Lipid Peroxidation Is Involved in the Activation of NF-κB by Tumor Necrosis Factor but Not Interleukin-1 in the Human Endothelial Cell Line ECV304

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-κB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-κB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-κB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-κB and stimulation of IκBα 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 toN-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-κB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-κB activation and IκBα 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-κB 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-κB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-κB activation by tumor necrosis factor but not interleukin-1.Taken together, the results argue against a role for H2O2 in NF-κB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-κB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation. It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-κB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-κB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-κB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-κB and stimulation of IκBα 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 toN-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-κB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-κB activation and IκBα 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-κB 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-κB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-κB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-κB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-κB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation. The inducible, higher eukaryotic transcription factor NF-κB has an important role in the regulation of a number of genes involved in immune and inflammatory responses. It is activated in many cell types by a wide range of stimuli including the proinflammatory cytokines interleukin-1 (IL-1) 1The abbreviations used are: IL-1, interleukin-1; BHA, butylated hydroxyanisole; t-BHP, tert-butyl hydroperoxide; DDTC, diethyldithiocarbamate; DFO, desferrioxamine; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; MDA, malondialdehyde; NAC, N-acetyl-l-cysteine; PDTC, pyrrolidine dithiocarbamate; PGA, pyroglutamic acid; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; TNF, tumor necrosis factor; FBS, fetal bovine serum; PBS, phosphate-buffered saline. and tumor necrosis factor (TNF) and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (reviewed in Ref. 1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar). In endothelial cells (ECs), activation of NF-κB is central to the regulation of many genes by IL-1 and TNF such as the cell adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (2Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1572) Google Scholar) and tissue factor (3Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Recently, NF-κB has been identified in an activated form in the ECs of atherosclerotic plaques (4Brand K. Page S. Rogler G. Bartsch A. Brandl R. Knuechel R. Page M. Kaltschmidt C. Baeuerle P.A. Neumeier D. J. Clin. Invest. 1996; 97: 1715-1722Crossref PubMed Scopus (714) Google Scholar), and it has been suggested that NF-κB may play a central role in the initiation of atherosclerosis (5Collins T. Lab. Invest. 1993; 68: 499-508PubMed Google Scholar). NF-κB exists in a latent form in the cytoplasm of unstimulated cells comprising a transcriptionally active dimer bound to an inhibitor protein, IκB. The currently known subunit members of the NF-κB family in mammals are p50, RelA (p65), c-Rel, p52, and RelB, while multiple forms of IκB also exist, namely IκBα, β, γ, and Bcl-3 (reviewed in Ref. 6Liou H.C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (517) Google Scholar). The predominant form of NF-κB activated in cells is a p50/RelA heterodimer, which is associated with IκBα in resting cells. Upon stimulation with agents such as IL-1 and TNF, IκBα is rapidly phosphorylated on two serine residues (Ser32 and Ser36), which targets the inhibitor protein for ubiquitination and subsequent degradation by the 26 S proteasome (reviewed in Ref. 7Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). This allows NF-κB to translocate to the nucleus and activate target genes by binding with high affinity to κB elements in their promoters. The phosphorylation and degradation of IκBα are tightly coupled events (7Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). Thus, it is likely that agents that activate NF-κB do so through the activation of a specific IκBα kinase or, alternatively, by inactivating a particular phosphatase. A high molecular mass kinase complex that phosphorylates IκBα on Ser32 and Ser36 has been identified (8Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). The upstream events that lead to phosphorylation of IκBα are unclear. A model has been proposed whereby diverse agents all activate NF-κB by causing oxidative stress (an increase in intracellular reactive oxygen intermediates (ROS)) (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar). In