Stress-activated protein kinases in the activation of rat hepatic stellate cells in culture

The signal cascades involved in the activation of hepatic stellate cells (HSC) are largely unknown. Factors initiating activation include tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, endothelin, and oxidative stress. In other cell types some of these have been reported to stimulate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We have therefore investigated the role of these kinases in HSC activation. HSC were isolated from male Wistar rats. Quiescent experiments were performed on day 2 HSC and transformed experiments on day 15 passage 1 HSC. Kinase activities were determined by immunoprecipitation and phosphorylation of specific substrate proteins and alpha-smooth muscle actin (SMA) expression by immunoblotting. The constitutive activity of p38 MAP kinase was higher in transformed versus quiescent cells. In quiescent cells TNFalpha stimulated p38 MAP kinase and JNK activities 12- and 4-fold respectively and this was halved by 2-mercaptoethanol, an indirect antioxidant. Endothelin-1 activated both kinases in quiescent cells via the endothelin-B receptor, while TGFbeta had no effect. Both 2-mercaptoethanol and a p38 inhibitor (SB202190) inhibited alpha-SMA expression by day 5 cells. The activation of p38 MAP kinase and JNK by TNFalpha and endothelin, together with the inhibition of this activation by 2-mercaptoethanol, provides indirect evidence supporting their role in HSC transformation. Direct evidence for a role for p38 MAP kinase is provided by the observations that its constitutive activity is higher in transformed versus quiescent cells and that its inhibitor reduces HSC activation in culture as assessed by alpha-SMA expression.