Transcriptome-Wide Profile of 25-Hydroxyvitamin D3 in Primary Immune Cells from Human Peripheral Blood.

Vitamin D3 is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25(OH)D3), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and <0.05 as a false discovery rate, in this ex vivo approach the maximal physiological concentration of 25(OH)D3 (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D3 regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH)2D3. The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D3. In conclusion, 100- and 1000-fold higher 25(OH)D3 concentrations than the reference 10 nM 1,25(OH)2D3 are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH)2D3.

Increased serum antimicrobial peptide LL-37 and HBD-2 combined with 25-hydroxyvitamin D3 deficiency in infants with pertussis.

INTRODUCTION: Most children with serious infection diseases suffer from malnutrition. Vitamin D participates in the immune response through endogenous antimicrobial peptides (AMPs) regulation. The aim of this study is to investigate the expression of 25-hydroxyvitamin D3 [25(OH)D3], AMPs [LL-37 and human ß-defensin 2 (HBD-2)] in the children with pertussis. METHODOLOGY: Serum levels of 25(OH)D3, LL-37, and HBD-2 were detected in 116 children with pertussis aged at 1-12 months (67 males and 49 females). Fifty healthy infants at similar age were employed as normal controls. RESULTS: The serum 25(OH)D3 levels in the children with mild (27.30 ± 5.98 ng/ml) and severe (24.40 ± 6.27 ng/ml) pertussis were significantly lower than that in the healthy group (30.16 ± 5.13 ng/ml; p <0.01). The vitamin D deficiency rates in children with mild (55.9%) and severe (78.12%) pertussis were significantly higher than that in the control group (34%; p < 0.01). The serum levels of LL-37 and HBD-2 were significantly higher in pertussis patients. Spearman rank correlation analysis did not show any correlation of 25-(OH)D3 with LL-37 or HBD-2. CONCLUSIONS: Most children with pertussis had vitamin D deficiency accompanied by elevated serum LL-37 and HBD-2 levels. However, the average level of 25(OH)D3 at 26.50 ng/ml in the infants with pertussis may not affect the immuno-regulatory ability; thus, the infants with pertussis still maintained a higher level of AMPs (LL-37 and HBD-2) against pertussis infection.

Generation of novel genetically modified rats to reveal the molecular mechanisms of vitamin D actions.

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that for 25(OH)D3, were also generated. Although Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH)2D3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system.

Family-based Association between Allele T of rs4646536 in CYP27B1 and vitamin D deficiency.

BACKGROUND: The circulating concentration of 25(OH)D is widely applied to indicate the vitamin D status, as the directly metabolic genes of 25(OH)D, CYP2R1, and CYP27B1 are associated with the concentration of 25(OH)D. However, the contributions of allelic transmission disequilibrium of single nucleotide polymorphisms (SNPs) in these genes to vitamin D deficiency remain unclear. We aimed at investigating the family-based association between SNPs of CYP2R1 and CYP27B1 and vitamin D deficiency. METHOD: First, SNPs selected in family-based study were screened by a pilot case-control study. Second, allelic transmissions of the selected SNPs were investigated with family-based study (n = 880). Finally, associations between selected SNPs and the concentration of 25(OH)D were verified in siblings (n = 120). RESULTS: The results of the pilot case-control study indicated that both CT and TT genotypes of rs4646536 in CYP27B1 could increase the susceptibility of vitamin D deficiency when compared with CC genotype. The adjusted ORs were 2.846 (95%CI 1.312-6.174, P = 0.008) and 2.609 (95%CI 1.197-5.687, P = 0.016), respectively. Furthermore, the results of family-based association test suggested that there was transmission disequilibrium for allele T of rs4646536 in vitamin D deficiency families. In addition, the concentration of 25(OH)D3 for CC genotype was higher than CT genotype between siblings (P = 0.016). CONCLUSIONS: Transmission disequilibrium of allele T of rs4646536 is associated with vitamin D deficiency.

Half the Genetic Variance in Vitamin D Concentration is Shared with Skin Colour and Sun Exposure Genes.

