Transcriptional activation of the alpha 1(I) procollagen gene and up-regulation of alpha 1(I) and alpha 1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice

Objective. To investigate the levels of expression of type I and type III c ollagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mic e and to examine the transcriptional regulation of the alpha 1(I) procollag en gene (COL1A1) in these cells. Methods. Dermal fibroblasts from Tsk2 mice and from normal age- and sex-mat ched control mice were studied. Steady-state levels of alpha 1(I) and alpha 1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot -blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs con taining portions of the COL1A1 promoter ligated to the chloramphenicol acet yltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift as says were performed with nuclear extracts prepared from normal and Tsk2 der mal fibroblasts. Results, Synthesis of collagen was almost 100% higher in Tsk2 dermal fibrob lasts than in control fibroblasts, Up-regulation of mRNA for 2 extracellula r matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the alpha 1(I) procollagen mRNA steady-state levels were 50% higher, and those of the alpha 1(III) procollagen mRNA 100% higher, in Tsk2 cells, The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of alpha 1(I) col lagen mRNA in Tsk2 cells were largely due to increased transcriptional acti vity of the corresponding gene. Electrophoretic mobility shift assays perfo rmed with a probe encompassing a relevant COL1A1 promoter region revealed i ncreased DNA-protein binding activities in nuclear extracts prepared from T sk2 fibroblasts compared with normal fibroblasts. Competition experiments u sing consensus Spl and nuclear factor 1 (NF-1) oligonucleotides and supersh ift experiments using anti-Spl and anti-NF-1 antibodies indicated that at l east 2 transcription factors, Spl and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts. Conclusion. Dermal fibroblasts from Tsk2 mice display increas ed collagen synthesis and up-regulation of alpha 1(I) and alpha 1(III) proc ollagen mRNA in vitro. The data also directly demonstrate the transcription al activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest th at the transcription factors Sp1 and NF-1 or their homologs play an importa nt role in the upregulated expression of this gene in Tsk2 fibroblasts, The se findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study o f the connective tissue alterations in this disease.