Essential Role of the NADPH Oxidase Subunit p47 phox in Endothelial Cell Superoxide Production in Response to Phorbol Ester and Tumor Necrosis Factor-α

Abstract

A phagocyte-type NADPH oxidase complex is a major source of endothelial reactive oxygen species (ROS) production, but its biochemical function and regulation remain unclear. In neutrophils, the p47 phox subunit is centrally involved in oxidase activation in response to agonists such as phorbol-12-myristate-13-acetate (PMA). We investigated the role of p47 phox in endothelial cell ROS production in response to PMA or tumor necrosis factor-α (TNFα) stimulation. To specifically address the role of p47 phox , we studied coronary microvascular endothelial cells (CMECs) isolated from p47 phox−/− mice and wild-type controls. p47 phox was absent in hearts of knockout mice whereas the essential oxidase subunit, p22 phox , was expressed in both groups. In the absence of agonist stimulation, the lack of p47 phox did not result in a reduction in NADPH-dependent ROS production in p47 phox−/− CMECs compared with wild-type CMECs. Prestimulation with PMA (100 ng/mL) or TNFα (100 U/mL) for 10 minutes significantly increased NADPH-dependent O 2 − production in wild-type CMECs, assessed either by lucigenin (5 μmol/L) chemiluminescence or dichlorohydrofluorescein (DCF) fluorescence. This response was completely lost in p47 phox−/− cells. Transfection of the full-length p47 phox cDNA into p47 phox−/− CMECs caused expression of p47 phox protein and restoration of the O 2 − response to PMA and TNFα. In wild-type CMECs, transfection of antisense p47 phox cDNA substantially reduced p47 phox expression and caused loss of the O 2 − response to PMA and TNFα. These data show that endothelial cell p47 phox is critical in the upregulation of NADPH oxidase activity by PMA and TNFα.