Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor beta1.
Transforming growth factor beta1 (TGF-beta1) is a multifunctional, profibrotic cytokine involved in cellular growth and differentiation. We have previously described a cell surface-associated H2O2-generating NADH:flavin:O2 oxidoreductase (referred to as NADH oxidase) activity in human lung fibroblasts induced by TGF-beta1 (Thannickal, V. J., and Fanburg, B. L. (1995) J. Biol. Chem. 270, 30334-30338). In this study, the potential for regulation of this novel TGF-beta1-activated oxidase in fibroblasts by protein tyrosine phosphorylation was examined. Immunoblots using anti-phosphotyrosine antibody demonstrated a time-dependent but delayed phosphorylation of two proteins of 115 and 103 kDa in cells stimulated with TGF-beta1 (2 ng/ml). Similar to the effect on TGF-beta1-induced H2O2 production, phosphorylation of these proteins was blocked by the addition of actinomycin D. The protein-tyrosine kinase inhibitors genistein and herbimycin A inhibited TGF-beta1-induced protein tyrosine phosphorylation, NADH oxidase activation, and H2O2 production in a dose-dependent manner. Catalase, diphenyliodonium (an inhibitor of flavoenzymes), and suramin (an inhibitor of receptor activation, added 4 h after TGF-beta1) had no effect on the induction of protein tyrosine phosphorylation. Phosphorylation of the 115- and 103-kDa proteins preceded the generation of H2O2 production and returned to control levels when H2O2 was undetectable at 48 h after TGF-beta1 exposure. These results suggest that protein tyrosine phosphorylation by a nonreceptor protein-tyrosine kinase(s) regulates the activity of the TGF-beta1-responsive H2O2-generating NADH oxidase in human lung fibroblasts. Additionally, this study demonstrates that TGF-beta1, which binds to a serine-threonine kinase receptor, is able to induce protein tyrosine phosphorylation in a delayed manner via a signaling pathway that requires transcriptional activation.
Thannickal
V J
VJ
Pulmonary and Critical Care Division, Department of Medicine, New England Medical Center, Tupper Research Institute, and Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Aldweib
K D
KD
Fanburg
B L
BL
D019898:Autocrine Communication; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D009097:Multienzyme Complexes; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; D014443:Tyrosine; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D011505:Protein-Tyrosine Kinases