The active hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], acts in target cells by binding to specific vitamin D receptors (VDR). The magnitude of the response elicited by 1,25(OH)2D3 is directly dependent on the concentration of VDR which, in turn, is regulated by a variety of factors. The premise of this grant is that the regulation of VDR gene expression is a critical mechanism that modulates the responsiveness of target cells to 1,25(OH)3D3 in a cell-specific manner. Regulation of VDR expression may be at the transcriptional or post-transcriptional level. This cell-specific mechanism of adaptation allows the response of the various organs to be differentially modulated to a general circulating level of 1,25(OH)2D#. This grant proposes to explore several categories of regulators of VDR gene expression, predominantly in human tissues, focusing on intestine and bone as critical target cells. Most studies will be performed in cultured cells but experiments using rat and human tissue removed at surgery are also planned. Preliminary data indicate that all of the regulators to be investigated substantially modulate the number of VDR in various target cells. The regulators to be studied include: 1) Glucocorticoids, which by down-regulation of VDR and anti-vitamin D action sin intestine and bone may contribute to glucocorticoid-induced osteoporosis. 2) Estrogens, which by up-regulation of intestinal and bone VDR may contribute to maintaining normal bone, a mechanism which when lost at the menopause may contribute to osteoporosis. 3) Serum and growth factors (EGF, IGF-1, Insulin) which increase cell proliferation as well as up-regulating VDR levels. 4) Activators of protein kinase C including phorbol esters which increase cell proliferation but decrease VDR to basal levels and inhibit serum and growth factor-up regulation. 5) Activators of protein kinase A including cAMP which up-regulate VDR even more than serum and growth factors. 6) Calcitropic hormones PTH and PTH-RP, which act on cells by stimulating protein kinase A and C and have important VDR regulatory activity. The mechanism of VDR regulation will be investigated at multiple levels including changes in the abundance, affinity and half-life of the VDR protein, accumulation of VDR mRNA and rate of transcription. Studies of the 5' upstream region of the VDR gene will employ a VDR promoter-CAT reporter plasmid transfected into various target cells to allow examination of the regulatory elements that modulate VDR gene expression at a molecular level. The long range goals of this research program included developing an understanding of the factors that regulate VDR gene expression and the molecular mechanisms by which that regulation takes place. Since VDR is regulated by a variety of hormones and factors, the regulation of this critical gene is an ideal model system for the study of the interactions of these factors as they regulate the expression of VDR. In addition to understanding the mechanism of 1,25(OH)2D3 action in the classic sphere of calcium homeostasis, this grant will also explore the expanded scope of vitamin D action as it relates to regulation of cell growth and differentiation as influenced by VDR modulation.
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