The c-Abl tyrosine kinase controls protein kinase Cdelta-induced Fli-1 phosphorylation in human dermal fibroblasts.

摘要

OBJECTIVE: We have previously demonstrated that in response to transforming growth factor beta (TGFbeta), Fli-1 activity is repressed through a series of sequential posttranslational modifications, consisting of protein kinase Cdelta (PKCdelta)-induced Thr312 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 TGFbeta. 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 PKCdelta adenovirus or were transiently transfected with a Bcr-Abl-overexpressing plasmid. Subcellular localization of PKCdelta was examined by immunocytochemistry. RESULTS: Western blot analysis of cell lysates demonstrated that the levels of phospho-Fli-1 (Thr312) 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 TGFbeta-induced phosphorylation of Fli-1. Additionally, we showed that c-Abl kinase activity was required for nuclear localization of PKCdelta. CONCLUSION: Our results demonstrate that in SSc fibroblasts, c-Abl is an upstream regulator of the profibrotic PKCdelta/phospho-Fli-1 pathway, via induction of PKCdelta nuclear localization. Additionally, the finding that Fli-1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-Abl/PKCdelta/phospho-Fli-1 pathway is constitutively activated in these cells. Thus, blocking the TGFbeta/c-Abl/PKCdelta/phospho-Fli-1 pathway could be an attractive alternative approach to therapy for scleroderma.