Dermal atrophy is a major conspicuous side effect of topical corticosteroid therapy and results from decreased collagen deposition. The decreased deposition of collagen in skin results from a decrease of procollagen synthesis which has been observed both in vivo and in fibroblast cell cultures. This laboratory has demonstrated that glucocorticoid-induced inhibition of procollagen synthesis occurs by a receptor-mediated glucocorticoid-induced down regulation of procollagen gene expression. A major objective of this proposal is to identify and functionally characterize DNA glucocorticoid regulatory elements in the 5'-flanking region and the first intron of the mouse pro-alpha2(1) gene and compare these sequences to glucocorticoid response elements. The 5' flanking or intronic DNA deletions will be ligated to the CAT (chloramphenicol acetyltransferase) gene. These DNA constructs will be transfected into embryonic mouse fibroblasts to determine the biological activity of sequences necessary for glucocorticoid-mediated down regulation of gene expression. We will synthesize glucocorticoid receptor in vitro from cDNA and determine its binding to DNA fragments of the 5'- flanking region and the first intron. The effect of glucocorticoid on steady state levels of the glucocorticoid receptor mRNA will also be determined. Glucocorticoid receptor may not bind directly to DNA and may involve cooperating regulatory protein(s) and possible known transcription factors through an indirect mechanism involving a regulatory gene, the product of which may regulate collagen gene expression. Accordingly, we will determine the effects of glucocorticoids on DNA binding proteins through gel retardation and stairway assays. In addition, the sequence(s) of the DNA binding elements will be determined by footprinting. It has been shown that Vitamin A blocks some of the glucocorticoid effects on collagen metabolism. An objective of this proposal will be to determine the ability of all-trans retinoic acid to reverse the molecular inhibitory effects of glucocorticoids on procollagen gene expression.
