Signalling pathways involved in isoprostane-mediated fibrogenic effects in rat hepatic stellate cells

Despite evidence supporting a potential role for F2-isoprostanes (F2-IsoPs) in liver fibrosis, their signalling mechanisms are poorly understood. We have previously provided evidence that F2-IsoPs stimulate hepatic stellate cell (HSC) proliferation and collagen hyperproduction by activation of a modified form of isoprostane receptor homologous to the classic thromboxane receptor (TP). In this paper, we examined which signal transduction pathways are set into motion by F2-IsoPs to exert their fibrogenic effects. HSC were isolated from the rat liver, cultured to their activated myofibroblast-like phenotype, and then treated with the isoprostane 15-F2-isoprostane (15-F2t-IsoP). Inositol trisphosphate (IP3) and adenosine 3’,5’-cyclic monophosphate (cAMP) levels were determined by commercial kits. Mitogen-activated protein kinases (MAPK) and cyclin D1 expression were assessed by western blotting. Cell proliferation and collagen synthesis were determined by measuring [3H]-thymidine and [3H]-proline incorporation, respectively. 15-F2t-IsoP elicited an activation of extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun HN2-terminal kinase (JNK), that are known to be also regulated by G-protein-coupled receptors. Preincubation with specific ERK (PD98059), p38 (SB203580) or JNK (SP600125) inhibitors prevented 15-F2t-IsoP-induced cell proliferation and collagen synthesis. 15-F2t-IsoP decreased cAMP levels within 30 minutes, suggesting the binding to TP isoform and activation of Gi protein. Also, 15-F2t-IsoP increased IP3 levels within few minutes, suggesting that Gq protein pathway is also involved. In conclusion, the fibrogenic effects of F2-IsoPs in HSC are mediated by downstream activation of MAPK, through TP binding that couples via both Gq and Gi proteins. Targeting TP receptor, or its downstream pathways, may contribute to prevent oxidative damage in liver fibrosis.