Negative modulation of α1(I) procollagen gene expression in human skin fibroblasts: Transcriptional inhibition by interferon-γ

Interferon-γ (IFN-γ), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of α1(I) procollagen gene (COL1A1) expression by recombinant IFN-γ. The results showed that IFN-γ stimulated the rapid accumulation of interferon regulated factor (IRF)-1 mRNA, followed by a delayed and dose-dependent inhibition of α1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF-1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre-mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter-chloramphenicol acetyltransferase reporter gene plasmids, IFN-γ selectively inhibited promoter activity and abrogated its stimulation induced by TGF-β. The inhibition by IFN-γ was not due to downregulation of TGF-β receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN-α and IFN-β by themselves had little effect on promoter activity, but IFN-α augmented the inhibitory effect of IFN-γ. Using a series of 5' deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN-γ response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF-1 but no homologs of previously characterized IFN- γ response elements. The putative IFN-γ response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN-γ. Gel mobility shift analysis showed that two distinct and specific DNA-protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN-γ response region. IFN-γ did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN-γ inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF-1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN-γ regulation of fibroblast function.