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 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.     

Direct regulation of HOXA10 by 1,25-(OH)2D3 in human myelomonocytic cells and human endometrial stromal cells

Vitamin D receptor (VDR) and the functionally active form of its ligand, 1,25-(OH)2D3, have been implicated in female reproduction function and myeloid leukemic cell differentiation. HOXA10 is necessary for embryo implantation and fertility, as well as hematopoeitic development. In this study, we identified a direct role of vitamin D in the regulation of HOXA10 in primary human endometrial stromal cells, the human endometrial stromal cell line (HESC), and in the human myelomonocytic cell line, U937. Treatment of primary endometrial stromal cells, or the cell lines HESC and U937 with 1,25-(OH)2D3 increased HOXA10 mRNA and protein expression. VDR mRNA and protein were detected in primary uterine stromal cells as well as HESC and U937 cells. We cloned the HOXA10 upstream regulatory sequence and two putative vitamin D response elements (VDRE) into luciferase reporter constructs and transfected primary stromal cells and HESC. One putative VDRE (P1: -385 to -434 bp upstream of HOXA10) drove reporter gene expression in response to treatment with 1,25-(OH)2D3. In EMSA, VDR demonstrated binding to the HOXA10 VDRE in the presence of 1,25-(OH)2D3. 1,25-(OH)2D3 up-regulates HOXA10 expression by binding VDR and interacting with a VDRE in the HOXA10 regulatory region. Direct regulation of HOXA10 by vitamin D has implications for fertility and myeloid differentiation.     

Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.

The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)(2)D(3), including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)(2)D(3). Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.     

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements.

The ability of vitamin D receptor-retinoid X receptor (VDR-RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXR beta have been used. The VDREs were from 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25-dihydroxyvitamin D3-24-hydroxylase, r24-OHase), and a 1,25-(OH)2D3 repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR-RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57 degrees, 56 degrees, 61 degrees, and 59 degrees, respectively for rOC, mOP, r24-Ohase, and hPTH). The distortions took place with or without a 1,25-(OH)2D3 ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR-RXR heterodimers induced a directed bend of 26 degrees, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR-RXR heterodimers induce DNA bending.     

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.     

1,25-Dihydroxyvitamin D(3) and 9-cis-retinoids are synergistic regulators of 24-hydroxylase activity in the rat and 1, 25-dihydroxyvitamin D(3) alters retinoic acid metabolism in vivo.

The RXR forms a heterodimer with the VDR to activate genes that are regulated by 1,25(OH)(2)D(3). In the absence of RXR's ligand, 9-cis-RA, RXR appears to be a silent partner to VDR. The effect of 9-cis-RA on VDR/RXR heterodimer formation and 1, 25(OH)(2)D(3)-mediated gene expression in vivo remains unclear. We examined the effect of exogenous 9-cis-RA or 9-cis-RA precursors, 9, 13-di-cis-RA and 9-cis-RCHO, on 1,25(OH)(2)D(3)-mediated induction rat renal 24-hydroxylase. The rats were treated as follows: (1) vehicle; (2) 1,25(OH)(2)D(3); (3) 1,25(OH)(2)D(3) + 9-cis-RA; (4) 1, 25(OH)(2)D(3) + 9,13-di-cis-RA; (5) 1,25(OH)(2)D(3) + 9-cis-RCHO; (6) 9-cis-RA; (7) 9,13-di-cis-RA; and (8) 9-cis-RCHO. 1, 25(OH)(2)D(3) was administered IP 18 h prior to sacrifice. The retinoids were administered every 4 h, starting 28 h prior to sacrifice. The last retinoid dose was administered 4 h prior to sacrifice. Treatment with 1,25(OH)(2)D(3) alone increased 24-hydroxylase from 35 +/- 6 (controls) to 258 +/- 44 pmol/min/g tissue. When 1,25(OH)(2)D(3) was administered with 9-cis-RA, 9, 13-di-cis-RA, or 9-cis-RCHO, 24-hydroxylases were 568 +/- 56, 524 +/- 56, and 463 +/- 62 pmol/min/g tissue, respectively. Furthermore, codosing of 1,25(OH)(2)D(3) and 9-cis-retinoids resulted in higher circulating concentrations of 9-cis-RA and 9,13-di-cis-RA when compared to rats dosed with 9-cis-retinoids alone. This was shown to be due to 1,25(OH)(2)D(3) increasing the half-life of 9,13-di-cis-RA by three to four times. These results show that 9-cis-RA can act synergistically with 1,25(OH)(2)D(3) in the regulation of 24-hydroxylase in vivo. Additionally, 1,25(OH)(2)D(3) regulates 9, 13-di-cis-RA metabolism in vivo.