Superoxide Production and Reactive Oxygen Species Signaling by Endothelial Nitric-oxide Synthase

Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O⨪2) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor α (TNFα) through a mechanism dependent on the production of O⨪2 and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFα promoter activity and TNFα production.Nω-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFα up-regulation. Likewise, Gln361eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFα. Similar to the effect of eNOS, a O⨪2 donor dose-dependently increased TNFα production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFα up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O⨪2 production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O⨪2, interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O⨪2 production.