Superoxide production and reactive oxygen species signaling by endothelial nitric-oxide synthase

Abstract

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 (O2/·-) 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 O2/·- 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. N(ω)-Methyl-L-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFα up- regulation. Likewise, Gln361 eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFα. Similar to the effect of eNOS, a O2/·- 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 O2/·- production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O2/·-, 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 O2/·- production.