title: Signaling pathways involved in isoprostane-mediated fibrogenic effects in rat hepatic stellate cells.
abstract: Despite evidence supporting a potential role for F2-isoprostanes (F2-IsoP's) in liver fibrosis, their signaling mechanisms are poorly understood. We have previously provided evidence that F2-IsoP's 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-IsoP's to exert their fibrogenic effects. HSCs were isolated from rat liver, cultured to their activated myofibroblast-like phenotype, and then treated with the isoprostane 15-F2t-isoprostane (15-F2t-IsoP). Inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined using commercial kits. Mitogen-activated protein kinase (MAPK) and cyclin D1 expression was assessed by Western blotting. Cell proliferation and collagen synthesis were determined by measuring [(3)H]thymidine and [(3)H]proline incorporation, respectively. 15-F2t-IsoP elicited an activation of extracellular-signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), which 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 min, suggesting binding to the TPβ isoform and activation of Giα protein. Also, 15-F2t-IsoP increased IP3 levels within a few minutes, suggesting that the Gq protein pathway is also involved. In conclusion, the fibrogenic effects of F2-IsoP's in HSCs are mediated by downstream activation of MAPKs, through TP binding that couples via both Gqα and Giα proteins. Targeting TP receptor, or its downstream pathways, may contribute to preventing oxidative damage in liver fibrosis.
authors.affiliation: Department of Molecular and Developmental Medicine, University of Siena, I-53100 Siena, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Acquaviva
authors.affiliation: Department of Molecular and Developmental Medicine, University of Siena, I-53100 Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vecchio
authors.affiliation: Department of Molecular and Developmental Medicine, University of Siena, I-53100 Siena, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Arezzini
authors.affiliation: Department of Molecular and Developmental Medicine, University of Siena, I-53100 Siena, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Comporti
authors.affiliation: Department of Molecular and Developmental Medicine, University of Siena, I-53100 Siena, Italy. Electronic address: concetta.gardi@unisi.it.
authors.forename: Concetta
authors.initials: C
authors.lastname: Gardi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015237:Dinoprost / Q000031:analogs & derivatives; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D028421:Isoprostanes / Q000378:metabolism* / Q000494:pharmacology; D008103:Liver Cirrhosis / Q000378:metabolism; D051381:Rats; D017482:Receptors, Thromboxane / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D028421:Isoprostanes; D017482:Receptors, Thromboxane; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost
keywords: 15-F2t-IsoP; AC; Collagen synthesis; DMEM; Dulbecco's modified Eagle's medium; ERK; F2-IsoP; F2-isoprostane; FSK; Free radicals; HSC; I-BOP; IP(3); Isoprostane receptor; JNK; Liver fibrosis; MAPK; PKA; PKC; PLC; TCA; TP; [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; adenosine 3′,5′-cyclic monophosphate; adenylyl cyclase; c-Jun NH(2)-terminal kinase; cAMP; extracellular-signal-regulated kinase; forskolin; hepatic stellate cell; inositol trisphosphate; mitogen-activated protein kinases; phospholipase C; protein kinase A; protein kinase C; thromboxane A2 receptor; trichloroacetic acid