Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells:: Role of p47phox phosphorylation and binding to TRAF4

Tumor necrosis factor alpha (TNF-alpha) receptor-associated factors (TRAFs) play important roles in TNF-alpha signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-a 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-alpha signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-alpha (100 U/ml; I to 60 min) induced rapid (within 5 min) p47(phox) phosphorylation. This was paralleled by a 2.7- +/- 0.5-fold increase in p47phox-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 O-2- production (all P < 0.05). TRAF4-p47(phox) binding was accompanied by a progressive increase in extracellular signal-regulated kinases I and 2 (ERK1/2) and p38(MAPK) activation, which was inhibited by an O-2- 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-alpha-induced ERK1/2 activation. In coronary microvascular EC from p47(phox-/-) mice, TNF-alpha-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-alpha 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-I expression in EC.