MECHANISM OF CULTURED CELL RESPONSES TO VITAMIN D3

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.