This study assessed the heritability of 25 hydroxyvitamin D3 (25(OH)D3) in a large twin cohort and the shared effect of sun exposure and skin colour on 25(OH)D3 variance. Study participants included 1604 twin pairs and their siblings (n = 4020). Twin correlations for 25(OH)D3 concentration were rMZ=0.79 (584 pairs) and rDZ = 0.52 (1020 pairs) consistent with an average h2 = 0.50 throughout the year. Significant phenotypic and genetic seasonal fluctuation was observed in 25(OH)D3 concentrations with heritability decreasing during the winter (h2 = 0.37) compared to summer (h2 = 0.62). Skin colour (measured both ordinally and quantitatively) and self-reported sun exposure were found to significantly affect 25(OH)D3 concentration. Twins with olive/dark skin had significantly lower 25(OH)D3 concentrations than those with fair/pale skin and multivariate genetic analysis showed that approximately half of the total additive genetic variation in 25(OH)D3 results from genes whose primary influence is on skin colour and sun exposure. Additionally, 37% of the total variance was attributed to shared environmental effects on vitamin D, skin colour and sun exposure measures. These results support a moderate estimate of vitamin D heritability and suggest significant influence of season, skin colour and sun exposure on the genetic variance.

Genetic and epigenetic factors influencing vitamin D status.

The global prevalence of vitamin D deficiency appears to be increasing, and the impact of this on human health is important because of the association of vitamin D insufficiency with increased risk of osteoporosis, cardiovascular disease and some cancers. There are few studies on the genetic factors that can influence vitamin D levels. In particular, the data from twin and family-based studies have reported that circulating vitamin D concentrations are partially determined by genetic factors. Moreover, it has been shown that genetic variants (e.g., mutation) and alteration (e.g., deletion, amplification, inversion) in genes involved in the metabolism, catabolism, transport, or binding of vitamin D to it receptor, might affect vitamin D level. However, the underlying genetic determinants of plasma 25-hydroxyvitamin D3 [25(OH)D] concentrations remain to be elucidated. Furthermore, the association between epigenetic modifications such as DNA methylation and vitamin D level has now been reported in several studies. The aim of current review was to provide an overview of the possible value of loci associated to vitamin D metabolism, catabolism, and transport as well epigenetic modification and environmental factors influencing vitamin D status.

Vitamin D-related gene polymorphisms do not influence the outcome and serum vitamin D level in pegylated interferon/ribavirin therapy combined with protease inhibitor for patients with genotype 1b chronic hepatitis C.

Although several vitamin D-related gene polymorphisms were reported to affect the outcome of pegylated interferon/ribavirin (PR) therapy in chronic hepatitis C patients, there are no reports on the impact of the vitamin D-related gene polymorphisms in PR therapy combined with protease inhibitor (PI). Vitamin D-related gene polymorphisms were determined in 177 genotype 1b-infected chronic hepatitis C patients who received 12 weeks of PR therapy with telaprevir, a first-generation PI, followed by 12 weeks of PR therapy. The sustained virologic response (SVR) rate was 83.1% (147 of 177 patients). The frequencies of vitamin D-related gene polymorphisms were: 83 non-TT and 94 TT genotypes for GC, 97 non-AA and 80 AA genotypes for DHCR7, 151 non-AA and 26 AA genotypes for CYP2R1, 162 non-GG and 15 GG genotypes for CYP27B1, and 105 non-GG and 72 GG genotypes for VDR gene. Multivariate analysis extracted IL28B TT genotype (P = 2.05 × 10(-6)) and serum 25(OH) D3 level (P = 0.024) as independent factors contributing to the achieving of SVR. The SVR rate in IL28B TT genotype patients with serum 25(OH) D3 level of < 25 ng/ml was significantly low compared to other patients. None of the vitamin D-related gene polymorphisms affected the treatment outcome and serum 25(OH) D3 level. In conclusions, the IL28B polymorphism and serum 25(OH) D3 level contributed significantly and independently to SVR in PR combined with PI for genotype 1b-infected chronic hepatitis C patients. However, none of vitamin D-related gene polymorphisms had an impact on the treatment outcome and serum 25(OH) D3 level.

Severe hypercalcemic crisis in an infant with idiopathic infantile hypercalcemia caused by mutation in CYP24A1 gene.

