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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]

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]

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]

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]

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]

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]