title: Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells.
abstract: It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kappaB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kappaB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kappaB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kappaB and stimulation of IkappaBalpha degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to N-acetyl-L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kappaB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kappaB activation and IkappaBalpha degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6, 6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kappaB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kappaB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kappaB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-kappaB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-kappaB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.
authors.affiliation: Department of Biochemistry, Trinity College, Dublin 2, Ireland.
authors.forename: A G
authors.initials: AG
authors.lastname: Bowie
authors.forename: P N
authors.initials: PN
authors.lastname: Moynagh
authors.forename: L A
authors.initials: LA
authors.lastname: O'Neill
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D002461:Cell Line, Transformed; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology*; D019169:Jurkat Cells; D007700:Kinetics; D015227:Lipid Peroxidation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D017262:Siderophores / Q000494:pharmacology; D013113:Spin Labels; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D003497:Cyclic N-Oxides; D007375:Interleukin-1; D016328:NF-kappa B; D011759:Pyrrolidines; D017262:Siderophores; D013113:Spin Labels; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C020972:pyrrolidine dithiocarbamic acid; D006861:Hydrogen Peroxide; D003676:Deferoxamine; D013755:Tetradecanoylphorbol Acetate; C003959:TEMPO