title: Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4.
abstract: 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.
authors.affiliation: Department of Cardiology, Guy's King's and St. Thomas' School of Medicine, King's College London, London, United Kingdom.
authors.forename: Jian-Mei
authors.initials: JM
authors.lastname: Li
authors.forename: Lampson M
authors.initials: LM
authors.lastname: Fan
authors.forename: Michael R
authors.initials: MR
authors.lastname: Christie
authors.forename: Ajay M
authors.initials: AM
authors.lastname: Shah
mesh_terms: 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*
chemical_list: 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
references.citation: Circ Res. 2000 Mar 17;86(5):494-501
references.pmid: 10720409
references.citation: Am J Physiol. 1998 Sep;275(3 Pt 1):L533-44
references.pmid: 9728048
references.citation: Circ Res. 2000 Aug 4;87(3):179-83
references.pmid: 10926866
references.citation: Arterioscler Thromb Vasc Biol. 2000 Aug;20(8):1903-11
references.pmid: 10938010
references.citation: Biochimie. 2000 Aug;82(8):727-32
references.pmid: 11018289
references.citation: Br J Pharmacol. 2001 Jan;132(1):270-6
references.pmid: 11156586
references.citation: Cell Signal. 2001 Jun;13(6):389-400
references.pmid: 11384837
references.citation: J Mol Cell Cardiol. 2001 Jun;33(6):1119-31
references.pmid: 11444917
references.citation: J Biol Chem. 2001 Nov 16;276(46):42728-36
references.pmid: 11559697
references.citation: Cardiovasc Res. 2001 Dec;52(3):477-86
references.pmid: 11738065
references.citation: Exp Cell Res. 2002 Jan 1;272(1):62-74
references.pmid: 11740866
references.citation: Circ Res. 2002 Feb 8;90(2):143-50
references.pmid: 11834706
references.citation: J Biol Chem. 2002 Feb 15;277(7):5468-75
references.pmid: 11733538
references.citation: Circ Res. 2002 May 17;90(9):1012-9
references.pmid: 12016268
references.citation: J Biol Chem. 2002 May 31;277(22):19952-60
references.pmid: 11893732
references.citation: J Biol Chem. 2002 Aug 2;277(31):28051-7
references.pmid: 12023963
references.citation: Eur J Biochem. 2002 Oct;269(19):4819-29
references.pmid: 12354113
references.citation: Ann N Y Acad Sci. 2002 Nov;973:505-7
references.pmid: 12485919
references.citation: J Biol Chem. 2003 Apr 4;278(14):12094-100
references.pmid: 12560337
references.citation: J Pharmacol Exp Ther. 2003 May;305(2):573-80
references.pmid: 12606638
references.citation: Cell. 2003 May 2;113(3):343-55
references.pmid: 12732142
references.citation: Circ Res. 2003 May 30;92(10):1089-97
references.pmid: 12714560
references.citation: Cytokine Growth Factor Rev. 2003 Jun-Aug;14(3-4):193-209
references.pmid: 12787559
references.citation: Leuk Res. 2003 Aug;27(8):687-94
references.pmid: 12801526
references.citation: Trends Immunol. 2003 Jun;24(6):327-34
references.pmid: 12810109
references.citation: Nat Rev Immunol. 2003 Sep;3(9):745-56
references.pmid: 12949498
references.citation: Clin Exp Pharmacol Physiol. 2003 Nov;30(11):860-6
references.pmid: 14678251
references.citation: Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195-9
references.pmid: 2550933
references.citation: J Invest Dermatol. 1992 Dec;99(6):683-90
references.pmid: 1361507
references.citation: J Clin Invest. 1993 Oct;92(4):1866-74
references.pmid: 7691889
references.citation: Annu Rev Physiol. 1995;57:827-72
references.pmid: 7778885
references.citation: Circ Res. 2000 Jul 7;87(1):26-32
references.pmid: 10884368