ACTIVATION OF AN H2O2-GENERATING NADH OXIDASE IN HUMAN LUNG FIBROBLASTS BY TRANSFORMING GROWTH-FACTOR-BETA-1

The cellular source(s) and mechanisms of generation of reactive oxygen species (ROS) in nonphagocytic cells stimulated by cytokines are uncl ear, In this study, we demonstrate that transforming growth factor bet a 1 (TGF-beta 1, 1 ng/ml) induces the release of H2O2 from human lung fibroblasts within 8 h following exposure to this cytokine, Elevation in H2O2 release peaked at 16 h (similar to 22 pmol/min/10(6) cells) an d gradually declined to undetectable levels at 48 h after TGF-beta 1 t reatment. NADH consumption by these cells was stimulated by TGF-beta 1 while that of NADPH remained unchanged. NADH oxidase activity as meas ured by diphenyliodonium (DPI)-inhibitable NADH consumption in TGF-bet a 1-treated cells followed a time course similar to that of H2O2 relea se. DPI, an inhibitor of the NADPH oxidase complex of neutrophils and other flavoproteins, also inhibited the TGF-beta 1-induced H2O2 produc tion, Inhibitors of other enzymatic systems involving flavoproteins th at may be responsible for the production of H2O2 in these cells, inclu ding xanthine oxidase, nitric oxide synthase, and both mitochondrial a nd microsomal electron transport systems, failed to inhibit TGF-beta 1 -induced NADH oxidation and H2O2 production. The delay (>4 h) followin g TGF-beta 1 exposure along with the inhibition of this process by cyc loheximide and actinomycin D suggest the requirement of new protein sy nthesis for induction of NADH oxidase activity in TGF-beta 1-stimulate d fibroblasts.