Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway.
Expression of endothelial nitric oxide synthase (eNOS) in transfected U-937 cells upregulates phorbol 12-myristate 13-acetate (PMA)-induced tumor necrosis factor-alpha (TNF-alpha) production through a superoxide (O(2)(-))-dependent mechanism. Because mitogen-activated protein kinases (MAPK) have been shown to participate in both reactive oxygen species signaling and TNF-alpha regulation, their possible role in eNOS-derived O(2)(-) signal transduction was examined. A redox-cycling agent, phenazine methosulfate, was found to both upregulate TNF-alpha (5.8 +/- 1.0 fold; P = 0.01) and increase the phosphorylation state of p42/44 MAPK (3.1 +/- 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. Although S-nitroso-N-acetylpenicillamine, a nitric oxide (NO) donor, also increased TNF-alpha production, NO exposure led to phosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-alpha production by eNOS transfection was associated with increases in activated p42/44 MAPK (P = 0.001), whereas levels of phosphorylated p38 MAPK were unaffected. Furthermore, cotransfection with Cu/Zn superoxide dismutase, which blocks TNF-alpha upregulation by eNOS, also abolished the effects on p42/44 MAPK. Expression of Gln(361)eNOS, a mutant that produces O(2)(-) but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, two NADPH binding site deletion mutants of eNOS that lack oxidase activity had no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathway inhibitor, blocked TNF-alpha upregulation by eNOS (P = 0.02). Thus O(2)(-) produced by eNOS increases TNF-alpha production via a mechanism that involves p42/44 MAPK activation.
Wang
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Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
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Nishanian
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Del Pilar Cintron
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Wesley
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Danner
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D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D017353:Gene Deletion; D006801:Humans; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D016897:Respiratory Burst / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D008773:Methylphenazonium Methosulfate; D009249:NADP; D009240:N-Formylmethionine Leucyl-Phenylalanine; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases