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Acute Tumor Necrosis Factor Alpha Signaling via NADPH Oxidase in Microvascular Endothelial Cells: Role of p47^phox Phosphorylation and Binding to TRAF4

Departments of Cardiology,¹ Medicine, Guy's King's and St. Thomas' School of Medicine, King's College London, London, United Kingdom²

Received 3 September 2004/ Returned for modification 2 October 2004/ Accepted 7 December 2004

Tumor necrosis factor alpha (TNF-) receptor-associated factors (TRAFs) play important roles in TNF- signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF- also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47^phox. Here, we investigated the potential interaction between p47^phox phosphorylation and TRAF4 binding and their relative roles in acute TNF- signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF- (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47^phox phosphorylation. This was paralleled by a 2.7- ± 0.5-fold increase in p47^phox-TRAF4 association, membrane translocation of p47^phox-TRAF4, a 2.3- ± 0.4-fold increase in p47^phox-p22^phox complex formation, and a 3.2- ± 0.2-fold increase in NADPH-dependent O₂^– production (all P < 0.05). TRAF4-p47^phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38^MAPK activation, which was inhibited by an O₂^– scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47^phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47^phox phosphorylation or binding to p22^phox but inhibited TNF--induced ERK1/2 activation. In coronary microvascular EC from p47^phox–/– mice, TNF--induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47^phox phosphorylation and TRAF4 are required for acute TNF- signaling. The increased binding between p47^phox and TRAF4 that occurs after p47^phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.

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