Vitamin D3 Is Transformed into 1,25(OH)2D3 by Triggering CYP3A11(CYP3A4) Activity and Hydrolyzing Midazolam.

BACKGROUND Vitamin D3 (VD3) is a commonly used supplement in clinical practice. Cytochrome P450 3A11 (CYP3A11) is the most important monomeric enzyme involved in metabolism of drugs. This study aimed to investigate effects of vitamin D3 (VD3) on CYP3A11 activity. MATERIAL AND METHODS Forty male Sprague-Dawley (SD) rats were randomly divided a Control group (peanut oil 0.1 ml/kg/d), a Low-VD3 group (100 IU/kg/d), a Medium-VD3 group (400 IU/kg/d), and a High-VD3 (1600 IU/kg/d) group. Blood samples were collected from the jugular vein after midazolam (MDZ) administration. CYP3A11 expressions in liver and colon were detected by Western blotting and immunohistochemistry (IHC) assay. The concentration of serum 25(OH)D3 and serum 1,25(OH)2D3 were evaluated using ELISA. Effects of different dosages of vitamin D3 on metabolism of MDZ were evaluated using high-performance liquid chromatography (HPLC). RESULTS Vitamin D3 significantly enhanced serum 25(OH)D3 and 1,25(OH)2D3 levels in rats compared to Control rats (p<0.05). Expressions of hepatic CYP3A11 were more than 10-fold higher in rats treated with vitamin D3 compared to Control rats (p<0.05). Expressions of colon CYP3A11 were 5-fold higher than in Control rats (p<0.05). CYP3A11 expressions in vitamin D3-treated groups were significantly higher compared to the Control group (p<0.05). MDZ levels were significantly higher in Vitamin D3-treated rats compared to that in Control rats (p<0.05). Concentrations of serum MDZ at every sampling point were remarkably lower in the vitamin D3-treated rats than in Control rats (p<0.05). CONCLUSIONS Vitamin D3 was transformed into 1,25(OH)2D3 by triggering CYP3A11 and CYP3A11 activity and by hydrolyzing MDZ.

Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice.

Factors that can modify the bioavailability of orally administered vitamin D are not yet widely known. Ergosterol is a common fungal sterol found in food which has a chemical structure comparable to that of vitamin D. This study aimed to investigate the effect of ergosterol on vitamin D metabolism. Therefore, 36 male wild type-mice were randomly subdivided into three groups (n = 12) and received a diet containing 25 µg vitamin D3 and either 0 mg (control), 2 mg or 7 mg ergosterol per kg diet for 6 weeks. To elucidate the impact of ergosterol on hepatic hydroxylation of vitamin D, human hepatoma cells (HepG2) were treated with different concentrations of ergosterol. Concentrations of vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in cells, livers and kidneys of mice and additionally 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in serum were quantified by LC-MS/MS. The concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in serum was analyzed by commercially-available enzyme immuno assay. The concentrations of cholesterol and triglycerides were analyzed in livers of mice by photometric assays. Analyses revealed that mice receiving 7 mg/kg ergosterol with their diet had 1.3-, 1.7- and 1.5-times higher concentrations of vitamin D3 in serum, liver and kidney, respectively, than control mice (P < 0.05), whereas no significant effects were observed in mice fed 2 mg/kg ergosterol. The hydroxylation of vitamin D remained unaffected by dietary ergosterol, since the concentration of 25-hydroxyvitamin D3 in serum and tissues and the concentrations of 1,25(OH)2D3 and 24,25(OH)2D3 in serum were not different between the three groups of mice. The lipid concentrations in liver were also not affected by dietary ergosterol. Data from the cell culture studies showed that ergosterol did not influence the conversion of vitamin D3 to 25(OH)D3. To conclude, ergosterol appears to be a modulator of vitamin D3 concentrations in the body of mice, without modulating the hydroxylation of vitamin D3 in liver.

24R,25-dihydroxyvitamin D3 modulates tumorigenicity in breast cancer in an estrogen receptor-dependent manner.

Vitamin D has long been prescribed as a supplement to breast cancer patients. This is partially motivated by data indicating that low serum vitamin D, measured as 25-hydroxyvitamin D3 [25(OH)D3], is associated with worsened cancer prognosis and decreased survival rates in cancer patients. However, clinical studies investigating the role of vitamin D supplementation in breast cancer treatment are largely inconclusive. One reason for this may be that many of these studies ignore the complexity of the vitamin D metabolome and the effects of these metabolites at the cellular level. Once ingested, vitamin D is metabolized into 37 different metabolites, including 25(OH)D3, which is the metabolite actually measured clinically, as well as 1,25(OH)2D3 and 24,25(OH)2D3. Recent work by our lab and others has demonstrated a role for 24R,25(OH)2D3, in the modulation of breast cancer tumors via an estrogen receptor α-dependent mechanism. This review highlights the importance of considering estrogen receptor status in vitamin d-associated prognostic studies of breast cancer and proposes a potential mechanism for 24R,25(OH)2D3 signaling in breast cancer cells.

