxyvitamin D 507 pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8 mg/dL using the intravenous preparation of calcium chloride administered via gastric tube. GENETIC STUDIES: A unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia. CONCLUSION: HVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.
Asunto(s)
Cloruro de Calcio/administración & dosificación , Raquitismo Hipofosfatémico Familiar , Mutación Puntual , Receptores de Calcitriol/genética , Fosfatasa Alcalina/sangre , Alopecia , Animales , Células COS , Calcifediol/sangre , Calcio/sangre , Chlorocebus aethiops , Raquitismo Hipofosfatémico Familiar/sangre , Raquitismo Hipofosfatémico Familiar/diagnóstico por imagen , Raquitismo Hipofosfatémico Familiar/tratamiento farmacológico , Raquitismo Hipofosfatémico Familiar/genética , Humanos , Lactante , Masculino , Fósforo/sangre , Radiografía , Receptores de Calcitriol/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Calcitriol/fisiología , Fibroblastos/metabolismo , Proteína Disulfuro Isomerasas/fisiología , Receptores de Calcitriol/fisiología , Núcleo Celular/metabolismo , Células Cultivadas , Raquitismo Hipofosfatémico Familiar/patología , Fibroblastos/efectos de la radiación , Humanos , Mutación Missense , Unión Proteica , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Estructura Terciaria de Proteína , Dímeros de Pirimidina/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Alopecia/genética , Mutación , Receptores de Calcitriol/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/fisiología , Alopecia/complicaciones , Animales , Calcio/metabolismo , Raquitismo Hipofosfatémico Familiar/complicaciones , Raquitismo Hipofosfatémico Familiar/genética , Raquitismo Hipofosfatémico Familiar/metabolismo , Folículo Piloso/metabolismo , Homeostasis , Humanos , Ratones , Ratones Noqueados , Receptores de Calcitriol/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
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).
Asunto(s)
Raquitismo Hipofosfatémico Familiar/genética , Heterocigoto , Mutación/genética , Receptores de Calcitriol/genética , Adolescente , Calcitriol/uso terapéutico , Niño , Preescolar , Ciclooxigenasa 2/genética , Análisis Mutacional de ADN , Raquitismo Hipofosfatémico Familiar/diagnóstico por imagen , Raquitismo Hipofosfatémico Familiar/tratamiento farmacológico , Raquitismo Hipofosfatémico Familiar/enzimología , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Genes Dominantes/genética , Humanos , Immunoblotting , Lactante , Proteínas Mutantes/metabolismo , Multimerización de Proteína , Radiografía , Receptores X Retinoide/genética , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Activación Transcripcional/genética , Vitamina D3 24-HidroxilasaRESUMEN
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.