25 Hydroxyvitamin D 1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasis.
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Keratinocytes express high levels of 25OHD 1alpha-hydroxylase (1OHase). The product of this enzyme, 1,25-dihydroxyvitamin D (1,25(OH)(2)D), promotes the differentiation of keratinocytes in vitro suggesting an important role for this enzyme in epidermal differentiation. To test whether 1OHase activity is essential for keratinocyte differentiation in vivo we examined the differentiation process in mice null for the expression of the 1alphaOHase gene (1alphaOHase(-/-)). Heterozygotes for the null allele were bred, and the progeny genotyped by PCR. The epidermis of the 1alphaOHase(-/-) animals and their wild-type littermates (1alphaOHase(+/+)) were examined by histology at the light and electron microscopic level, by immunocytochemistry for markers of differentiation, and by function examining the permeability barrier using transepidermal water loss (TEWL). No gross epidermal phenotype was observed; however, immunocytochemical assessment of the epidermis revealed a reduction in involucrin, filaggrin, and loricrin-markers of differentiation in the keratinocyte and critical for the formation of the cornified envelope. These observations were confirmed at the electron microscopic level, which showed a reduction in the F (containing filaggrin) and L (containing loricrin) granules and a reduced calcium gradient. The functional significance of these observations was tested using TEWL to evaluate the permeability barrier function of the epidermis. Although TEWL was normal in the basal state, following disruption of the barrier using tape stripping, the 1alphaOHase(-/-) animals displayed a markedly delayed recovery of normal barrier function. This delay was associated with a reduction in lamellar body secretion and a failure to reform the epidermal calcium gradient. Thus, the 25OHD 1OHase is essential for normal epidermal differentiation, most likely by producing the vitamin D metabolite, 1,25(OH)(2)D, responsible for inducing the proteins regulating calcium levels in the epidermis that are critical for the generation and maintenance of the barrier. (+info)
Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase.
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The synthesis of bioactive vitamin D requires hydroxylation at the 1 alpha and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1 alpha-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. (+info)
Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.
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OBJECTIVES: Many patients with Crohn's disease (CD) have low bone mineral density (BMD) that may not be solely attributable to glucocorticoid use. We hypothesised that low BMD in patients with CD is associated with elevated circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D). We further hypothesised that this was secondary to increased synthesis of 1,25(OH)(2)D by inflammatory cells in the intestine. The aim of this study was to examine the relationship between 1,25(OH)(2)D levels and BMD in patients with CD. METHODS: An IRB approved retrospective review of medical records from patients with CD (n = 138) or ulcerative colitis (UC, n = 29). Measurements of vitamin D metabolites and immunoreactive parathyroid hormone (iPTH) were carried out. BMD results were available for 88 CD and 20 UC patients. Immunohistochemistry or real time reverse transcription-polymerase chain reaction (RT-PCR) for the enzyme 1alpha-hydroxylase was performed on colonic biopsies from patients with CD (14) or UC (12) and normal colons (4). RESULTS: Inappropriately high levels of serum 1,25(OH)(2)D (>60 pg/ml) were observed in 42% of patients with CD compared with only 7% in UC, despite no differences in mean iPTH. Serum 1,25(OH)(2)D levels were higher in CD (57 pg/ml) versus UC (41 pg/ml) (p = 0.0001). In patients with CD, there was a negative correlation between 1,25(OH)(2)D levels and lumbar BMD (r = -0.301, p = 0.005) independent of therapeutic glucocorticoid use. 1,25(OH)(2)D levels also correlated with CD activity. Lastly, immunohistochemistry and RT-PCR demonstrated increased expression of intestinal 1alpha-hydroxylase in patients with CD. CONCLUSIONS: These data demonstrate that elevated 1,25(OH)(2)D is more common in CD than previously appreciated and is independently associated with low bone mineral density. The source of the active vitamin D may be the inflamed intestine. Treatment of the underlying inflammation may improve metabolic bone disease in this subgroup of patients. (+info)
A promoter polymorphism of the CYP27B1 gene is associated with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus in Germans.
