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Objective: We investigated the impact of vitamin D3 (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor {VDR) in mouse ovarian follicles at different Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0,25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcrip-tase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR. Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages. Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed. [ABSTRACT FROM AUTHOR]

Purpose: Vitamin D-dependent rickets type 1b (VDDR1b) is a very rare autosomal recessive disorder caused by mutations in CYP2R1 that produces 25-hydroxylase. To date only five mutations in CYP2R1 have been identified. This study has reported the genetic results and the clinical characteristics of a family with VDDR1b and compared this family to the other families with VDDR1b in literature. Methods: After two probands were diagnosed with VDDR1b, all other family members were evaluated. Serum calcium, phosphorus, alkaline phosphatase, parathyroid hormone, 25-hydroxy vitamin D, and 1.25-dihydroxy vitamin D levels were measured in all family members. All individuals were evaluated radiographically, and a genetic analysis was done in all family members. The other families with VDDR1b in literature were reviewed. Results: Two novel mutations [c.367 + 1G > C and p.E339Q (c.1015G > C)] were identified. The clinic and laboratory findings were strikingly different among the members of this family regardless of the mutation and the number of alleles affected. The families having different mutations in literature had also extensive variation in both the clinical and the laboratory findings. Conclusion: The current study further expands CYP2R1 mutation spectrum. The findings of both the current and the previous studies suggest that VDDR1b is a more complex disorder than the known autosomal recessive inheritance model and the phenotype may show an extensive variation regardless of the mutation type and the gene dosage. [ABSTRACT FROM AUTHOR]

Objective: To assess whether varicocele affects testicular 25-hydroxylase activity. Methods: Twenty normozoospermic patients with bilateral varicocele (grade III according to the Dubin and Amelar classification) without indications to undergo varicocele repair (normal sperm parameters and testicular volume; no scrotal pain) were consecutively enrolled and followed-up for four years. Serum levels of parathyroid hormone (PTH), calcium, and 25-hydroxy-cholecalciferol [25(OH)D] along with serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), total testosterone (TT), conventional sperm parameters, sperm DNA fragmentation (SDF) rate, and testicular volume (TV) were measured annually for three years. PTH, calcium, and 25(OH)D serum levels over time were compared with those of age- and body mass index (BMI)-matched control group of twenty varicocelectomized patients. Main results: Both intra- and between-group analyses showed that serum PTH levels increased significantly over time in parallel with a significant decline in 25(OH)D levels. Serum calcium levels did not change significantly. At the same time, signs of mild Leydig and Sertoli cell dysfunction were found, such as an increase in gonadotropins and decreased TT and VT. However, conventional sperm parameters and SDF rate did not change significantly. Conclusion: This prospective controlled study provides the first evidence of a negative impact of bilateral grade III varicocele on testicular 25-hydroxylase activity. Accordingly, the patients included in this study showed a significant increase in PTH and a decrease in 25(OH)D levels over time. Patients with varicocele deserve endocrinologic counseling.

Background: Vitamin D is a fat-soluble cholesterol derivative found in two forms, vitamin D2, and vitamin D3. Cytochrome P450 2R1 (CYP2R1) encoded by the CYP2R1 gene is the major hydroxylase that activates vitamin D by catalyzing the formation of 25-hydroxyvitamin D (25(OH)D). Methods: We collected 89 (100%) subjects, 46 of which (51.69%) had a documented severe deficiency of 25(OH)D (30 ng/mL). We performed Sanger sequencing of three selected fragments of the CYP2R1 gene (Ch11: 14878000–14878499; Ch11: 14880058–14880883 and Ch11: 14885321–14886113) that affect the binding of substrates to this enzyme and analyzed the possible involvement of genetic variation in these regions with an increased risk of vitamin D deficiency in healthy Polish individuals. Results: Two substitutions were found within the three fragments. Bioinformatic analysis suggested that one of these (NC_000011.10: g.14878291G>A) may influence the structure and function of CYP2R1. Conclusions: Variant NC_000011.10: g.14878291G>A may have a perturbing effect on heme binding in the active site of CYP2R1 and on the function of 25-hydroxylase and probably affects the concentration of 25(OH)D in vivo. We intend to perform functional verification in a larger patient population to confirm and extend these results.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo - and in vitro -developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo - and in vitro -developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo -developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro -developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro -developed antral follicles, which was restored to levels comparable to those of in vivo -developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary. [ABSTRACT FROM AUTHOR]

