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.     

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.     

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.     

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.     

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).     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation

Both calcium and 1,25(OH)(2)D promote the differentiation of keratinocytes in vitro. The autocrine or paracrine production of 1,25(OH)(2)D by keratinocytes combined with the critical role of the epidermal calcium gradient in regulating keratinocyte differentiation in vivo suggest the physiologic importance of this interaction. The interactions occur at a number of levels. Calcium and 1,25(OH)(2)D synergistically induce involucrin, a protein critical for cornified envelope formation. The involucrin promoter contains an AP-1 site essential for calcium and 1,25(OH)(2)D induction and an adjacent VDRE essential for 1,25(OH)(2)D but not calcium induction. Calcium regulates coactivator complexes that bind to the Vitamin D receptor (VDR). Nuclear extracts from cells grown in low calcium contain an abundance of DRIP(205), whereas calcium induced differentiation leads to reduced DRIP(205) and increased SRC 3 which replaces DRIP in its binding to the VDR. In vivo models support the importance of 1,25(OH)(2)D-calcium interactions in epidermal differentiation. The epidermis of 1alphaOHase null mice fails to form a normal calcium gradient, has reduced expression of proteins critical for barrier function, and shows little recovery of the permeability barrier when disrupted. Thus in vivo and in vitro, calcium and 1,25(OH)(2)D interact at multiple levels to regulate epidermal differentiation.     

Vitamin D binding protein and monocyte response to 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D: analysis by mathematical modeling

Vitamin D binding protein (DBP) plays a key role in the bioavailability of active 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and its precursor 25-hydroxyvitamin D (25OHD), but accurate analysis of DBP-bound and free 25OHD and 1,25(OH)(2)D is difficult. To address this, two new mathematical models were developed to estimate: 1) serum levels of free 25OHD/1,25(OH)(2)D based on DBP concentration and genotype; 2) the impact of DBP on the biological activity of 25OHD/1,25(OH)(2)D in vivo. The initial extracellular steady state (eSS) model predicted that 50 nM 25OHD and 100 pM 1,25(OH)(2)D), <0.1% 25OHD and <1.5% 1,25(OH)(2)D are 'free' in vivo. However, for any given concentration of total 25OHD, levels of free 25OHD are higher for low affinity versus high affinity forms of DBP. The eSS model was then combined with an intracellular (iSS) model that incorporated conversion of 25OHD to 1,25(OH)(2)D via the enzyme CYP27B1, as well as binding of 1,25(OH)(2)D to the vitamin D receptor (VDR). The iSS model was optimized to 25OHD/1,25(OH)(2)D-mediated in vitro dose-responsive induction of the vitamin D target gene cathelicidin (CAMP) in human monocytes. The iSS model was then used to predict vitamin D activity in vivo (100% serum). The predicted induction of CAMP in vivo was minimal at basal settings but increased with enhanced expression of VDR (5-fold) and CYP27B1 (10-fold). Consistent with the eSS model, the iSS model predicted stronger responses to 25OHD for low affinity forms of DBP. Finally, the iSS model was used to compare the efficiency of endogenously synthesized versus exogenously added 1,25(OH)(2)D. Data strongly support the endogenous model as the most viable mode for CAMP induction by vitamin D in vivo. These novel mathematical models underline the importance of DBP as a determinant of vitamin D 'status' in vivo, with future implications for clinical studies of vitamin D status and supplementation.     

Antagonistic action of a 25-carboxylic ester analogue of 1alpha, 25-dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators

A 25-carboxylic ester analogue of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)), ZK159222, was described as a novel type of antagonist of 1alpha,25-(OH)(2)D(3) signaling. The ligand sensitivity of ZK159222, in facilitating complex formation between 1alpha,25-(OH)(2)D(3) receptor (VDR) and the retinoid X receptor (RXR) on a 1alpha,25-(OH)(2)D(3) response element (VDRE), was approximately 7-fold lower when compared with 1alpha,25-(OH)(2)D(3). However, ZK159222 was not able to promote a ligand-dependent interaction of the VDR with the coactivator proteins SRC-1, TIF2, and RAC3, neither in solution nor in a complex with RXR on DNA. Functional analysis in HeLa and COS-7 cells demonstrated a 10-100-fold lower ligand sensitivity for ZK159222 than for 1alpha, 25-(OH)(2)D(3) and, most interestingly, a potency that was drastically reduced compared with 1alpha,25-(OH)(2)D(3). A cotreatment of 1alpha,25-(OH)(2)D(3) with a 100-fold higher concentration of ZK159222 resulted in a prominent antagonistic effect both in functional in vivo and in in vitro assays. These data suggest that the antagonistic action of ZK159222 is due to a lack of ligand-induced interaction of the VDR with coactivators with a parallel ligand sensitivity, which is sufficient for competition with the natural hormone for VDR binding.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.