24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo.

BACKGROUND: Epidemiological studies indicate high serum 25(OH)D3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH)2D3. However, 1α,25(OH)2D3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH)2D3, as an alternative non-calcemic vitamin D3 supplement. METHODS: NOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH)2D3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH)2D3in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7. RESULTS: 24R,25(OH)2D3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH)2D3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH)2D3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH)2D3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs. CONCLUSION: These results indicate that 24R,25(OH)2D3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis. GENERAL SIGNIFICANCE: 24R,25(OH)2D3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH)2D3 effects on chondrocytes. Thus, 24R,25(OH)2D3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies.

MicroRNA-31 Negatively Regulates Interleukin-34 Expression In Vitro.

Interleukin-34 (IL-34) is a recently discovered cytokine that promotes tissue macrophage maturation and differentiation. We previously found that 1α,25-Dihydroxyvitamin D3 up-regulated IL-34 expression in SH-SY5Y neural cells. However, whether microRNA regulates IL-34 expression is not completely clear. By using on-line TargetScan and MiRanda software, we found that there was only one conserved microRNA-31 (miR-31) binding site in the 3' untranslated region (3'UTR) of IL-34 mRNA. Intriguingly, using qPCR we demonstrated that miR-31 levels were negatively correlated to IL-34 mRNA levels in different cell lines. By examining the effect of miR-31 on IL-34 3' UTR reporter luciferase activity and on IL-34 mRNA and argonaute RISC catalytic component 2 (AGO2) binding, it was found that miR-31 bound directly to IL-34 3'UTR and regulated the post-transcriptional expression of IL-34 in MGC-803 cells. Moreover, a miR-31 mimic significantly reduced IL-34 expression levels while a miR-31 inhibitor up-regulated IL-34 expression in KYSE-45 and HT-29 cells. Taken together, these results show that miR-31 negatively regulates IL-34 expression by directly binding to the IL-34 3' UTR in vitro.

Lithocholic Acid Is a Vitamin D Receptor Ligand That Acts Preferentially in the Ileum.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the biological action of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], and regulates calcium and bone metabolism. Lithocholic acid (LCA), which is a secondary bile acid produced by intestinal bacteria, acts as an additional physiological VDR ligand. Despite recent progress, however, the physiological function of the LCA−VDR axis remains unclear. In this study, in order to elucidate the differences in VDR action induced by 1,25(OH)2D3 and LCA, we compared their effect on the VDR target gene induction in the intestine of mice. While the oral administration of 1,25(OH)2D3 induced the Cyp24a1 expression effectively in the duodenum and jejunum, the LCA increased target gene expression in the ileum as effectively as 1,25(OH)2D3. 1,25(OH)2D3, but not LCA, increased the expression of the calcium transporter gene Trpv6 in the upper intestine, and increased the plasma calcium levels. Although LCA could induce an ileal Cyp24a1 expression as well as 1,25(OH)2D3, the oral LCA administration was not effective in the VDR target gene induction in the kidney. No effect of LCA on the ileal Cyp24a1 expression was observed in the VDR-null mice. Thus, the results indicate that LCA is a selective VDR ligand acting in the lower intestine, particularly the ileum. LCA may be a signaling molecule, which links intestinal bacteria and host VDR function.

Regulation of vitamin D metabolism following disruption of the microbiota using broad spectrum antibiotics.

Vitamin D, 25hydroxyvitamin D (25D), and 24,25dihydroxyvitamin D (24,25D) were measured before and after broad spectrum antibiotic (Abx) treatment for 2 wks. Abx treatments increased 25D and 24,25D levels suggesting that the microbiota or Abx were altering vitamin D metabolism. Increased 25D, but not 24,25D, following Abx treatments were found to be dependent on toll like receptor signaling. Conversely, the effects of Abx on 24,25D levels required that the vitamin D receptor (VDR) be expressed in tissues outside of the hematopoietic system (kidney) and not the immune system. Fibroblast growth factor (FGF)23 increased following Abx treatment and the effect of Abx treatment on FGF23 (like the effect on 24,25D) was not present in VDR knockout (KO) mice. The Abx mediated increase in 24,25D was due to changes to the endocrine regulation of vitamin D metabolism. Conversely, 25D levels went up with Abx treatment of the VDR KO mice. Host sensing of microbial signals regulates the levels of 25D in the host.

