GLUCOCORTICOIDS COORDINATELY REGULATE TYPE-I COLLAGEN PRO-ALPHA-1 PROMOTER ACTIVITY THROUGH BOTH THE GLUCOCORTICOID AND TRANSFORMING GROWTH-FACTOR-BETA RESPONSE ELEMENTS - A NOVEL MECHANISM OF GLUCOCORTICOID REGULATION OF EUKARYOTIC GENES

Glucocorticoids have previously been shown to decrease Type I collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type I procollagen gene expression. These latter studies have included uridine incorporation into pro alpha 1(I) and pro alpha 2(I) mRNAs and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking region of the pro alpha 1(I) collagen gene and the reporter gene, chloramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibroblasts that dexamethasone down regulates the promoter activity of the pro alpha 1(I) collagen gene. The glucocorticoid-mediated down-regulation of procollagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whole ColCat 3.6 plasmid did not eliminate the effect. The possibility existed that another cis-element in the 5' flanking region of the proc alpha 1(I) collagen gene was also required for the glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate collagen pro alpha 1(I) and pro alpha 2(I) gene activities. Dexamethasone treatment of non-transfected skin fibroblasts did result in a decrease of transforming growth factor-beta (TGF-beta) secretion into the media. In addition, CAT activity was decreased by dexamethasone and increased by TGF-beta. The decrease of CAT activity by dexamethasone was brought back to control value by the addition of exogenous TGF-beta to the culture media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid receptor binding to the GRE and TGF-beta activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the interaction of these TGF-beta molecules with cellular membrane receptors and subsequent transduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expression through both the GRE and TGF-beta elements. Depression of procollagen gene expression by glucocorticoids through the TGF-beta element is mediated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the basis for a novel mechanism of glucocorticoid-mediated regulation of eukaryotic genes containing the TGF-beta element. (C) 1995 Wiley-Liss, Inc.