A negative association between blood lead and vitamin D concentrations has been reported, however, experimental data on the effect of lead (Pb) on vitamin D metabolism is scarce. We investigated the effects of Pb on serum vitamin D metabolites, vitamin D activating enzymes and vitamin D receptor (VDR) in rats. Newborn Wistar rat pups were exposed to 0.2% Pb-acetate via their dams' drinking water from post-natal day (PND) 1 to 21 and directly in drinking water until PND30. Serum 25-hydroxyvitamin D was analyzed with LC-MS/MS and 1,25-dihydroxyvitamin D with an immunoassay. Tissue expression of vitamin D activating enzymes and VDR were measured by Western blot and immunohistochemistry. Serum 25-hydroxyvitamin D was significantly decreased at both PND21 and PND30, whereas 1,25-dihydroxyvitamin D was decreased (p < 0.05) only at PND21 in the Pb-exposed rats. Expression of renal 1-α-hydroxylase was decreased by Pb only at PND21 (p < 0.05) but the brain 1-α-hydroxylase was not affected. Hepatic 25-hydroxylase expression was significantly decreased at PND21 but significantly increased at PND30 by Pb exposure. VDR expression in the brain was increased at both PND21 and PND30 (p < 0.05). These results suggest that Pb interferes with vitamin D metabolism by affecting the expression of its metabolizing enzymes. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10 nM 1α,25(OH) 2 D 3 for 6 and 24 h and found that 1α,25(OH) 2 D 3 increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10 nM 1α,25(OH) 2 D 3 led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH) 2 D 3 could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD 3 , cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD 3 levels were quantified by ELISA. We found that 25OHD 3 levels increased in a concentration-dependent manner. Treatment of BEC with 10 μM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo , we intranasally delivered 1 μM 1α,25(OH) 2 D 3 into mice. After 6 h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with newly developed CF treatments. [ABSTRACT FROM AUTHOR]

ABSTRACT Vitamin D requires a two-step activation by hydroxylation: The first step is catalyzed by hepatic 25-hydroxylase ( CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α-hydroxylase ( CYP27B1, 12q13.1), which produces the active hormonal form of 1,25-(OH)2D. Mutations of CYP2R1 have been associated with vitamin D-dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss-of-function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss-of-function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25-OH-D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25-(OH)2D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa-hydroxyvitamin D3 or alfacalcidol (1α-OH-D3) treatment, and we observed a dramatic increase in the 1,25-(OH)2D serum concentration, which indicated the role of accessory 25-hydroxylase enzymes. Lastly, in patients who received calcifediol (25-OH-D3), we documented normal 24-hydroxylase activity ( CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25-OH-D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

A negative association between blood lead and vitamin D concentrations has been reported, however, experimental data on the effect of lead (Pb) on vitamin D metabolism is scarce. We investigated the effects of Pb on serum vitamin D metabolites, vitamin D activating enzymes and vitamin D receptor (VDR) in rats. Newborn Wistar rat pups were exposed to 0.2% Pb-acetate via their dams’ drinking water from post-natal day (PND) 1 to 21 and directly in drinking water until PND30. Serum 25-hydroxyvitamin D was analyzed with LC-MS/MS and 1,25-dihydroxyvitamin D with an immunoassay. Tissue expression of vitamin D activating enzymes and VDR were measured by Western blot and immunohistochemistry. Serum 25-hydroxyvitamin D was significantly decreased at both PND21 and PND30, whereas 1,25-dihydroxyvitamin D was decreased (p < 0.05) only at PND21 in the Pb-exposed rats. Expression of renal 1-α-hydroxylase was decreased by Pb only at PND21 (p < 0.05) but the brain 1-α-hydroxylase was not affected. Hepatic 25-hydroxylase expression was significantly decreased at PND21 but significantly increased at PND30 by Pb exposure. VDR expression in the brain was increased at both PND21 and PND30 (p < 0.05). These results suggest that Pb interferes with vitamin D metabolism by affecting the expression of its metabolizing enzymes.

