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.
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.
V J
VJ
Thannickal
K D
KD
Aldweib
B L
BL
Fanburg
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