Acetaldehyde induced collagen synthesis by inhibition of PPARgamma trascriptional activity via hydrogen peroxide-mediated activation of c-Abl non-receptor tyrosine kinase in human hepatic stellate cells

Accumulating evidence have indicated that acetaldehyde (Ac- CHO) is one of the main mediator of fibrogenesis in alcoholic liver disease. Although AcCHO can stumulate synthesis of fibrillar collagens in human hepatic stellate cells (HSC), the molecular events directly involved in the activation of collagen genes are still debate. PPARgamma is a member of the nuclear receptor superfamily of ligand-dependent transcription factor that is expressed in HSC where it has been shown have a key role in the maintenance of resting phenotype. Furthermore receptor activation by specific ligands inhibits collagen gene expression in vitro and in vivo. In consideration of the recent observation that AcCHO inhibits PPARs trascriptional activity in hepatoma cells, aim of this study was to evaluate the effect of AcCHO on PPARgamma trascriptional activity and the correlation with AcCHO-induced collagen gene expression. The activity of a transfected PPARgamma-responsive reporter gene (ARE7-Tk-LUC) was used as index of PPARgamma function in activated HSC. AcCHO treatment inhibited ligand dependent and independent PPARgamma trascriptional activity in a dose dependent manner. This effect was not associated with an impairement of the ability of the receptor to bind the specific PPARresponsive elements evaluated by EMSA, but it was correlated to an increased phosphorylation of a MAP kinase site at the serine 84 of the human PPARgamma 1. Confirming in vivo phosphorylation experiments with [32P]-orthophospate, transfection of the PPARgSer84Ala mutant completely prevent the effect of AcCHO on PPARgamma activity and in parallel abrogate the induced collagen gene expression by AcCHO. The inhibitory effect of AcCHO on PPARgamma activity was blocked by inhibitor of ERK1/2 pathway, PD98059 and UO180, and by PKCdelta inhibitors, rottlerin and deltaV1-1. Furthermore PEG-catalase completely abrogated the effect of AcCHO on PPARgamma and block the activation of either ERK1/2 or PKCdelta, suggesting that hydrogen peroxyde is involved in the signalling pathways controlling PPARgamma phosphorylation by AcCHO. Finally we tested whether c-Abl kinase, that is implicated in oxidative stress response, is a mediator of PPARgamma inactivation by AcCHO. Both the c-Abl inhibitor, imatinib mesylate, and c-Abl dominant negative completely abrogated the effect of AcCHO both on PPARgamma and on collagen synthesis. Furtermore PPARgSer84Ala mutant expression prevented the profibrogenic signals mediated by c-Abl activation. In conclusion the induction of collagen expression by AcCHO in HSC is dependent on PPARgamma phoshorylation via H202-mediated activation c-Abl/PKDdelta/ ERK1/2 signalling pathway