Background Polymorphic markers of vitamin D pathway genes have been associated with asthma traits in different White populations. This study investigated the relationship between asthma phenotypes and single nucleotide polymorphisms ( SNPs) of vitamin D receptor ( VDR), vitamin D binding protein ( GC), two 25-hydroxylases ( CYP2R1 and CYP27A1), and 1α-hydroxylase ( CYP27B1) in Hong Kong Chinese children. Methods 23 SNPs of the five vitamin D pathway genes were successfully genotyped in 914 asthmatic children and 1231 non-allergic controls. Genotypic and haplotypic associations with asthma phenotypes (diagnosis, spirometric indices, total IgE, and eosinophil percentage) were analyzed by multivariate regression. Generalized multifactor dimensionality reduction was used to detect epistatic interactions between SNPs for asthma phenotypes. Results Several SNPs of CYP27A1, CYP27B1, GC, and CYP2R1 were associated with asthma or spirometric indices, although only the association between FEV1 and CYP2R1 rs7935792 passed Bonferroni correction (p = 2.73 × 10−4). Patients with CC genotype of rs7935792 had higher FEV1 than those with the other two genotypes. Asthma was also associated with TT haplotype of CYP27A1 and AGGATA haplotype of CYP2R1 (p = 0.021 and 0.024, respectively). Besides, strong association was found between FEV1 and GATAG of CYP2R1 (β = 13.37, p = 4.83 × 10−4). GMDR failed to identify any 2-locus to 4-locus interaction that modulated asthma or spirometric indices. Conclusions Several SNPs and haplotypes of CYP2R1 are associated with asthma diagnosis and FEV1 in children. Asthma is also modestly associated with a CYP27A1 haplotype. These two 25-hydroxylase genes may be genetic determinants for asthma phenotypes in children. [ABSTRACT FROM AUTHOR]

Objective: To assess whether varicocele affects testicular 25-hydroxylase activity. Methods: Twenty normozoospermic patients with bilateral varicocele (grade III according to the Dubin and Amelar classification) without indications to undergo varicocele repair (normal sperm parameters and testicular volume; no scrotal pain) were consecutively enrolled and followed-up for four years. Serum levels of parathyroid hormone (PTH), calcium, and 25-hydroxy-cholecalciferol [25(OH)D] along with serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), total testosterone (TT), conventional sperm parameters, sperm DNA fragmentation (SDF) rate, and testicular volume (TV) were measured annually for three years. PTH, calcium, and 25(OH)D serum levels over time were compared with those of age- and body mass index (BMI)-matched control group of twenty varicocelectomized patients. Main results: Both intra- and between-group analyses showed that serum PTH levels increased significantly over time in parallel with a significant decline in 25(OH)D levels. Serum calcium levels did not change significantly. At the same time, signs of mild Leydig and Sertoli cell dysfunction were found, such as an increase in gonadotropins and decreased TT and VT. However, conventional sperm parameters and SDF rate did not change significantly. Conclusion: This prospective controlled study provides the first evidence of a negative impact of bilateral grade III varicocele on testicular 25-hydroxylase activity. Accordingly, the patients included in this study showed a significant increase in PTH and a decrease in 25(OH)D levels over time. Patients with varicocele deserve endocrinologic counseling. [ABSTRACT FROM AUTHOR]

