A haplotype of the CYP27B1 promoter is associated with autoimmune Addison's disease but not with Graves' disease in a UK population.
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Previous studies have suggested an association between alleles of the CYP27B1 (1-alpha hydroxylase) gene and autoimmune conditions. We have examined three single nucleotide polymorphisms (SNPs) that are located in the 5' region and promoter of the CYP27B1 gene for association in a cohort of Graves' disease and autoimmune Addison's disease subjects from the UK. Genomic DNA samples from white patients with autoimmune Addison's disease (n = 104) and healthy controls (n = 464) were genotyped by PCR-RFLP analysis for the SNPs at positions -1260 and -1077 5' of the coding CYP27B1 sequences. The -1260 SNP was also examined in a cohort of patients with Graves' disease (n = 446). Chi 2 testing of contingency tables was used to determine the significance of genotypes and alleles. Haplotype frequencies and linkage disequilibrium measures were estimated using the UNPHASED and HAPLOVIEW packages. Alleles at the three CYP27B1 markers were in tight linkage disequilibrium with each other and all showed association with autoimmune Addison's disease. The maximum evidence for association was with the -1260 C allele (76.0% in Addison's subjects versus 64.9% in controls; P = 0.003; odds ratio 1.71 (5-95% confidence intervals, 1.20-2.44). A global test of significance for the common -1918 T, -1260 C and -1077 G haplotype was significant in Addison's subjects compared with controls (P = 0.01). In contrast, there was no association of alleles at the -1260 SNP with Graves' disease. We are able to confirm that a CYP27B1 promoter allele is associated with autoimmune Addison's disease, and extend this finding to include an associated promoter haplotype. (+info)
Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1.
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We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3. (+info)
Vitamin D(3) metabolism in human glioblastoma multiforme: functionality of CYP27B1 splice variants, metabolism of calcidiol, and effect of calcitriol.
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PURPOSE: A better understanding of the vitamin D(3) metabolism is required to evaluate its potential therapeutic value for cancers. Here, we set out to contribute to the understanding of vitamin D(3) metabolism in glioblastoma multiforme. EXPERIMENTAL DESIGN: We did nested touchdown reverse transcription-PCR (RT-PCR) to identify CYP27B1 splice variants and real-time RT-PCR to quantify the expression of CYP27B1. A cell line was treated with calcitriol to determine the effect on the expression of CYP27B1, 1alpha,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24), and vitamin D(3) receptor (VDR). We generated three antibodies for the specific detection of CYP27B1 and splice variants. High-performance TLC was done to determine the endogenous CYP27B1 activity and the functionality of CYP27B1 splice variants. Using WST-1 assay, we determined the effect of vitamin D(3) metabolites on proliferation. RESULTS: We report a total of 16 splice variants of CYP27B1 in glioblastoma multiforme and a different expression of CYP27B1 and variants between glioblastoma multiforme and normal tissues. We found preliminary evidence for enzymatic activity of endogenous CYP27B1 in glioblastoma multiforme cell cultures but not for the functionality of the splice variants. By adding calcitriol, we found a proliferative effect for some cell lines depending on the dose of calcitriol. The administration of calcitriol led to an elevated expression of CYP27B1 and CYP24 but left the expression of the VDR unaltered. CONCLUSIONS: Our findings show that glioblastoma multiforme cell lines metabolize calcidiol. In addition, we show various effects mediated by calcitriol. We found a special vitamin D(3) metabolism and mode of action in glioblastoma multiforme that has to be taken into account in future vitamin D(3)-related therapies. (+info)
Vitamin D deficiency enhances the growth of MC-26 colon cancer xenografts in Balb/c mice.
