Requirement of hydrogen peroxide generation in TGF-β1 signal transduction in human lung fibroblast cells: Involvement of hydrogen peroxide and Ca²⁺ in TGF-β1-induced IL-6 expression

Stimulation of human lung fibroblast cells with TGF-β1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-L-cysteine (NAC). TGF-β1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-β1-induced IL-6 gene expression, and direct H₂O₂ treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-β1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca²⁺ influx is stimulated by TGF-β1 treatment. EGTA suppressed TGF- β1- or H₂O₂- induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-β1- or H₂O₂-induced IL-6 and AP-1 activation. In addition, TGF-β1 or H₂O₂ increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-β1-induced IL-6 expression. Taken together, these results indicate that TGF-β1 induces a transient increase of intracellular H₂O₂ production, which regulates downstream events such as Ca²⁺ influx, MAPK, and AP-1 activation and IL-6 gene expression.