TYROSINE PHOSPHORYLATION REGULATES H2O2 PRODUCTION IN LUNG FIBROBLASTS STIMULATED BY TRANSFORMING-GROWTH-FACTOR BETA-1

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional, p rofibrotic cytokine involved in cellular growth and differentiation. W e have previously described a cell surface-associated H2O2-generating NADH:flavin:O-2 oxidoreductase (referred to as NADIR oxidase) activity in human lung fibroblasts induced by TGF-beta 1 (Thannickal, V. J., a nd Fanburg, B. L. (1995) J. Biol. Chem. 270, 30334-30338). In this stu dy, the potential for regulation of this novel TGF-beta 1-activated ox idase in fibroblasts by protein tyrosine phosphorylation was examined. Immunoblots using anti-phosphotyrosine antibody demonstrated a time-d ependent but delayed phosphorylation of two proteins of 115 and 103 kD a in cells stimulated with TGF-beta 1 (2 ng/ml), Similar to the effect on TGF-beta 1-induced H2O2 production, phosphorylation of these prote ins was blocked by the addition of actinomycin D. The protein-tyrosine kinase inhibitors genistein and herbimycin A inhibited TGF-beta 1-ind uced protein tyrosine phosphorylation, NADH oxidase activation, and H2 O2 production in a dose-dependent manner. Catalase, diphenyliodonium ( an inhibitor of flavoenzymes), and suramin (an inhibitor of receptor a ctivation, added 4 h after TGF-beta 1) had no effect on the induction of protein tyrosine phosphorylation, Phosphorylation of the 115- and 1 03-kDa proteins preceded the generation of H2O2 production and returne d to control levels when H2O2 was undetectable at 48 h after TGF-beta 1 exposure, These results suggest that protein tyrosine phosphorylatio n by a nonreceptor protein-tyrosine kinase(s) regulates the activity o f the TGF-beta 1-responsive H2O2-generating NADH oxidase in human lung fibroblasts, Additionally, this study demonstrates that TGF-beta 1, w hich binds to a serine-threonine kinase receptor, is able to induce pr otein tyrosine phosphorylation in a delayed manner via a signaling pat hway that requires transcriptional activation.