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-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 p38(MAPK) 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.
Li
Jian-Mei
JM
Department of Cardiology, Guy's King's and St. Thomas' School of Medicine, King's College London, London, United Kingdom.
Fan
Lampson M
LM
Christie
Michael R
MR
Shah
Ajay M
AM
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D008833:Microcirculation / Q000166:cytology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000502:physiology*; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D026082:Singlet Oxygen / Q000378:metabolism; D053449:TNF Receptor-Associated Factor 4; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011506:Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D053449:TNF Receptor-Associated Factor 4; C506367:TRAF4 protein, human; C506368:Traf4 protein, mouse; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D026082:Singlet Oxygen; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
Circ Res. 2000 Mar 17;86(5):494-501
10720409
Am J Physiol. 1998 Sep;275(3 Pt 1):L533-44
9728048
Circ Res. 2000 Aug 4;87(3):179-83
10926866
Arterioscler Thromb Vasc Biol. 2000 Aug;20(8):1903-11
10938010
Biochimie. 2000 Aug;82(8):727-32
11018289
Br J Pharmacol. 2001 Jan;132(1):270-6
11156586
Cell Signal. 2001 Jun;13(6):389-400
11384837
J Mol Cell Cardiol. 2001 Jun;33(6):1119-31
11444917
J Biol Chem. 2001 Nov 16;276(46):42728-36
11559697
Cardiovasc Res. 2001 Dec;52(3):477-86
11738065
Exp Cell Res. 2002 Jan 1;272(1):62-74
11740866
Circ Res. 2002 Feb 8;90(2):143-50
11834706
J Biol Chem. 2002 Feb 15;277(7):5468-75
11733538
Circ Res. 2002 May 17;90(9):1012-9
12016268
J Biol Chem. 2002 May 31;277(22):19952-60
11893732
J Biol Chem. 2002 Aug 2;277(31):28051-7
12023963
Eur J Biochem. 2002 Oct;269(19):4819-29
12354113
Ann N Y Acad Sci. 2002 Nov;973:505-7
12485919
J Biol Chem. 2003 Apr 4;278(14):12094-100
12560337
J Pharmacol Exp Ther. 2003 May;305(2):573-80
12606638
Cell. 2003 May 2;113(3):343-55
12732142
Circ Res. 2003 May 30;92(10):1089-97
12714560
Cytokine Growth Factor Rev. 2003 Jun-Aug;14(3-4):193-209
12787559
Leuk Res. 2003 Aug;27(8):687-94
12801526
Trends Immunol. 2003 Jun;24(6):327-34
12810109
Nat Rev Immunol. 2003 Sep;3(9):745-56
12949498
Clin Exp Pharmacol Physiol. 2003 Nov;30(11):860-6
14678251
Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195-9
2550933
J Invest Dermatol. 1992 Dec;99(6):683-90
1361507
J Clin Invest. 1993 Oct;92(4):1866-74
7691889
Annu Rev Physiol. 1995;57:827-72
7778885
Circ Res. 2000 Jul 7;87(1):26-32
10884368