Placental vitamin D metabolism and its associations with circulating vitamin D metabolites in pregnant women.

Background: Little is known about placental vitamin D metabolism and its impact on maternal circulating vitamin D concentrations in humans.Objective: This study sought to advance the current understanding of placental vitamin D metabolism and its role in modulating maternal circulating vitamin D metabolites during pregnancy.Design: Nested within a feeding study, 24 healthy pregnant women (26-29 wk of gestation) consumed a single amount of vitamin D (511 IU/d from diet and a cholecalciferol supplement) for 10 wk. Concentrations of placental and blood vitamin D metabolites and placental messenger RNA (mRNA) abundance of vitamin D metabolic pathway components were quantified. In addition, cultured human trophoblasts were incubated with 13C-cholecalciferol to examine the intracellular generation and secretion of vitamin D metabolites along with the regulation of target genes.Results: In placental tissue, 25-hydroxyvitamin D3 [25(OH)D3] was strongly correlated (r = 0.83, P < 0.001) with 24,25-dihydroxyvitamin D3 Moreover, these placental metabolites were strongly correlated (r ≤ 0.85, P ≤ 0.04) with their respective metabolites in maternal circulation. Positive associations (P ≤ 0.045) were also observed between placental mRNA abundance of vitamin D metabolic components and circulating vitamin D metabolites [i.e., LDL-related protein 2 (LRP2, also known as megalin) with 25(OH)D3 and the C3 epimer of 25(OH)D3 [3-epi-25(OH)D3]; cubilin (CUBN) with 25(OH)D3; 25-hydroxylase (CYP2R1) with 3-epi-25(OH)D3; 24-hydroxylase (CYP24A1) with 25(OH)D3, 3-epi-25(OH)D3, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]; and 1α-hydroxylase [(CYP27B1) with 3-epi-25(OH)D3 and 1,25(OH)2D3]. Notably, in vitro experiments with trophoblasts showed increased production and secretion of 25(OH)D3 and higher CYP24A1 gene transcript abundance in response to cholecalciferol treatment.Conclusions: The numerous associations of many of the placental biomarkers of vitamin D metabolism with circulating vitamin D metabolites among pregnant women [including a CYP27B1-associated increase in 1,25(OH)2D3] and the evidence of trophoblast production and secretion of vitamin D metabolites, especially 25(OH)D3, suggest that the placenta may play an active role in modulating the vitamin D metabolite profile in maternal circulation in human pregnancy. This trial was registered at clinicaltrials.gov as NCT03051867.

Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells.

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1-3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1ß, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4nmol/L), 24R,25(OH)2D3 (14.4nmol/L) and 24S,25(OH)2D3 (57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Dietary calcium does not interact with vitamin D3 in terms of determining the response and catabolism of serum 25-hydroxyvitamin D during winter in older adults.

BACKGROUND: Interactions between calcium and vitamin D may have implications for the regulation of serum 25-hydroxyvitamin D [25(OH)D] and its catabolism and, consequently, the vitamin D dietary requirement. OBJECTIVE: We investigated whether different calcium intakes influenced serum 25(OH)D and indexes of vitamin D activation and catabolism during winter and in the context of both adequate and inadequate vitamin D intakes. DESIGN: A 15-wk winter-based, randomized, placebo-controlled, double-blind vitamin D3 intervention (20 µg/d) study was carried out in free-living men and women aged ≥50 y (n = 125) who were stratified according to calcium intakes [moderate-low (<700 mg/d) or high (>1000 mg/d) intake]. The serum 25(OH)D concentration was the primary outcome, and serum calcium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)2D], 24,25-dihydroxyvitamin D [24,25(OH)2D], the ratio of 24,25(OH)2D to 25(OH)D, vitamin D-binding protein, and free 25(OH)D were exploratory outcomes. RESULTS: A repeated-measures ANOVA showed there was no significant (P = 0.2) time × vitamin D treatment × calcium intake grouping interaction effect on the mean serum 25(OH)D concentration over the 15-wk intervention period. Serum 25(OH)D concentrations increased (P ≤ 0.005) and decreased (P ≤ 0.002) in vitamin D3 and placebo groups, respectively, and were of similar magnitudes in subjects with calcium intakes <700 mg/d (and even <550 mg/d) compared with >1000 mg/d. The response of serum PTH, 1,25(OH)2D, 24,25(OH)2D, the ratio of 24,25(OH)2D to 25(OH)D, and free 25(OH)D significantly differed in vitamin D3 and placebo groups but not by calcium intake grouping. CONCLUSIONS: We found no evidence of a vitamin D sparing effect of high calcium intake, which has been referred to by some authors as "vitamin D economy." Thus, recent dietary vitamin D requirement estimates will cover the vitamin D needs of even those individuals who have inadequate calcium intakes.

