The broad objective of the proposed continuing research is to elucidate the molecular functions of the nuclear vitamin D receptor (VDR), which is known to cooperate with the retinoid X receptor (RXR) in order to mediate the actions of the vitamin D hormone (1,25(OH)2D3) to prevent rickets and osteopenia by effecting normal calcium and phosphate homeostasis. Effort for the requested renewal period will be concentrated on characterizing new roles for VDR that are postulated to be relevant to suppressing the promotion of colon cancer, and to facilitating normal hair cycling and skin cell integrity. Actions of VDR in colon and keratinocyte cells are hypothesized to be executed by novel VDR ligands that are structurally divergent from 1,25(OH)2D3. One of these, lithocholic acid, likely relevant in colon, is identified in preliminary data. The proposed hair follicle VDR ligand will be detected utilizing a cell-based reporter gene assay and characterized by HPLC purification and negative-ion nanoelectrospray mass spectrometry. Furthermore, the central hypothesis being tested is that several of the extraosseous (non-calcemic) functions of VDR are analogous to those of SXR (the human steroid and xenobiotic receptor, also referred to as PXR), and involve the sensing of xenobiotic lipids and their detoxification via the induction of cytochrome P450s (CYPs). Specifically, intestinal CYP3A4, as well as renal CYP24 and CYP3A23 gene expression will be investigated as VDR targets in cotransfected cultured cells.
A second Aim will be to extend the concepts of extraosseous VDR ligands and gene targets to the identification of unique VDR-interacting proteins (VIPs) at these sites, with candidates such as the hairless (Hr) gene product in skin. The characterization of VIPs will enhance our understanding of the biochemical mechanisms whereby VDR represses or induces the transcription of target genes in skin, and those encoding enzymes for the metabolism of xenobiotics. Cell- and CYP promoter-specific VIPs will be screened by employing the yeast two-hybrid system and CYP vitamin D responsive element-VDR-RXR differential affinity chromatography, respectively, with functional relevance verified by mammalian two-hybrid and chromatin immunoprecipitation (CHIP) assays.
The final Aim i s to probe the functional impact of common polymorphisms in the 5' and 3' regions of the human VDR gene on CYP induction and the non-calcemic actions of VDR. It is conceivable that VDR gene polymorphisms could influence the sensitivity of VDR to xenobiotic ligands that induce CYPs, as is the case for SXR/PXR. Therefore, by investigating novel VDR ligands, cell- and promoter-specific VIPs, and the role of common human VDR gene polymorphisms in terms of the affect of genotype on transcriptional activation phenotype, it should be possible to clarify the participation of this nuclear receptor in xenobiotic detoxification, as well as in the pathophysiology and treatment of abnormal hair cycling, and hyperproliferative diseases of epithelial cells.