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BACKGROUND: CYP27B1 hydroxylase catalyzes the conversion of 25 hydroxyvitamin D(3) (25OHD(3)) to 1,25(OH)(2)D(3), the most active natural vitamin D metabolite, which plays a role in the regulation of immunity and cell proliferation. We therefore investigated two single nucleotide polymorphisms in the CYP27B1 hydroxylase gene for an association with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus. METHODS: Patients with Addison's disease (n=124), Hashimoto's thyroiditis (n=139), Graves' disease (n=334), type 1 diabetes mellitus (n=252) and healthy controls (n=320) were genotyped for the promoter (-1260) C/A polymorphism and for the intron 6 (+2838) C/T polymorphism of the CYP27B1 gene. Patients and controls were compared using genotype-wise and allele-wise X(2) testing. RESULTS: A significant association was found between allelic variation of the promoter (-1260) C/A polymorphism and Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus (P=0.0062, P=0.0173, P=0.0094 and P=0.0028 respectively). Significant differences were also observed for the intron 6 (+2838) C/T polymorphism (P=0.0058) in Hashimoto's thyroiditis but not for the other autoimmune endocrine diseases. CONCLUSIONS: The CYP27B1 promoter (-1260) C/A polymorphism appears to be associated with endocrine autoimmune diseases but the CYP27B1 intron 6 (+2838) C/T polymorphism appears to be associated only with Hashimoto's thyroiditis. These results imply a regulatory difference of the CYP27B1 hydroxylase to predispose to endocrine autoimmunity. (+info)
The novel azole R126638 is a selective inhibitor of ergosterol synthesis in Candida albicans, Trichophyton spp., and Microsporum canis.
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R126638 is a novel triazole with in vitro activity similar to that of itraconazole against dermatophytes, Candida spp., and Malassezia spp. In animal models of dermatophyte infections, R126638 showed superior antifungal activity. R126638 inhibits ergosterol synthesis in Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum canis at nanomolar concentrations, with 50% inhibitory concentrations (IC(50)s) similar to those of itraconazole. The decreased synthesis of ergosterol and the concomitant accumulation of 14 alpha-methylsterols provide indirect evidence that R126638 inhibits the activity of CYP51 that catalyzes the oxidative removal of the 14 alpha-methyl group of lanosterol or eburicol. The IC(50)s for cholesterol synthesis from acetate in human hepatoma cells were 1.4 microM for itraconazole and 3.1 microM for R126638. Compared to itraconazole (IC(50) = 3.5 microM), R126638 is a poor inhibitor of the 1 alpha-hydroxylation of 25-hydroxyvitamin D(3) (IC(50) > 10 microM). Micromolar concentrations of R126638 and itraconazole inhibited the 24-hydroxylation of 25-hydroxyvitamin D(3) and the conversion of 1,25-dihydroxyvitamin D(3) into polar metabolites. At concentrations up to 10 microM, R126638 had almost no effect on cholesterol side chain cleavage (CYP11A1), 11 beta-hydroxylase (CYP11B1), 17-hydroxylase and 17,20-lyase (CYP17), aromatase (CYP19), or 4-hydroxylation of all-trans retinoic acid (CYP26). At 10 microM, R126638 did not show clear inhibition of CYP1A2, CYP2A6, CYP2D6, CYP2C8, CYP2C9, CYP2C10, CYP2C19, or CYP2E1. Compared to itraconazole, R126638 had a lower interaction potential with testosterone 6 beta hydroxylation and cyclosporine hydroxylation, both of which are catalyzed by CYP3A4, whereas both antifungals inhibited the CYP3A4-catalyzed hydroxylation of midazolam similarly. The results suggest that R126638 has promising properties and merits further in vivo investigations for the treatment of dermatophyte and yeast infections. (+info)
Increased expression of 25-hydroxyvitamin D-1alpha-hydroxylase in dysgerminomas: a novel form of humoral hypercalcemia of malignancy.
