Figure 5

Bosentan reversed the pro-fibrotic phenotype of systemic sclerosis (SSc) dermal fibroblasts via increasing the DNA binding of Fli1. ( A-C ) Whole cell lysates (WCL) prepared from SSc fibroblasts treated with endothelin-1 (ET-1) for 24 hours (A) or bosentan (BOS) for 48 hours (B, C) were subjected to immunoblotting ( D ) Protein kinase C (PKC)-δ levels were examined by immunoblotting in cytoplasmic extracts (CE) and nuclear extracts (NE) prepared from SSc fibroblasts treated or untreated with bosentan for 48 hours. ( E-G ) SSc fibroblasts were treated or untreated with bosentan for 48 hours. Fli1 phosphorylation levels and total Fli1 levels were determined by immunoblotting (E). Fli1 occupancy of the human α2 (I) collagen (COL1A2) promoter was determined by chromatin immunoprecipitation and reverse transcription (RT) real-time PCR (F). Fli1 mRNA levels were determined by RT real-time PCR (G). The graph represents fold change in type I collagen protein levels quantified by densitometry (A, B), Fli1 occupancy of the COL1A2 promoter (F), and Fli1 mRNA levels (G) in comparison to unstimulated controls, which were arbitrarily set at 1. Equal amounts of loading were confirmed by immunoblotting for β-actin in cytoplasmic extracts and for TATA binding protein (TBP) in nuclear extracts. The values below each blot represent the relative levels of target molecules normalized by loading controls with densitometry. α1(I), α1(I) procollagen; α2(I), α2(I) procollagen; AU, arbitrary unit.