Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.     

Loss of CYP3A7 gene induction by 1,25-dihydroxyvitamin D3 is caused by less binding of VDR to the proximal ER6 in CYP3A7 gene

Cytochrome P450 3A4 and 3A7 (CYP3A4 and CYP3A7, respectively) are predominant forms in the human adult and fetal liver, respectively. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to be a potent inducer of CYP3A4 in human colon carcinoma Caco-2 via vitamin D receptor (VDR). However, whether CYP3A7 is inducible by 1,25(OH)(2)D(3) has not yet been elucidated. In the present study, we examined the effect of 1,25(OH)(2)D(3) on CYP3A7 gene expression in Caco-2 cells, which express CYP3A4 and CYP3A7 mRNAs. 1,25(OH)(2)D(3) hardly induced the expression of CYP3A7 mRNA in contrast to the marked induction of CYP3A4 mRNA. Reporter assay using 5'-franking region CYP3A4 and CYP3A7 genes also revealed that 1,25(OH)(2)D(3) activates CYP3A4 promoter, but not CYP3A7 promoter, which has two mutations in the proximal ER6 site compared with CYP3A4 promoter. In addition, we found that the binding of VDR to the proximal ER6 in CYP3A7 gene was markedly less than that to the proximal ER6 in CYP3A4 gene using gel shift assay. Taken together, the decrease of VDR binding to the proximal ER6 caused by the mutation results in the loss of CYP3A7 gene activation by 1,25(OH)(2)D(3).