Influence of estrogen therapy on calcium, phosphorus, and other regulatory hormones in postmenopausal women: the MESA study.

BACKGROUND: Estrogen therapy (ET) is associated with lower serum calcium and phosphorus concentrations and is known to increase bone mineral density (BMD). Other biomarkers of mineral metabolism may help understand the biological basis of these actions. METHODS: We studied 2767 postmenopausal women in the Multi-Ethnic Study of Atherosclerosis, 862 (31%) of whom were using ET. We measured serum concentrations of calcium, phosphorus, 25-hydroxyvitamin D, 24,25-dihydoxyvitamin D, and fibroblast growth factor-23 and urinary fractional excretion of calcium (FEca) and phosphorus (FEphos). We examined the associations of ET with each biomarker. In addition, we tested whether the adjustment for biomarkers attenuated the association of ET with lumbar BMD measured by abdominal computed tomography in a subset of 810 women. RESULTS: In adjusted models, women who used ET were younger in age [62 (SD 8) vs 66 (9) y, P < .001], had lower mean serum calcium [-13 mg/dL (95% confidence interval [CI] -0.17, -0.10), P < .001] and lower FEca [-0.15% (95% CI -0.21, -0.09), P < .001]. Mean serum phosphorus was lower [-0.19 mg/dL (95% CI -0.23, -0.15), P < .001] and FEphos [0.56% (95% CI 0.16, 0.96), P = .007] was higher in women on ET. Mean 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were higher [1.52 ng/dL (95% CI 0.57, 2.47), P = .002, and 0.26 ng/mL (95% CI 0.03, 0.48), P = .03, respectively] in women who used ET. Mean PTH and fibroblast growth factor-23 did not differ significantly by the use of ET. ET use was strongly associated with higher lumbar BMD [12.75 mg/cm³ (95% CI 7.77-17.73), P < .001]; however, mineral metabolism measures did not meaningfully alter this association. CONCLUSIONS: In a multiethnic cohort of postmenopausal women, ET use was associated with lower serum calcium, lower FEca, lower serum phosphorus, and higher FEphos, suggesting these associations are attributable to increased calcium intake into bone and increased urinary phosphorus excretion. ET use was also associated with greater concentrations of vitamin D metabolites. ET-associated differences in these mineral metabolism measures did not meaningfully attenuate the strong association between ET use and lumbar BMD.

Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles.

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the K(m) for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The K(m) for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (k(cat) /K(m) ) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D(2) and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable k(cat) values of 50-71 min(-1) , similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K(m) values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane.

Enhancement of vitamin D metabolites in the eye following vitamin D3 supplementation and UV-B irradiation.

PURPOSE: This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure. METHODS: Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy. RESULTS: 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3. CONCLUSIONS: There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

Regulation of vitamin D metabolism.

Fundamental to understanding the way in which perturbations in the vitamin D endocrine system can affect human health is an appreciation of the steps involved in the production of the well-recognized active hormonal form, 1,25-dihydroxyvitamin D(3). Thus this paper focuses first on the nature and regulation of the two enzymes responsible for the production of 1,25-dihydroxyvitamin D(3), the 25-hydroxylase in the liver and the 1α-hydroxylase in the kidney. The most important regulators of the 1α-hydroxylase in the kidney are 1,25-dihydroxyvitamin D(3) itself, parathyroid hormone and FGF23. The extent and importance of extra-renal, 1,25-dihydroxyvitamin D(3) synthesis is then considered. Finally the features of the 24R-hydroxylase, which produces 24R,25-dihydroxyvitamin D(3) in the kidney and is induced by and inactivated, 1,25-dihydroxyvitamin D(3)in target cells are described.

24R,25-Dihydroxyvitamin D3, lysophosphatidic acid, and p53: a signaling axis in the inhibition of phosphate-induced chondrocyte apoptosis.

