IDENTIFICATION OF ELEMENTS IN THE PROMOTER REGION OF THE ALPHA-1(I) PROCOLLAGEN GENE INVOLVED IN ITS UP-REGULATED EXPRESSION IN SYSTEMIC-SCLEROSIS

Objective. To identify regulatory elements in the promoter region of t he al(I) procollagen gene (COL1A1) involved in the transcriptional act ivation of this gene in systemic sclerosis (SSc), and to identify the transcription factors interacting with these regulatory elements. Meth ods. Dermal fibroblasts from 6 patients with diffuse SSc of recent ons et and from 6 healthy individuals were studied. The transcriptional re gulation of COL1A1 was examined by transient transfections with deleti on constructs containing portions of the COL1A1 promoter. The DNA bind ing activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays. A proc edure was established to allow the quantitative determination of the a mount of DNA binding proteins interacting with the COL1A1 promoter, em ploying DNA binding protein and DNA titration experiments analyzed by gel mobility shift assays. Results. Maximal chloramphenicol acetyltran sferase activity was observed with a -174-bp to +42-bp COL1A1 promoter construct in both normal and SSc cells; however, the activity driven by this construct was 70-260% higher in SSc fibroblasts. Most of the t ranscriptional activity of the COL1A1 promoter was contained in a mini mal promoter region encompassing -174 bp to -84 bp, Electrophoretic mo bility shift assays performed with oligonucleotides corresponding to t he regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promote r revealed marked increases in the intensities of DNA-protein complexe s formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts, Comp etition experiments showed that each of these regions contained elemen ts recognized by Spl and nuclear factor 1 (NF-1) binding proteins. A q uantitative determination of DNA binding activity recognizing the Spl binding element within the -129/-107-bp region showed that it was 23.6 nM in SSc fibroblasts compared with 6.9 nM in normal fibroblasts, Con clusion. The results demonstrate that a short region in the proximal p romoter of COL1A1 containing 2 tandem NF-1/Sp1 elements displays up-re gulated transcriptional activity in SSc fibroblasts, and that SSc fibr oblasts contain 3.4-fold greater DNA binding activity recognizing thes e elements than normal cells.