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Vitamin D deficiency has been associated with increased risk of colon cancer in epidemiologic and prospective clinical studies. In vitro and in vivo studies demonstrated that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its analogs inhibit colon cancer cell proliferation. Few studies have evaluated the effect of vitamin D deficiency on the development and growth of colon cancer. To assess the antiproliferative effects of 25-hydroxyvitamin D [25(OH)D] and 1,25(OH)2D3 in vitro, we cultured MC-26 (a colon cancer cell line) in the presence of 25(OH)D3 and 1,25(OH)2D3 and performed [3H]thymidine incorporation. The proliferation of MC-26 was significantly inhibited by both 25(OH)D3 and 1,25(OH)2D3. To determine the effect of vitamin D deficiency on colon cancer proliferation, Balb/c mice were rendered vitamin D deficient by feeding them a vitamin D-deficient diet for 3 mo. A group of vitamin D-sufficient mice was given the same diet with supplemental vitamin D. The mice were injected with MC-26 colon cancer cells and the tumors were measured daily for 20 d. Vitamin D-sufficient mice had 40% smaller tumors than vitamin D-deficient mice. The tumors were evaluated for mRNA expression of the vitamin D receptor (VDR) and 25-hydroxvitamin D-1alpha-hydroxylase (1alpha-OHase) by quantitative RT-PCR. The expression of the mRNA for the VDR and the 1alpha-OHase was 37- and 6-fold higher, respectively, in the vitamin D-sufficient mice compared with the vitamin D-deficient mice. We conclude that vitamin D deficiency enhances the growth of colon cancer in mice. The tumor expression of VDR and 1alpha-OHase indicates possible autocrine/paracrine cell growth regulation by vitamin D. (+info)
Ligand-induced transrepression by VDR through association of WSTF with acetylated histones.
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We have previously shown that the novel ATP-dependent chromatin-remodeling complex WINAC is required for the ligand-bound vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D3 1alpha-hydroxylase (1alpha(OH)ase) gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1alpha(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated his tones, which appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1alpha(OH)ase gene expression. (+info)
25-Hydroxyvitamin D3 1alpha-hydroxylase expression in breast cancer and use of non-1alpha-hydroxylated vitamin D analogue.
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INTRODUCTION: The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated. METHODS: The expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells. RESULTS: 1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3. CONCLUSION: These results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed. (+info)
CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1alpha-hydroxylase promoter activity in the skin.
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The hormonally active form of vitamin D(3),1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1alpha-hydroxylase gene is replaced by the genes for beta-galactosidase (lacZ) and neomycin resistance. Null mice produced no detectable 1alpha-hydroxylase transcript. The mice grew normally when maintained on a balanced diet containing 1,25(OH)(2)D(3) but rapidly developed rickets when phosphorus and 1,25(OH)(2)D(3) were restricted. Rickets was curable through administration of 1,25(OH)(2)D(3) but not its biological precursor, 25-hydroxyvitamin D(3). Upon administration of a diet low in calcium and devoid of any form of vitamin D(3), beta-galactosidase activity was detected in the kidneys of the -/- and +/- mice and in placentas harvested from -/- females bred with -/- males. No beta-galactosidase activity was detected in skin sections or in primary keratinocyte cultures from -/- animals. Our results demonstrate we have generated 1alpha-hydroxylase null mice that display phenotypes characteristic of vitamin D-dependency rickets type I. From the histochemical analysis of reporter gene expression in these mice, we conclude that acute 1,25(OH)(2)D(3) deficiency in otherwise healthy animals does not stimulate local production of 1,25(OH)(2)D(3) in the skin. These findings stand in contrast to previously published reports of 1,25(OH)(2)D(3) production in keratinocytes. (+info)
Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process.
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Fibroblast growth factor 23 null mice (Fgf-23-/-) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging-like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Furthermore, increased vitamin D activities in homozygous mutants are associated with severe atherosclerosis and widespread soft tissue calcifications; ablation of vitamin D activity from Fgf-23-/- mice, by genetically deleting the 1alpha(OH)ase gene, eliminates atherosclerosis and ectopic calcifications and significantly rescues premature aging-like features of Fgf-23-/- mice, resulting in prolonged survival of Fgf-23-/-/1alpha(OH)ase-/- double mutants. Our results indicate a novel role of Fgf-23 in developing premature aging-like features through regulating vitamin D homeostasis. Finally, our data support a new model of interactions among Fgf-23, vitamin D, and klotho, a gene described as being associated with premature aging process. (+info)