Collagen α 1 (I) Gene Contains an Element Responsive to Tumor Necrosis Factor-α Located in the 5' Untranslated Region of Its First Exon

Abstract

The aims of the present study were to identify the cis-acting element through which tumor necrosis factor-α (TNFα) inhibits collagen alpha ₁(I) gene transcription and the trans-acting factors involved in this effect in cultured hepatic stellate cells. Deletion analysis of the collagen α₁(I) promoter demonstrated that TNFα inhibited gene expression through an element located between - 59 and + 116 bp relative to the transcription start site. DNase I protection assays revealed a footprint between + 68 and + 86 bp of the collagen first exon, the intensity of which decreased when the DNA probe was incubated with nuclear protein from TNFα-treated hepatic stellate cells. This footprint contained a G + C-rich box. Transfection experiments demonstrated that mutations in this G + C-rich element abrogated the inhibitory effect of TNF alpha on the collagen alpha₁(I) promoter. Gel retardation experiments using a radiolabeled oligonucleotide containing sequences of this region confirmed that TNFα treatment decreased the formation of two complexes between nuclear proteins and DNA. These complexes were efficiently blocked with an oligonucleotide containing an Sp1-binding site and were supershifted with specific Sp1 and Sp3 antibodies. These results suggest that TNFα inhibits collagen α₁(I) gene expression by decreasing the binding of Sp1 to a G + C-rich box in the 5' untranslated region of its first exon.