Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: Involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression

Stimulation of human lung fibroblast cells with TGF-beta 1 resulted in a tr ansient burst of reactive oxygen species with maximal increase at 5 min aft er treatment. This reactive oxygen species increase was inhibited by the an tioxidant, N-acetyl-L-cysteine (NAC), TGF-beta 1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antio xidants including NAC, glutathione, and catalase reduced TGF-beta 1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-beta 1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported t hat Ca2+ influx is stimulated by TGF-beta 1 treatment. EGTA suppressed TGF- beta 1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expre ssion, with simultaneously modulating AP-1 activity in the same pattern. PD 98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extra cellular signal-related kinase kinase 1, suppressed TGF-beta 1- or H2O2-ind uced IL-6 and AP-1 activation. In addition, TGF-beta 1 or H2O2 increased MA PK activity which was reduced by EGTA and NAG, suggesting that MAPK is invo lved in TGF-beta 1-induced IL-6 expression. Taken together, these results i ndicate that TGF-beta 1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, an d AP-1 activation and IL-6 gene expression.