Ras-dependent and -independent regulation of oxygen species by mitogenic growth factors and TGF-β1

Mitogenic growth factors and transforming growth factor beta 1 (TGF-beta 1) induce the generation of reactive oxygen species (ROS) in non phagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta 1 to activate a cell surface-associated NADH:flavin:O-2 oxidoreductase (NADH oxidase) that generates 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 site 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 generation of intracellular O-2(.-) without detectable NADH oxidase activity or extracellular H2O2 release. In contrast, TGF-beta 1 does not stimulate intracellular O-2(.-) production and the delayed induction of extracellular H2O2 release is not associated with O-2(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O-2(.-) generation but has no effect on TGF-beta 1-induced NADH oxidase activation/H2O2 production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta 1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/ cytokines may have different biological effects depending on the primary reactive species generated and site of production.