Essential role of the NADPH oxidase subunit p47phox in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-α
Essential role of the NADPH oxidase subunit p47phox in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-α
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DOI：
10.1161/hh0202.103615
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发表时间：
2002-02-08
影响因子：
20.1
通讯作者：
Shah, AM
中科院分区：
文献类型：
--
作者：
Li, JM;Mullen, AM;Shah, AM
吞噬细胞型NADPH氧化酶复合物是内皮细胞活性氧（ROS）产生的主要来源，但其生化功能和调控尚不清楚。在嗜中性粒细胞中，p47（phox）亚单位主要参与氧化酶激活，以响应激动剂，如佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）。我们研究了p47（phox）在PMA或肿瘤坏死因子-α（TNF-α）刺激下内皮细胞ROS产生中的作用。为了明确p47（phox）的作用，我们研究了从p47（phox-/-）小鼠和野生型对照中分离的冠状动脉微血管内皮细胞（CMECs）。p47（phox）在敲除小鼠的心脏中不存在，而必需氧化酶亚基p22（phox）在两组中均表达。在没有激动剂刺激的情况下，与野生型CMECs相比，p47（phox）的缺乏并没有导致p47（phox-/-）CMECs中NADPH依赖性ROS产生的减少。通过光泽精（5 mumol/L）化学发光或二氯氢荧光素（DCF）荧光检测，PMA（100 ng/mL）或TNF α（100 U/mL）预刺激10分钟可显著增加野生型CMECs中NADPH依赖性O-2（-）的产生。这种反应在p47（phox-/-）细胞中完全丧失。将p47（phox）全长cDNA转染p47（phox-/-）CMECs可引起p47（phox）蛋白的表达和对PMA和TNF α的O-2（-）反应的恢复。在野生型CMECs中，转染反义p47（phox）cDNA显著降低了p47（phox）表达，并导致对PMA和TNF α的O-2（-）反应丧失。这些数据表明，内皮细胞p47（phox）在PMA和TNF α上调NADPH氧化酶活性中是关键的。
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-alpha (TNFalpha) 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 TNFalpha (100 U/mL) for 10 minutes significantly increased NADPH-dependent O-2(-) production in wild-type CMECs, assessed either by lucigenin (5 mumol/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 TNFalpha. 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 TNFalpha. These data show that endothelial cell p47(phox) is critical in the upregulation of NADPH oxidase activity by PMA and TNFalpha.