UNLABELLED: We report on a male infant presenting at 4 months of age with failure to thrive, dehydration, hypotonia, lethargy, and vomiting. Laboratory and imaging tests revealed severe hypercalcemia (5.8 mmol/l), suppressed parathyroid hormone (0.41 pmol/l), hypercalciuria (8.0 mmol/mmol creatinine), elevated 25-hydroxyvitamin D3 (over 600 nmol/l), and nephrocalcinosis. These symptoms are characteristic of idiopathic infantile hypercalcemia (IIH, MIM 143880). Conservative therapy (parenteral rehydration, diuretics, corticosteroids, bisphosphonates, and vitamin D prophylaxis withdrawal) was not able to improve the symptoms and laboratory values, and acute hemodiafiltration was necessary to normalize hypercalcemia. Clinical symptoms resolved rapidly after normalization of serum calcium levels. Molecular genetic testing revealed a homozygous mutation (R396W) in the CYP24A1 gene (MIM 126065) encoding 25-hydroxyvitamin D3 24-hydroxylase, which is the key enzyme responsible for 1,25-dihydroxyvitamin D3 degradation. The CYP24A1 gene mutation leads to the increased sensitivity of the patients to even prophylactic doses of vitamin D and to the development of severe symptomatic hypercalcemia in patients with IIH. CONCLUSION: Our patient is only the thirteenth patient with IIH caused by mutation in the CYP24A1 gene and the first one needing acute hemodiafiltration for severe symptomatic hypercalcemic crisis. In all patients with suspected IIH the DNA analysis for CYP24A1 gene mutations should be performed regardless of the type of vitamin D supplementation and serum levels of vitamin D.

Activity of 25-hydroxylase in human gingival fibroblasts and periodontal ligament cells.

BACKGROUND: We previously demonstrated that 25-hydroxyvitamin D(3) concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D(3) can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D(3) that resulted in the production of 1α,25-dihydroxyvitamin D(3). Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D(3) and 1α,25-dihydroxyvitamin D(3) production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D(3) synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1ß and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation.

CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the ß3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the ß3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.

The role of cubilin gene polymorphisms and their influence on 25(OH)D3 and 1,25(OH)2D3 plasma levels in type 1 diabetes patients.

BACKGROUND: Megalin and cubilin bind and internalize the 25(OH)D3-DBP complex in the kidney. Once the complex is internalized, 25(OH)D3 is released and activated to 1,25(OH)2D3 the ligand for the vitamin D receptor (VDR). Supporting the important role of cubilin in this process recent findings showed that cubilin deficiency results in decrease of 25(OH)D3 and 1,25(OH)2D3 plasma levels. METHODS: Two hundred T1D patients and healthy controls (n=200) were genotyped for five polymorphisms (rs3740168, rs3740165, rs1801233, rs1801229 and rs2796835) within the cubilin gene. The polymorphisms were analyzed by RFLP or real time PCR. Statistic analyses were performed by using allele-wise and genotype-wise chi2 testing by using BiAS software. A p-value<0.05 was considered as significant. RESULTS: We found that the genotype "AA" of the rs3740165 was more frequent in T1D patients compared to healthy controls (26.7% vs. 5.1%, p=4x10(-7)). Nevertheless no association between the rs3740165 polymorphism and the 25(OH)D3 or 1,25(OH)2D3 plasma levels was found. No association with the other studied polymorphisms was observed. CONCLUSION: Thus our findings reveal a novel association of the cubilin rs3740165 polymorphism with type 1 diabetes. Nevertheless how exactly this polymorphism could increase the risk to develop type 1 diabetes is subject for further investigations.

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1.

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

Metabolism of vitamin D3 in human osteoblasts: evidence for autocrine and paracrine activities of 1 alpha,25-dihydroxyvitamin D3.

