Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP.
These results suggest that EM703 inhibits the transcription of type I collagen in both normal and SSc fibroblasts, and that the transcription is inhibited through the CCAAT box of the COL1A1 promoter.
Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF.
We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF).
These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts.
The numbers of cells and grains per cell per square millimeter expressing alpha 1(I) procollagen mRNA in fibrotic zone SSc skin were significantly elevated compared with normal control skin (both P < 0.01).
Electrophoretic mobility shift assays performed with oligonucleotides corresponding to the regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promoter revealed marked increases in the intensities of DNA-protein complexes formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts.
Transcription of the COL1A1 gene in SSc fibroblasts was induced 2-3-fold over that in controls in both monolayer and lattice cultures, accounting in part for the elevated steady-state level.
These results suggest that only a specific subset of fibroblasts are activated in SSc, as evidence by an increased proportion of cells with high levels of alpha 1(I) procollagen mRNA.