Effects of 1,25 and 24,25 Vitamin D on Corneal Epithelial Proliferation, Migration and Vitamin D Metabolizing and Catabolizing Enzymes.
ABSTRACT: This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.
Project description:Purpose:We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes. Methods:Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results:VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin ?4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin ?4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions:VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin ?4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.
Project description:Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3 Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element-binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1-deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2-deleted strain did not affect 1,25(OH)2D3 induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.
Project description:The 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) together with parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) regulates calcium (Ca2+) and phosphate (Pi) homeostasis, 1,25(OH)2D3 synthesis is mediated by hydroxylases of the cytochrome P450 (Cyp) family. Vitamin D is first modified in the liver by the 25-hydroxylases CYP2R1 and CYP27A1 and further activated in the kidney by the 1?-hydroxylase CYP27B1, while the renal 24-hydroxylase CYP24A1 catalyzes the first step of its inactivation. While the kidney is the main organ responsible for circulating levels of active 1,25(OH)2D3, other organs also express some of these enzymes. Their regulation, however, has been studied less.Here we investigated the effect of several Pi-regulating factors including dietary Pi, PTH and FGF23 on the expression of the vitamin D hydroxylases and the vitamin D receptor VDR in renal and extrarenal tissues of mice. We found that with the exception of Cyp24a1, all the other analyzed mRNAs show a wide tissue distribution. High dietary Pi mainly upregulated the hepatic expression of Cyp27a1 and Cyp2r1 without changing plasma 1,25(OH)2D3. FGF23 failed to regulate the expression of any of the studied hydroxylases at the used dosage and treatment length. As expected, renal mRNA expression of Cyp27b1 was reduced and Cyp24a1 was increased in response to 1,25(OH)2D3 treatment. However, the 25-hydroxylases were rather unaffected by 1,25(OH)2D3 treatment.The analyzed vitamin D hydroxylases are regulated in a tissue and treatment-specific manner.
Project description:The biological activity of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] remains controversial, but it has been suggested that it contributes to fracture healing. Cyp24a1-/- mice, synthesizing no 24R,25(OH)2D3, show suboptimal endochondral ossification during fracture repair, with smaller callus and reduced stiffness. These defects were corrected by 24R,25(OH)2D3 treatment, but not by 1,25-dihydroxyvitamin D3. Microarrays with Cyp24a1-/- callus mRNA identified FAM57B2 as a mediator of the 24R,25(OH)2D3 effect. FAM57B2 produced lactosylceramide (LacCer) upon specific binding of 24R,25(OH)2D3. Fam57b inactivation in chondrocytes (Col2-Cre Fam57bfl/fl) phenocopied the callus formation defect of Cyp24a1-/- mice. LacCer or 24R,25(OH)2D3 injections restored callus volume, stiffness, and mineralized cartilage area in Cyp24a1-null mice, but only LacCer rescued Col2-Cre Fam57bfl/fl mice. Gene expression in callus tissue suggested that the 24R,25(OH)2D3/FAM57B2 cascade affects cartilage maturation. We describe a previously unrecognized pathway influencing endochondral ossification during bone repair through LacCer production upon binding of 24R,25(OH)2D3 to FAM57B2. Our results identify potential new approaches to ameliorate fracture healing.
Project description:1?,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1?-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1?,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFN? and IL1?). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1?,3?-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.
Project description:Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1?,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.
Project description:The pandemic increase in obesity is inversely associated with vitamin D levels. While a higher BMI was causally related to lower 25-hydroxyvitamin D (25(OH)D), no evidence was obtained for a BMI lowering effect by higher 25(OH)D. Some of the physiological functions of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol or calcitriol) via its receptor within the adipose tissue have been investigated such as its effect on energy balance, adipogenesis, adipokine, and cytokine secretion. Adipose tissue inflammation has been recognized as the key component of metabolic disorders, e.g., in the metabolic syndrome. The adipose organ secretes more than 260 different proteins/peptides. However, the molecular basis of the interactions of 1,25(OH)2D3, vitamin D binding proteins (VDBPs) and nuclear vitamin D receptor (VDR) after sequestration in adipose tissue and their regulations are still unclear. 1,25(OH)2D3 and its inactive metabolites are known to inhibit the formation of adipocytes in mouse 3T3-L1 cell line. In humans, 1,25(OH)2D3 promotes preadipocyte differentiation under cell culture conditions. Further evidence of its important functions is given by VDR knock out (VDR(-/-)) and CYP27B1 knock out (CYP27B1 (-/-)) mouse models: Both VDR(-/-) and CYP27B1(-/-) models are highly resistant to the diet induced weight gain, while the specific overexpression of human VDR in adipose tissue leads to increased adipose tissue mass. The analysis of microarray datasets from human adipocytes treated with macrophage-secreted products up-regulated VDR and CYP27B1 genes indicating the capacity of adipocytes to even produce active 1,25(OH)2D3. Experimental studies demonstrate that 1,25(OH)2D3 has an active role in adipose tissue by modulating inflammation, adipogenesis and adipocyte secretion. Yet, further in vivo studies are needed to address the effects and the effective dosages of vitamin D in human adipose tissue and its relevance in the associated diseases.
Project description:Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4?,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4?,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1?,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4?,25(OH)2D3 and decrease in 1?,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4?-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.
Project description:Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D3 status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n?=?40 and White, n?=?20). Plasma 25(OH)D3 levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)2D3. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D3 status was normal and not significantly different between ethnicities. Neither 25(OH)D3 status nor 1,25(OH)2D3 supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)2D3-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D3 status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)2D3-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.
Project description:The classical pathway of vitamin D activation follows the sequence D3?25(OH)D3?1,25(OH)2D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. Some of these metabolites are hydroxylated by CYP27B1 at C1?, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 are non-calcemic, while the addition of an OH at C1? confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)2D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both ROR? and ROR? receptors. Specifically, they have high docking scores using crystal structures of ROR? and ROR? LBDs. Furthermore, 20(OH)D3 and 20,23(OH)2D3 have been tested in a cell model that expresses a Tet-on ROR? or ROR? vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of ROR?/?. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing ROR?/?. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of ROR?/?. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)2D3 and VDR.