1,25-Dihydroxyvitamin D3 (1,25(OH2D3) is considered to be the functional metabolite of vitamin D3 which stimulates the expression of hormone-specific genes via genomic action. Recently, this sterol has been identified in the regulation of such diverse biologic phenomena as protein synthesis and secretion, enzyme activity, and changes in cell morphology and differentiation. We propose to utilize several mammalian cultured cell lines in which appropriate bioresponses to vitamin D3 have been identified, including human intestine cells (407), human leukemic cells (HL-60), pig kidney cells (LLC-PK1), rat osteosarcoma cells (ROS 17/2.8), and mouse fibroblasts (3T6), to elucidate the mechanism(s) of the sterol's action. We will initially examine the characteristics of interaction between 1,25(OH)2D3 and the intact cell, its binding to specific cytoplasmic receptors, and its effects on transcriptional parameters. Regulation of biologic activity by 1,25(OH)2D3 will be examined at 4 levels: i) molecular (CaBP, collagen, and actin synthesis), ii) enzymatic (vitamin D3-24-OHase), iii) plasma membrane (Ca++ transport), and iv) cellular control (morphology and differentiation). We will investigate the effects of vitamin D3 metabolites on cultured cell responses, and attempt to correlate the extent of the response with both the administered vitamin D3 metabolite concentration, receptor presence, and the degree of receptor occupancy. Inhibitor studies (actinomycin D, butyrate, cycloheximide) will be utilized to further define the level at which the cultured cell responds to 1,25(OH)2D3. Finally, DNA binding site-specific antireceptor antibodies will be utilized in microinjection studies as selective blockers of the receptor-mediated nuclear action of the hormone, thus permitting evaluation of receptor requirements and identification of non-genomic ativities of 1,25(OH)2D3. This approach is expected to further define the relationship between vitamin D3 and newly identified cellular bioresponses, and in particular, the mechanism by which these activities are promoted. We feel that the proposed experiments may reveal important clues as to the regulation of processes involved in cell differentiation, a phenomenon crucially important to our understanding of human cancers.
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