Glucocorticoids coordinately regulate type I collagen proα1 promoter activity through both the glucocorticoid and transforming growth factor β response elements: A novel mechanism of glucocorticoid regulation of eukaryotic genes

Abstract

Glucocorticoids have previously have 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 pro alpha 1 (I) collagen gene was also required for the collagen glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate in a decrease of transforming growth factor-beta (TGF-beta) secretion into the 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-mediator regulation of eukaryotic genes containing the TGF-beta element.

AP-1 overexpression impairs corticosteroid inhibition of collagen production by fibroblasts isolated from asthmatic subjects

“…We found that TGF-␤ induced procollagen mRNA expression, protein levels, and promoter activity from NBF and ABF but cannot be antagonized by Dex treatment. A GR response element is present in the TGF-␤ gene promoter and is thus susceptible to CS treatment (31,35). We found that the expression of TGF-␤ was reduced by CS in normal cells but not in cells from asthmatics.…”

The impact of chronic in vivo glucocorticoid excess on the functional characteristics of human skin fibroblasts obtained from patients with endogenous Cushing’s syndrome

“…In fact, it is well recognized that acute in vivo and in vitro exposure to GCs grossly diminished type I procollagen synthesis at the mRNA and protein level in human dermal fibroblasts (8,19). Repression of procollagen genes has been attributed to a co-ordinate action of GCs through both GC-and TGF-b-responsive elements (20). On the other hand, in previous studies, expression of MMP-1 and -2 was found to be reduced following treatment with GCs (8,21).…”

Species differences in cis-elements of the Pro?1(I) procollagen promoter and their binding proteins

“…A probe containing the conserved GRE halfsite at position Ϫ655 of the rat promoter binds to a nuclear protein from rat fibroblasts [Meisler et al, 1995], but it was not tested with isolated GR. Therefore, we used gel shift assays to determine whether the 40 bp rGRE probe bound to recombinant human GR, and for comparison also tested nuclear proteins from rat and human cells.…”

“…Other regions in the pro␣1(I) promoter also were analyzed to determine if there were additional species differences in transcription factor binding sites. The analysis revealed that the upstream region containing a glucocorticoid response element (GRE) at positions Ϫ655/ Ϫ650 in the rat gene [Meisler et al, 1995], differed significantly in the human promoter. Therefore, a detailed study of the binding of glucocorticoid receptor (GR) and nuclear proteins to this region of the human and rat promoters also was carried out.…”

“…It also has been reported, however, that a 174 bp minimal promoter is sufficient for optimal transcription [Jimenez et al, 1994], which would suggest that the Ϫ190/Ϫ170 sequence analogous to the mouse A1 region, may not be involved in human promoter activity. Regions further upstream also modulate transcriptional activity of, or confer tissue specific expression to, the human [Slack et al, 1990], mouse [Rossert et al, 1995], and rat promoters [Dhalla et al, 1997;Dodig et al, 1996;Meisler et al, 1995].…”