Circulating 1 alpha,25-dihydroxyvitamin D(3) (1,25D) derives from renal conversion of 25-hydroxyvitamin D(3) (25D), by the 25D 1 alpha-hydroxylase (CYP27B1). Blood 25D levels, but not 1,25D levels, are the best indicator of vitamin D status and predict fracture risk in the elderly. We examined the extent to which osteoblasts can metabolize 25D. Well-characterized human primary osteoblasts and osteosarcoma (OS) cell lines were examined for the expression and regulation of genes associated with vitamin D metabolism, using real-time PCR. Primary osteoblasts and OS cell lines were found to express CYP27B1 mRNA and secreted detectable 1,25D in response to 25D. Of the OS cell lines tested, HOS expressed the most CYP27B1 mRNA and secreted the highest levels of 1,25D. All osteoblastic cells examined up-regulated expression of the catabolic regulator of 1,25D, the 25-hydroxyvitamin D-24-hydroxylase (CYP24), when incubated with either 1,25D or 25D. Exposure to physiological levels of 25D resulted in up-regulated transcription of the 1,25D responsive genes, osteocalcin (OCN), osteopontin (OPN) and RANKL. Specific knockdown of CYP27B1 in HOS cells using siRNA resulted in up to 80% reduction in both 1,25D secretion and the transcription of OCN and CYP24, strongly implying that the 25D effect in osteoblasts is preceded by conversion to 1,25D. Incubation with 25D, like 1,25D, inhibited primary osteoblast proliferation and promoted in vitro mineralization. Finally, we detected expression by osteoblasts of receptors for vitamin D binding protein (DBP), cubilin and megalin, suggesting that osteoblasts are able to internalize DBP-25D complexes in vivo. Together, our results suggest that autocrine, and perhaps paracrine, pathways of vitamin D(3) metabolism may regulate key osteoblast functions independently of circulating, kidney derived 1,25D. Our results are therefore consistent with the reported benefits of maintaining a healthy vitamin D status in the elderly to reduce the risk of fractures.

Statistical genetic analysis of plasma levels of vitamin D: familial study.

The present study attempted to evaluate the relative importance of familial/genetic factors in interindividual variation of plasma concentrations of vitamin D (calcidiol). Ninety-five nuclear pedigrees (187 males and 168 females, aged 18-91 and 18-86 years old respectively), from the Chuvasha Autonomy, Russia, were assessed for calcidiol and sex hormones. Age-adjusted 25-hydroxy-vitamin D (25OHD) measures showed very high pair-wise correlations between all members of families, which suggest that there may be a genetic factor transmitted within the families. To test this assumption, complex segregation analysis of non-adjusted 25OHD data was undertaken, in which genotype-sex specific dependence of the trait on age was incorporated. The analysis showed that some 44% of the 25OHD variance was attributable to all genetic and non-genetic factors incorporated into the model. About 22% of the variation was accounted for by a putative major gene effect.

Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing.

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.

Selective expression of a normal action of the 1,25-dihydroxyvitamin D3 receptor in human skin fibroblasts with hereditary severe defects in multiple actions of that receptor.

We evaluated three actions of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in human skin fibroblasts to test for heterogeneity in hormone-response coupling. In fibroblasts from normal subjects the 1,25-(OH)2D3 concentrations for half-maximal effect (EC50) were: for mitogenic effect 0.0001-0.0005 nM, for antimitogenic effect 1 nM, and for induction of 25-OHD3 24-hydroxylase (24-OHase) 5 nM. To evaluate the effects of mutations presumed to be in the gene for the 1,25-(OH)2D3 receptor we examined cell lines representing four kindreds with hereditary resistance to 1,25-(OH)2D3 ("mutant" cell lines). In one mutant cell line all three 1,25-(OH)2D3 actions were severely abnormal. In one mutant cell line 24-OHase induction and mitogenic action were undetectable, but EC50 and maximal effect were normal for antimitogenic action of 1,25-(OH)2D3. In two mutant cell lines 24-OHase induction and antimitogenic actions were undetectable or severely impaired but mitogenic action were undetectable or severely impaired but mitogenic action was normal in EC50 and normal or increased in maximal effect. The mitogenic and antimitogenic actions in normal cells showed a similar profile of potency ratios for 1,25-(OH)2D3 and six analogues. Whenever a mutant cell showed a normal or even an abnormal mitogenic or antimitogenic effect of 1,25-(OH)2D3, these effects showed potency ratios similar to wild type, suggesting mediation by a similar 1,25-(OH)2D3 receptor. We conclude that three 1,25-(OH)2D3 actions show important differences in hormone response coupling indicated by differences in EC50 for 1,25-(OH)2D3 and by different consequences of receptor mutations.