Résumé: La 1,25-dihydroxyvitamine D3 (1,25(OH)2D3) est connue pour son effet endocrine sur l’homéostasie calcique. De manière plus récente, a été découverte une action immunomodulatrice paracrine ou autocrine liée à la synthèse locale de 1,25(OH)2D3 par les cellules du système immunitaire, qui expriment le récepteur de la vitamine D (VDR) et les enzymes nécessaires à son métabolisme (1α-, 25- et 24-hydroxylases). Ces effets immunomodulateurs, mis en évidence dans les modèles animaux et les expériences de culture cellulaire, s’exercent de manière directe et indirecte, concernent les lymphocytes T et B ainsi que les cellules présentatrices d’antigène (cellules dendritiques et macrophages). Ils interviennent dans l’immunité innée et adaptative. L’effet global est un switch d’une réponse Th1/Th17 vers un profil Th2/Treg. Ces effets immunomodulateurs de la vitamine D pourraient expliquer la relation épidémiologique entre le statut vitaminique D et de nombreuses pathologies auto-immunes et inflammatoires suggérée par des études observationnelles (dans la polyarthrite rhumatoïde, le lupus, les entéropathies inflammatoires, le diabète de type 1, mais aussi les infections, cancers, rejets de greffes et maladies cardiovasculaires). Un effet thérapeutique de la supplémentation en vitamine D a également été mis en évidence dans les modèles animaux correspondant à ces pathologies. Ainsi, la vitamine D est un enjeu majeur de santé publique et pourrait représenter un espoir thérapeutique dans les maladies dysimmunitaires. [ABSTRACT FROM AUTHOR]

Abstract: Calcitriol, or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a well-known endocrine regulator of calcium homeostasis. More recently, local calcitriol production by immune cells was shown to exert autocrine or paracrine immunomodulating effects. Immune cells that produce calcitriol also express the vitamin D receptor (VDR) and the enzymes needed to metabolize vitamin D3 (1α-, 25-, and 24-hydroxylases). Studies of animal models and cell cultures showed both direct and indirect immunomodulating effects involving the T cells, B cells, and antigen-presenting cells (dendritic cells and macrophages) and affecting both innate and adaptive immune responses. The overall effect is a switch from the Th1/Th17 response to the Th2/Treg profile. The immunomodulating effects of vitamin D may explain the reported epidemiological associations between vitamin D status and a large number of autoimmune and inflammatory diseases. Such associations have been suggested by observational studies not only in rheumatoid arthritis, lupus, inflammatory bowel disease, and type 1 diabetes; but also in infections, malignancies, transplant rejection, and cardiovascular disease. In animal models for these diseases, vitamin D supplementation has been found to produce therapeutic effects. Thus, vitamin D is a key focus for public health efforts and may hold promise for the treatment of dysimmune diseases. [ABSTRACT FROM AUTHOR]

Background: Vitamin D is a fat-soluble cholesterol derivative found in two forms, vitamin D2, and vitamin D3. Cytochrome P450 2R1 (CYP2R1) encoded by the CYP2R1 gene is the major hydroxylase that activates vitamin D by catalyzing the formation of 25-hydroxyvitamin D (25(OH)D). Methods: We collected 89 (100%) subjects, 46 of which (51.69%) had a documented severe deficiency of 25(OH)D (<10 ng/mL) and 43 (48.31%) in the control group with documented optimum levels of 25(OH)D (>30 ng/mL). We performed Sanger sequencing of three selected fragments of the CYP2R1 gene (Ch11: 14878000–14878499; Ch11: 14880058–14880883 and Ch11: 14885321–14886113) that affect the binding of substrates to this enzyme and analyzed the possible involvement of genetic variation in these regions with an increased risk of vitamin D deficiency in healthy Polish individuals. Results: Two substitutions were found within the three fragments. Bioinformatic analysis suggested that one of these (NC_000011.10: g.14878291G>A) may influence the structure and function of CYP2R1. Conclusions: Variant NC_000011.10: g.14878291G>A may have a perturbing effect on heme binding in the active site of CYP2R1 and on the function of 25-hydroxylase and probably affects the concentration of 25(OH)D in vivo. We intend to perform functional verification in a larger patient population to confirm and extend these results. [ABSTRACT FROM AUTHOR]

Abstract: vitamin D is 25-hydroxylated in the liver, before being activated by 1α-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848–56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087min−1), vitamin D2 (0.16min−1), and 1α-hydroxyvitamin D3 (2.2min−1). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4min−1) more efficiently than vitamin D2 (0.86min−1). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2. [Copyright &y& Elsevier]

