The vitamin D response element-binding protein. A novel dominant-negative regulator of vitamin D-directed transactivation

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a non-vitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin d-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamin d-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominant-negative regulator of vitamin D action.     

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism

Using vitamin D-resistant New World primates as model of natural diversity for sterol/steroid action and metabolism, two families of novel intracellular vitamin D regulatory proteins have been discovered and their human homologs elucidated. The first family of proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), initially considered to function only as pre-mRNA-interacting proteins, have been demonstrated to be potent cis-acting, trans-dominant regulators of vitamin D hormone-driven gene transactivation. The second group of proteins bind 25-hydroxylated vitamin D metabolites. Their overexpression increases vitamin D receptor (VDR)-directed target gene expression. We found that these intracellular vitamin D binding proteins (IDBPs) are homologous to proteins in the heat shock protein-70 family. Our ongoing studies indicate directly or indirectly through a series of protein interactions that the IDBPs interact with hydroxylated vitamin D metabolites and facilitate their intracellular targeting.     

A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.     

The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity: studies in human skin fibroblasts bearing vitamin D receptor mutations

If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.     

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.     

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.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

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.     

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.