The c-Abl Tyrosine Kinase Controls Protein Kinase Cδ-Induced Fli-1 Phosphorylation in Human Dermal Fibroblasts

Objective. We have previously demonstrated that in response to transforming growth factor beta(TGF beta), Fli-1 activity is repressed through a series of sequential posttranslational modifications, consisting of protein kinase C delta (PKC delta)-induced Thr(312) phosphorylation, acetylation by p300/CREB binding protein-associated factor, and detachment from the collagen promoter. The purpose of this study was to further investigate the upstream events that lead to Fli-1 phosphorylation in response to TGF beta. Methods. Dermal fibroblasts were isolated from systemic sclerosis (SSc) patients and healthy control subjects matched for age, sex, and ethnicity. Western blotting was used to analyze protein levels and real-time quantitative reverse transcription-polymerase chain reaction analysis was used to measure messenger RNA expression. Cells were transduced with constitutively active PKC delta adenovirus or were transiently transfected with a Bcr-Abl-overexpressing plasmid. Subcellular localization of PKC delta was examined by immunocytochemistry. Results. Western blot analysis of cell lysates demonstrated that the levels of phospho-Fli-1 (Thr(312)) were up-regulated in SSc fibroblasts, correlating with increased levels of type I collagen and c-Abl protein. Experiments using a constitutively activated form of c-Abl, small interfering RNA against c-Abl and the specific tyrosine kinase inhibitor imatinib, demonstrated the requirement of c-Abl for the TGF beta-induced phosphorylation of Fli-1. Additionally, we showed that c-Abl kinase activity was required for nuclear localization of PKC delta. Conclusion. Our results demonstrate that in SSc fibroblasts, c-Abl is an upstream regulator of the profibrotic PKC delta/phospho-Fli-1 pathway, via induction of PKC delta nuclear localization. Additionally, the finding that Fli-1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-Abl/PKC delta/phospho-Fli-1 pathway is constitutively activated in these cells. Thus, blocking the TGF beta/c-Abl/PKC delta/phospho-Fli-1 pathway could be an attractive alternative approach to therapy for scleroderma.