Abstract: Previous studies have suggested that hepatic production of 25-hydroxyvitamin D3 may be suppressed by 1α,25-dihydroxyvitamin D3. However, the molecular details of these observations have not been clarified. In the current study, the 5′-flanking DNA sequence of CYP2D25, a porcine microsomal vitamin D 25-hydroxylase, was isolated and analyzed. The CYP2D25 promoter contains a putative vitamin D response element (VDRE). The promoter activity was markedly suppressed by 1α,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in presence of vitamin D receptor (VDR). The data suggest that VDR-mediated inhibition of 25-hydroxylase(s) by vitamin D3 metabolites at the transcriptional level may play an important role in the regulation of 25-hydroxyvitamin D3 production in liver and other tissues. [Copyright &y& Elsevier]

Epidemiological evidence suggests an inverse relationship between prostate cancer and serum vitamin D levels. We examined the ability of cholecalciferol (vitamin D3), a calcitriol precursor, to inhibit or reverse cellular changes associated with malignant transformation and invasion and explored its mechanisms of action. The RWPE2-W99 human prostate epithelial cell line, which forms slow-growing tumors in nude mice, was used because it mimics the behavior of the majority of primary human prostate cancers. Cholecalciferol, at physiological levels: (i) inhibited anchorage-dependent and -independent growth; (ii) induced differentiation by decreasing vimentin expression with a concomitant decrease in motility/chemotaxis; (iii) decreased MMP-9 and MMP-2 activity with concomitant decrease in invasion; and (iv) exerted its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptor-α (RXR-α), and androgen receptor (AR) in a dose-dependent manner. Furthermore, we found that RWPE2-W99 prostate cancer cells, similar to RWPE-1 cells (Tokar and Webber. Clin Exp Metast 2005; 22: 265–73), constitutively express the enzyme 25-hydroxylase CYP27A1 which is markedly up-regulated by cholecalciferol. Cholecalciferol has effects similar to those of calcitriol on growth, MMP activity, and VDR. The ability of CYP27A1 to catalyze the conversion of cholecalciferol to 25(OH)D3 and of 25(OH)D3 to calcitriol has been reported. RWPE2-W99 cells, similar to RWPE-1 cells, appear to have the rare ability to locally convert cholecalciferol to the active hormone calcitriol. Because it can inhibit cellular changes associated with malignant transformation and invasion, we propose that cholecalciferol may be an effective agent for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

The 20–30 year latency period for prostate cancer provides an important opportunity to prevent the development of invasive cancer. A logical approach for chemoprevention to reduce incidence is to identify agents, such as, vitamin D, which can inhibit cell proliferation and induce differentiation, are safe, and readily available to the public at low cost. Epidemiological evidence suggests that vitamin D deficiency is associated with increased risk for prostate cancer. We examined the ability and mechanisms of action of cholecalciferol (vitamin D3), a precursor of the most biologically active hormone calcitriol, to block or reverse premalignant changes. The immortalized, non-tumorigenic, RWPE-1 human prostate epithelial cell line, was used. Results show that cholecalciferol, at physiological levels: (i) inhibits anchorage-dependent growth (ii) induces differentiation by increasing PSA expression and (iii) exerts its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptors (RXRs), and androgen receptor (AR). Furthermore, we discovered that human prostate epithelial cells constitutively express appreciable levels of 25-hydroxylase CYP27A1 protein, the enzyme which catalyzes the conversion of cholecalciferol to 25(OH)D3, and that CYP27A1 is up-regulated by cholecalciferol. Recent studies show that human mitochondrial CYP27A1 can also catalyze 1α-hydroxylation of 25(OH)D3 to calcitriol. The presence of 25-hydroxylase in human prostate epithelial cells has not previously been shown. Since human prostate epithelial cells have the necessary enzymes and the rare ability to locally convert cholecalciferol to the active hormone calcitriol, we propose that they are a prime target for chemoprevention of prostate cancer with cholecalciferol whose safety is well established as a supplement in vitamins and fortified foods. [ABSTRACT FROM AUTHOR]

Objective: We investigated the impact of vitamin D3 (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor (VDR) in mouse ovarian follicles at different stages., Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0, 25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcriptase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR., Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages., Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed.

Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower k cat than CYP27A1 but a much lower K m for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (k cat /K m ), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH) 2 D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH) 2 D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH) 2 D3]. The 20,25(OH) 2 D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound., (Copyright © 2017 Elsevier Ltd. All rights reserved.)