Ras-dependent and -independent regulation of oxygen species by mitogenic growth factors and TGF-beta 1

Mitogenic growth factors and transforming growth factor beta 1 (TGF-beta 1) induce the generation of reactive oxygen species (ROS) in non phagocytic c ells, but their enzymatic source(s) and regulatory mechanisms are largely u nknown. We previously reported on the ability of TGF-beta 1 to activate a c ell surface-associated NADH:flavin:O-2 oxidoreductase (NADH oxidase) that g enerates extracellular H2O2. In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta 1 with respect to the primary reactive species produced (O-2(.-) vs. H2O2), the s ite of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha tan EGF receptor ligand) are able to rapidly (within 5 min) induce the generat ion of intracellular O-2(.-) without detectable NADH oxidase activity or ex tracellular H2O2 release. In contrast, TGF-beta 1 does not stimulate intrac ellular O-2(.-) production and the delayed induction of extracellular H2O2 release is not associated with O-2(.-) production. Expression of dominant-n egative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O-2(.-) generation but has no effe ct on TGF-beta 1-induced NADH oxidase activation/H2O2 production. These res ults demonstrate that there are at least two distinctly different ROS-gener ating enzymatic systems in lung fibroblasts regulated by mitogenic growth f actors and TGF-beta 1 via Ras-dependent and -independent mechanisms, respec tively. In addition, these findings suggest that endogenous production of R OS by growth factors/ cytokines may have different biological effects depen ding on the primary reactive species generated and site of production.