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Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic disorder usually associated with increased synthesis of parathyroid hormone-related peptide (PTHrP). Unlike non-cancer forms of hypercalcemia, HHM does not routinely involve increased circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Dysgerminomas are a notable exception to this rule, previous reports having described hypercalcemia with elevated serum 1,25(OH)2D3. To investigate the etiology of this form of HHM we have characterized expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)2D3, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), in a collection of 12 dysgerminomas. RT-PCR analyses indicated that mRNA for 1alpha-hydroxylase was increased 222-fold in dysgerminomas compared to non-tumor ovarian tissue. Parallel enzyme assays in tissue homogenates showed that dysgerminomas produced fivefold higher levels of 1,25(OH)2D3 compared to normal ovarian tissue. Immunolocalization studies indicated that 1alpha-hydroxylase was expressed by both tumor cells and by macrophages within the inflammatory cell infiltrate associated with dysgerminomas. The immunological nature of the increased 1,25(OH)2D3 production observed in dysgerminomas was further emphasized by correlation between expression of 1alpha-hydroxylase and the endotoxin recognition factors CD14 and toll-like receptor 4 (TLR4). These data suggest that inflammatory mechanisms associated with dysgerminomas are the underlying cause of the increased expression and activity of 1alpha-hydroxylase associated with these tumors. We further postulate that this autocrine/paracrine action of 1alpha-hydroxylase may lead to increased circulating levels of 1,25(OH)2D3 and a form of HHM which is distinct from that seen with PTHrP-secreting tumors. (+info)
Inhibition of proliferation and induction of apoptosis by 25-hydroxyvitamin D3-3beta-(2)-Bromoacetate, a nontoxic and vitamin D receptor-alkylating analog of 25-hydroxyvitamin D3 in prostate cancer cells.
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The 25-hydroxyvitamin D(3) (25-OH-D(3)) is a nontoxic and low-affinity vitamin D receptor (VDR)-binding metabolic precursor of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We hypothesized that covalent attachment of a 25-OH-D(3) analog to the hormone-binding pocket of VDR might convert the latter into transcriptionally active holo-form, making 25-OH-D(3) biologically active. Furthermore, it might be possible to translate the nontoxic nature of 25-OH-D(3) into its analog. We showed earlier that 25-hydroxyvitamin D(3)-3-bromoacetate (25-OH-D(3)-3-BE) alkylated the hormone-binding pocket of VDR. In this communication we describe that 10(-6) mol/L of 25-OH-D(3)-3-BE inhibited the growth of keratinocytes, LNCaP, and LAPC-4 androgen-sensitive and PC-3 and DU145 androgen-refractory prostate cancer cells, and PZ-HPV-7 immortalized normal prostate cells with similar or stronger efficacy as 1,25(OH)(2)D(3). But its effect was strongest in LNCaP, PC-3, LAPC-4, and DU145 cells. Furthermore, 25-OH-D(3)-3-BE was toxic to these prostate cancer cells and caused these cells to undergo apoptosis as shown by DNA-fragmentation and caspase-activation assays. In a reporter assay with COS-7 cells, transfected with a 1alpha,25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase)-construct and VDR-expression vector, 25-OH-D(3)-3-BE induced 24-OHase promoter activity. In a "pull down assay" with PC-3 cells, 25-OH-D(3)-3-BE induced strong interaction between VDR and general transcription factors, retinoid X receptor, and GRIP-1. Collectively, these results strongly suggested that the cellular effects of 25-OH-D(3)-3-BE were manifested via 1,25(OH)(2)D(3)/VDR signaling pathway. A toxicity study in CD-1 mice showed that 166 microg/kg of 25-OH-D(3)-3-BE did not raise serum-calcium beyond vehicle control. Collectively, these results strongly suggested that 25-OH-D(3)-3-BE has a strong potential as a therapeutic agent for androgen-sensitive and androgen-refractory prostate cancer. (+info)
Vitamin D and breast cancer: insights from animal models.
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1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3[, the biologically active form of vitamin D that interacts with the vitamin D receptor (VDR), is a coordinate regulator of proliferation, differentiation, and survival of breast cancer cells. Therefore, vitamin D compounds that bind and activate VDRs offer promise as therapeutic agents for the treatment of established breast cancer. In addition, epidemiologic, clinical, and animal studies suggested that vitamin D status is important for protection against the development of breast cancer. To elucidate potential biological mechanisms through which vitamin D status might be associated with breast cancer risk, basic research studies focused on defining the molecular effects of vitamin D signaling in the normal mammary gland. Both VDR and vitamin D 1-hydroxylase, the enzyme that generates 1,25(OH)2D3, are expressed and dynamically regulated in the normal mammary gland. Furthermore, studies with mice lacking VDRs established that vitamin D participates in negative growth control of the normal mammary gland and that disruption of VDR signaling is associated with abnormal ductal morphologic features, increased incidence of preneoplastic lesions, and accelerated mammary tumor development. These studies support the concept that suboptimal generation of 1,25(OH)2D3 in the mammary gland might sufficiently deregulate VDR-mediated gene expression to sensitize mammary cells to transformation. In light of these observations, studies to define the most appropriate biomarkers of vitamin D status in relation to protection against breast cancer among human subjects are urgently needed. (+info)