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH)(2)D(3)], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH)(2)D(3) are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53. However, the exact mechanism by which 24R,25(OH)(2)D(3) and LPA exert their effects is not fully understood. It was found that both 24R,25(OH)(2)D(3) and LPA attenuate Pi-induced caspase-3 activity. The actions of 24R,25(OH)(2)D(3) and LPA were dependent upon G(αi), LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI(3)K) signaling, and nuclear export. 24R,25(OH)(2)D(3) decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH)(2)D(3) inhibits Pi-induced apoptosis through Ca(2+), PLD, and PLC signaling and through LPA-LPA1/3-G(αi)-PI(3)K-mdm2-mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity.

24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells.

Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH)2D3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH)2D3 blocked this effect. Further studies indicated that 24R,25(OH)2D3 acts via at least two independent pathways. 24R,25(OH)2D3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH)2D3 acts by increasing PKC activity. 24R,25(OH)2D3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH)2D3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1alpha,25(OH)2D3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH)2D3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

Interrelation of parathyroid hormone and vitamin D metabolites in adolescents from the UK and The Gambia.

Parathyroid hormone (PTH) is used as a marker of vitamin D (VD) status. However, PTH depends on many other factors. The 24,25-dihydroxy VD (24,25VD) concentration may be a sensitive marker because its production is reduced in VD deficiency. The relationship between VD metabolites, their ratio and PTH was investigated in adolescents from the UK and The Gambia with different calcium intakes and VD status. In the UK, there was a significant positive (+ve) association between 25VD and both 1,25-dihydroxy VD (1,25VD) and 24,25VD and a negative (-ve) association with PTH. The 24,25:25VD ratio was consistent across the 25VD concentration range. There was a +ve association between PTH and 1,25:25VD, (1,25+24,25):25VD or 1,25:24,25VD, a -ve association with 24,25VD and none with 1,25VD or 24,25:25VD. Using LnPTH and 1,25:25VD ratio (but not 1,25VD:24,25VD or 25VD:24,25VD) increased uniformity between groups and strength of relationships compared to PTH and 1,25 or 25VD alone. In The Gambia, there was a significant -ve relationship between 25VD and PTH and none with 1,25VD. There was a +ve association between 1,25VD or 1,25:25VD and PTH. The more uniform prediction of PTH by the 1,25VD:25VD ratio may be because this better reflects the extent to which PTH-induced 1,25VD production can be met by VD supply. Further validation is needed.

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1).

CYP27B1 catalyzes the 1alpha-hydroxylation of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3, the hormonally active form of vitamin D3. To further characterize mouse CYP27B1, it was expressed in Escherichia coli, purified and its activity measured on substrates incorporated into phospholipid vesicles, which served as a model of the inner mitochondrial membrane. 25-Hydroxyvitamin D3 and 25-hydroxyvitamin D2 in vesicles underwent 1alpha-hydroxylation with similar kinetics, the catalytic rate constants (k(cat)) were 41 and 48mol/min/mol P450, respectively, while K(m) values were 5.9 and 4.6mmol/mol phospholipid, respectively. CYP27B1 showed inhibition when substrate concentrations in the membrane were greater than 4 times K(m), more pronounced with 25-hydroxyvitamin D3 than 25-hydroxyvitamin D2. Higher catalytic efficiency was seen in vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin than for dimyristoyl phosphatidylcholine vesicles. CYP27B1 also catalyzed 1alpha-hydroxylation of vesicle-associated 24R,25-dihydroxyvitamin D3 and 20-hydroxyvitamin D3, and 25-hydroxylation of 1alpha-hydroxyvitamin D3 and 1alpha-hydroxyvitamin D2, but with much lower efficiency than for 25(OH)D3. This study shows that CYP27B1 can hydroxylate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 associated with phospholipid membranes with the highest activity yet reported for the enzyme. The expressed enzyme has low activity at higher concentrations of 25-hydroxyvitamin D in membranes, revealing that substrate inhibition may contribute to the regulation of the activity of this enzyme.

A kinetic overview of the receptors involved in 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 signaling: a systems biology approach.

The vitamin D endocrine system modulates an arsenal of important biological functions in more than 30 different tissues in short- and long-term perspectives. Two membrane receptors and one nuclear receptor are suggested to be involved in the vitamin D signaling system, but the function and physiological relevance of the receptors are debated. The complexity of the vitamin D endocrine system makes it necessary to combine experimental data with in silico simulations to get a holistic view of vitamin D-dependent regulation of tissue and cell physiology. This review focus on binding characteristics for the three putative vitamin D receptors and proposes a future systems biology approach including mathematical modeling that will be helpful together with experimental methods in depicting antitumoral and other biological effects promoted by the vitamin D endocrine system.