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.     

Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene.

Mitochondrial cytochrome P450(24) expression in the vitamin D-degradation pathway is induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The molecular basis of this enzyme regulation was investigated by isolating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction process. Constructs containing different lengths of 5'-flanking region of the gene were linked to a luciferase reporter gene and transiently co-transfected with a human vitamin D receptor (hVDR) expression vector (pRSV-hVDR) into COS-1 cells. These experiments showed that the flanking region from -298 to -122 directed a 24-fold increase in luciferase activity in response to 1,25-(OH)2D3 provided that the cells were co-transfected with pRSV-hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)2D3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase promoter. Mutagenesis revealed that the sequence from position -150 to -136 is required for induction by 1,25-(OH)2D3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) reported for other genes. Gel shift mobility assays showed this region specifically bound a nuclear protein complex from 1,25-(OH)2D3 treated COS-1 cells that had been co-transfected with pRSV-hVDR. The retarded band was specifically competed with the well characterized VDRE from the mouse osteopontin gene. A VDRE at position -150 to -136 in the promoter of the rat P450(24) gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.     

Analysis of the 5-lipoxygenase promoter and characterization of a vitamin D receptor binding site

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and transforming growth factor beta (TGFbeta) potently induce 5-lipoxygenase (5-LO) in myeloid cells. We analyzed vitamin D receptor (VDR) binding to putative vitamin D response elements within the 5-LO promoter and analyzed its function by reporter gene analysis. Binding of VDR and retinoid X receptor to the promoter region was shown in DNase I footprinting, electrophoretic mobility shift and chromatin immunoprecipitation assays. However, the identified VDR binding region did not mediate induction of reporter gene activity by 1,25(OH)(2)D(3)/TGFbeta, neither in the 5-LO promoter context nor with the thymidine kinase (tk) promoter. Insertion of the rat atrial natriuretic factor VDRE in reporter plasmids containing the 5-LO promoter diminished induction by 1,25(OH)(2)D(3)/TGFbeta as compared with the tk promoter. Similarly, low inductions were obtained when cells were transiently or stably transfected with constructs containing various 5-LO promoter regions. Concerning basal promoter activity, we identified a positive regulatory region (-779 to -229), which includes the VDR binding region, in 5-LO-positive MonoMac6 cells. In summary, the VDR/RXR complex binds to putative VDREs in the 5-LO promoter, but other sequences outside the 5-LO promoter seem to be responsible or additionally required for the prominent induction of 5-LO mRNA expression by 1,25(OH)(2)D(3) and TGFbeta.     

Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.     

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.     

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).     

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.