Intracellular signaling pathways activated by reactive oxygen species regulate proliferation and collagen synthesis of human hepatic stellate cells

Reactive oxygen species (ROS) stimulate human hepatic stellate cells (HSC) proliferation and collagen synthesis. Intracellular signaling elements (such as ERKlR and PI3K) have a role in transducing signals from the cell surface to the nucleus and in mediating a fibrogenic behaviour in HSC. We thus verified which intracellular signaling pathways are activated by ROS and associated with HSC proliferation and collagen synthesis. The xanthine/xanthine oxidase system (K/X0) was used as a superoxide anion donor. Phosphorylation of ERKU2 and pp70S6K (a downstream component of PI3K) was evaluated by Western blot. HSC proliferation and matrix synthesis were evaluated by 3H-TdR incorporation and ELISA for type I collagen, respectively. HSC incubation with x/x0 was associated with increased ERK1/2 and pp70S6K phosphorylation, well evident at 10 and 30 min and maintained up to 60 min for ERKU2. These effects were completely abolished by tyrphostin A9, a selective inhibitor of the PDGF receptor tyrosine kinase, and PD98059, a MEK inhibitor. The PI3K inhibitor wortmannin reduced pp70S6K phosphorylation with no effect on ERKlR, while the PKC inhibitor calphostin C decreased protein phosphorylation only in the later phase. Inhibition of these mitogenic in a parallel reduction in X/X0-induced HSC proliferation, whereas X/KO-induced stimulation of type I collagen accumulation was inhibited only by calphostin C and PD98059. In conclusion, this study indicates that ROS activate several signaling pathways which differentially regulate HSC proliferation and collagen synthesis, thus providing new selective targets for the specific inhibition of these effects in human HSC