Oxidative stress induce collagen synthesis by c-Abl activation in human hepatic stellate cells

Accumulating data support the hypothesis that oxidative stress plays a key role in the development of hepatic fibrosis. However, the molecular events directly involved in oxidative stressinduced collagen gene expression are still debatable. Aim of the study was to investigate the role of c-Abl in oxidative stressinduced collagen synthesis in Hepatic Stellate cells. Methods. Human hepatic stellate cells were isolated from wedge sections unsuitable for transplantation. After pronase and collagenase digestion, stellate cells were purified over continuous Nycodenz gradient and cultured on plastic. Xanthine/xanthine oxidase (x/xo) complex was used to induce oxidative stress by producion of superoxide anion. Collagen I production in the culture media was evaluated by ELISA, mRNA levels were analyzed by Northen Blot. Induction of collagen I gene transcription was assessed by use of a luciferase reporter. Kinase activity of c-Abl was evaluated by pull-down assays. ERK1/2 phosphorylation was detected by western blotting. Kinase active and Kinase dead c-Abl mutants were obtained by site directed mutagenesis. Results. Oxidative stress by x/xo induced procollagen I production in cultured human hepatic stellate cells, both at the mRNA and protein level. Xanthine/xanthine oxidase treatment induced an early activation of c-Abl kinase activity and ERK1/2 phosphorylation. The c-Abl inhibitor Imatinib mesylate impaired the x/xo induction ERK1/2 phosphorylation and collagen production. The inhibitory action of Imatinib was mimicked by overexpression of a C-Abl dominant negative mutant. Conversely, Imatinib mesylate failed to reduce the x/xo induced-ERK phosphorylation and collagen synthesis in cells expressing a constitutive kinase-active c-Abl mutant. Conclusions. These results highlight the importance of c-Abl as a mediator of the pro-fibrogenic effects of oxidative stress through the ERK1/2 pathway, and suggest that c-Abl could represent a therapeutic target for the treatment of liver fibrosis