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Molecular and Cellular Biology, March 2005, p. 2320-2330, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2320-2330.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Acute Tumor Necrosis Factor Alpha Signaling via NADPH Oxidase in Microvascular Endothelial Cells: Role of p47phox Phosphorylation and Binding to TRAF4

Jian-Mei Li,1 Lampson M. Fan,1 Michael R. Christie,2 and Ajay M. Shah1*

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-{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-{alpha} 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 p47phox. Here, we investigated the potential interaction between p47phox 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; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- ± 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- ± 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- ± 0.2-fold increase in NADPH-dependent O2– production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK activation, which was inhibited by an O2– scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-{alpha}-induced ERK1/2 activation. In coronary microvascular EC from p47phox–/– 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 p47phox phosphorylation and TRAF4 are required for acute TNF-{alpha} signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.

* Corresponding author. Mailing address: Department of Cardiology, GKT School of Medicine, Bessemer Rd., London SE5 9PJ, United Kingdom. Phone: 44-020-7346 3865. Fax: 44-020-7346 4771. E-mail: ajay.shah{at}kcl.ac.uk.

Molecular and Cellular Biology, March 2005, p. 2320-2330, Vol. 25, No. 6
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.6.2320-2330.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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