Characterization of a DNA binding site that mediates the stimulatory effect of cyclosporin-A on type III collagen expression in renal cells

Background. Previous work from our laboratory demonstrated upregulation of type III collagen by cyclosporin A (CsA) in a cellular model of renal fibro blasts 'in vitro', suggesting that a mechanism of gene transcriptional acti vation might be responsible for collagen accumulation in renal fibrosis res ulting from chronic CsA treatment. Methods. We analysed in the same cellular model: (i) COL3A1 mRNA expression by RT-PCR; (ii) COL3A1 promoter activity by transfection of renal fibrobla sts with constructs containing promoter fragments of different length fused to a reporter gene; (iii) expression of transcription factors by western b lot analysis; (iv) DNA-protein binding by gel retardation assays with nucle ar extracts from CsA-treated and untreated cells; and (v) site-directed mut agenesis of COL3A1 promoter to verify the role of a short DNA segment as Cs A responsive element. Results. CsA induced a 3-5-fold increase in COL3A1 mRNA that was paralleled by a stimulation of the COL3A1 promoter. Degradation of COL3A1 mRNA was co mparable in CsA-treated and -untreated cells. The target region was first l imited to a 178 bp fragment from -117 to +61 (pFV1). By gel retardation, ut ilizing several oligonucleotides that covered the whole length of pFV1, we detected a factor able to bind the promoter DNA (oligo 31) in nuclear extra cts after 3 h treatment with CsA. The binding was absent in untreated cells and it was not detected when a 10-base mutation was introduced in oligonuc leotide 31. Finally, the same substitution mutation at the site of binding of this factor abolished the stimulatory effect of CsA on COL3A1 promoter. Some transcription factors, whose potential binding sites are included in t he above promoter fragment, were induced by CsA treatment either soon (3 h) or late (24-72 h) after treatment and were detected by western blot analys is. Conclusions. CsA induces the synthesis of type III collagen by stimulating a pathway leading to activation of COL3A1 promoter and upregulation of COL3 A1 mRNA. A short promoter fragment, proximal to the transcription start sit e, is the target of CsA stimulation.