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PKC&zgr; Regulates TNF-&agr;–Induced Activation of NADPH Oxidase in Endothelial Cells

Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKC&zgr;, has been implicated in the mechanism of tumor necrosis factor-&agr; (TNF-&agr;)–induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKC&zgr; in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47phox and membrane-bound gp91phox in human pulmonary artery endothelial (HPAE) cells. TNF-&agr; failed to activate oxidant generation in lung vascular endothelial cells derived from p47phox−/− and gp91phox−/− mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-&agr; resulted in the phosphorylation of p47phox and its association with gp91phox. Inhibition of PKC&zgr; by multiple pharmacological and genetic approaches prevented the TNF-&agr;–induced phosphorylation of p47phox, and its translocation to the membrane. PKC&zgr; was shown to colocalize with p47phox, and inhibition of PKC&zgr; activation prevented the interaction of p47phox with gp91phox induced by TNF-&agr;. Furthermore, inhibition of association of p47phox with gp91phox prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKC&zgr; in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.