Distinct signaling pathways exist for small lipophilic hormones such as the sex and adrenal steroids, thyroid hormone, and the vitamins A and D. These signaling pathways function to convey information from the cellular environment to the nucleus of the cell where they elicit unique transcriptional responses. Most cellular responses are initiated through interaction of a hormonal ligand with its unique receptor and culminate in receptor-mediated modifications in gene expression. Like other hormones, vitamin D is also capable of promoting both the differentiation of cells from precursors populations and regulating the expression of genes involved in differentiated cell function. The integration of these fundamental actions by vitamin D in target tissues likely accounts for its focal as well as global role as a calciotropic hormone. Since the ability of tissues to response to an external vitamin D stimulus revolves around the expression of the VDR gene, the goal of this project is to determine the molecular basis for its expression. We will focus on bone forming cells where the anabolic actions of vitamin D on both differentiation and differentiated-cell functions are well established. We will utilized primary human osteoblastic cells to establish the fundamentals of VDR regulation at the protein, mRNA, and transcriptional levels. We will focus upon basal expression of VDR as well as the gene's profile of inducibility by 1,25(OH)2D3 as well as bone relevant regulators such as PTH, retinoids, and glucocorticoids. While our efforts in these studies are directed towards bone cells, VDR expression studies will also be carried out in intestinal-like HT-29 cells for comparison. Having established VDR expression and regulation in bone cells, the transcriptional component will be explored through an analysis of the activity and regulatory properties of the cloned human promoter for the VDR. We will use molecular approaches to identify determinants of both basal as well as inducible expression. We anticipate characterizing factors important to the expression and regulation of the VDR in bone cells such as PEBP2alphaA1/Cbfa1. Finally, we plan to prepare transgenic animals to assess the capacity of the cloned 4.0 kb hVDR promoter to direct faithful expression of firefly luciferase in bone cells and other tissues. Several additional mouse strains carrying mutant hVDR promoters will also be prepared to assess the role of specific cis elements. Our expectation is to more fully understand important determinants of human VDR regulation in bone cells as well as other cells at the transcriptional level. These determinants may be exploited together with novel vitamin D analogues in the treatment of bone diseases exemplified by osteoporosis and hypeproliferative diseases such as psoriasis and cancer.
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