Characterization of a GC-rich Region Containing Sp1 Binding Site(s) as a Constitutive Responsive Element of the α2(I) Collagen Gene in Human Fibroblasts

Abstract

To analyze regulatory elements in the human alpha 2(I) collagen gene (COL1A2) promoter, a series of deletion mutants from -323 to -186 base pairs was tested in transient transfection assays in human fibroblasts. A strong positive responsive element was mapped to a GC-rich region located between base pairs -303 and -271. This region contains three binding sites (GC-boxes) resembling recognition sites for the transcription factor Sp1. Substitution mutations in the GC-boxes abolished binding to the GC-rich region in gel shift analyses and resulted in 90% reduction of promoter activity in transient transfection assays. We demonstrated that transcription factor Sp1 is essential for binding based on the following observations. 1) Sp1 consensus binding site alone competes by binding to the GC-rich region in the DNase I protection assay; 2) both Sp1 consensus binding site and Sp1 antibodies prevent the formation of a DNA-protein complex in the mobility shift assay; 3) anti-Sp1 antibodies recognize a component of the complex competed for by Sp1 consensus binding site.

Oncostatin M Stimulates Transcription of the Human α2(I) Collagen Gene via the Sp1/Sp3-binding Site

“…Deletions to bp Ϫ148 did not significantly alter the level of inducibility, but further deletion to bp Ϫ108 abolished OSM stimulation. In agreement with previous data, deletion of the Sp1-binding sites (three GC boxes) between bp Ϫ353 and Ϫ264 significantly decreased basal promoter activity (17). Subsequent deletion to bp Ϫ186 had no additional effect on basal promoter activity, whereas deletion to bp Ϫ148 increased basal promoter activity ϳ2-fold compared with the deletion to Ϫ186.…”

“…Plasmid Constructions-The deletion and substitution mutant plasmids (with the exception of the Ϫ148 end point deletion construct) have been previously described (17,18). The Ϫ148 ␣2(I) collagen promoter deletion construct was generated using polymerase chain reaction technology.…”

“…DNA mobility shift assay was performed as described previously (17). Briefly, the binding reaction was performed on ice for 30 min in binding buffer (10 mM HEPES (pH 7.9), 50 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl 2 , 4% glycerol, 0.5 mM EDTA, 0.7 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 2 g/ml leupeptin, and 2 g/ml pepstatin) containing 50,000 cpm labeled probe, 2 g of poly(dI-dC)⅐poly(dI-dC), and nuclear extracts containing 5 g of protein.…”

“…Our laboratory is involved in studies of the molecular mechanisms underlying the regulation of expression of the human ␣2(I) collagen gene in healthy and fibrotic human fibroblasts (17)(18)(19). The mechanism of modulation of type I collagen by OSM is of special interest because of a previous report that indicated a differential response of systemic sclerosis scleroderma and healthy skin fibroblasts to OSM.…”

Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators

“…Notably, we show here that TGF-b induced a dose-dependent increase of p300 accumulation in the ®broblasts. Taken together, these observations suggest that the activation state and relative cellular abundance of Smad3, p300/CBP, and probably additional factors implicated on collagen transcription such as Sp1 (Tamaki et al, 1995;Li et al, 1995) collectively determine the level of COL1A2 transcription in ®broblasts.…”