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 hav e demonstrated that glucocorticoids decrease Type I procollagen gene e xpression. These latter studies have included uridine incorporation in to pro alpha 1(I) and pro alpha 2(I) mRNAs and nuclear run-off experim ents. Using the ColCat 3.6 plasmid, which contains part of the 5' flan king 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 ge ne. The glucocorticoid-mediated down-regulation of procollagen gene ex pression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasm id. 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 po ssibility existed that another cis-element in the 5' flanking region o f the proc alpha 1(I) collagen gene was also required for the glucocor ticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate collagen pro alpha 1(I) and pro a lpha 2(I) gene activities. Dexamethasone treatment of non-transfected skin fibroblasts did result in a decrease of transforming growth facto r-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 fibrob lasts decreased glucocorticoid receptor binding to the GRE and TGF-bet a activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the i nteraction of these TGF-beta molecules with cellular membrane receptor s and subsequent transduction is dramatically decreased resulting in l ess 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 me diated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gen e(s). The present studies provide the basis for a novel mechanism of g lucocorticoid-mediated regulation of eukaryotic genes containing the T GF-beta element. (C) 1995 Wiley-Liss, Inc.