particular, H2O2 has been implicated as a common second messenger in the various pathways leading to NF-κB activation (10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar). This hypothesis is based on several of lines of evidence. First, in some cell types H2O2 has been shown to be released in response to agents that also activate NF-κB (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 11Los M. Schenk H. Hexel K. Baeuerle P.A. Droge W. Schulze-Osthoff K. EMBO J. 1995; 14: 3731-3740Crossref PubMed Scopus (297) Google Scholar). Second, direct addition of H2O2 to culture medium has been shown to activate NF-κB in some cell lines (12Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3420) Google Scholar, 13Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1269) Google Scholar). Third, overexpression of the H2O2-metabolizing enzyme catalase in a mouse epidermal cell line was shown to attenuate the activation of NF-κB by TNF and okadaic acid, while overexpression of the H2O2-producing enzyme superoxide dismutase potentiated the activation, suggesting a role for H2O2 in these pathways to NF-κB (10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Other work has suggested that some of these observations are cell-specific. H2O2 had no stimulatory effect on NF-κB in a number of other cell types (14Israel N. Gougerot-Pocidalo M.A. Aillet F. Virelizier J.C. J. Immunol. 1992; 149: 3386-3393PubMed Google Scholar, 15Bradley J.R. Johnson D.R. Pober J.S. Am. J. Pathol. 1993; 142: 1598-1609PubMed Google Scholar, 16Brennan P. O'Neill L.A.J. Biochim. Biophys. Acta. 1995; 1260: 167-175Crossref PubMed Scopus (209) Google Scholar, 17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar), while Suzuki et al.(18Suzuki Y.J. Mizuno M. Packer L. Biochem. Biophys. Res. Commun. 1995; 210: 537-541Crossref PubMed Scopus (42) Google Scholar) showed that in COS-1 cells, overexpression of catalase did not block activation of NF-κB by either TNF or PMA. It has also been suggested that oxidative stress facilitates but does not mediate NF-κB activation (14Israel N. Gougerot-Pocidalo M.A. Aillet F. Virelizier J.C. J. Immunol. 1992; 149: 3386-3393PubMed Google Scholar). Another line of evidence implicating oxidative stress and H2O2 as central to NF-κB activation has been the effect of antioxidants in inhibiting NF-κB activation in response to diverse stimuli. Two compounds in particular have been extensively used, the glutathione precursor and radical scavengerN-acetyl-l-cysteine (NAC) and the putative antioxidant pyrrolidine dithiocarbamate (PDTC). However, the effect of NAC on NF-κB is also somewhat cell-specific, in that although it has proved inhibitory in some cells (12Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3420) Google Scholar, 13Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1269) Google Scholar), we and others have reported NAC-insensitive pathways to NF-κB (16Brennan P. O'Neill L.A.J. Biochim. Biophys. Acta. 1995; 1260: 167-175Crossref PubMed Scopus (209) Google Scholar, 17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar, 19Suzuki Y.I. Mizuno M. Packer L. J. Immunol. 1994; 153: 5008-5015PubMed Google Scholar). PDTC seems to be a better general inhibitor of NF-κB and, in fact, has been proposed as a specific universal inhibitor of NF-κB that acts independently of the activating agent and cell type used (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar). However, in addition to its radical scavenging and metal-chelating properties (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 20Sunderman F.W. Ann. Clin. Lab. Sci. 1991; 21: 70-81PubMed Google Scholar), PDTC can also exert a pro-oxidant effect in some cells by increasing oxidized glutathione levels (21Nobel C.S.I. Kimland M. Lind B. Orrenius S. Slater A.F.G. J. Biol. Chem. 1995; 270: 26202-26208Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 22Brennan P. O'Neill L.A.J. Biochem. J. 1996; 320: 975-981Crossref PubMed Scopus (123) Google Scholar), which also leads to an inhibition of NF-κB (22Brennan P. O'Neill L.A.J. Biochem. J. 1996; 320: 975-981Crossref PubMed Scopus (123) Google Scholar, 23Mihm S. Galter D. Droge W. FASEB J. 1995; 9: 246-252Crossref PubMed Scopus (153) Google Scholar). Information on the role of oxidative stress and H2O2 in cytokine stimulation of NF-κB in ECs remains limited, mainly coming from studies using PDTC or NAC to perturb particular genes downstream of NF-κB activation (24Weber C. Erl W. Pietsch A. Strobel M. Ziegler-Heitbrock H.W.L. Weber P.C. Arterioscler. Thromb. 1994; 14: 1665-1673Crossref PubMed Google Scholar, 25Orthner C. Rodgers G.M. Fitzgerald L.A. Blood. 1995; 86: 436-443Crossref PubMed Google Scholar, 26Marui N. Offermann M.K. Swerlick R. Kunsch C. Rosen C.A. Ahmad M. Alexander R.W. Medford R.M. J. Clin. Invest. 1993; 92: 1866-1874Crossref PubMed Scopus (978) Google Scholar). Given the importance of NF-κB in ECs together with the often cell-specific nature of the effect of H2O2 and antioxidants on NF-κB activation, we decided to investigate the role of H2O2 and oxidative stress in NF-κB activation in ECs using both primary and transformed ECs. Our results show that although H2O2 activates NF-κB in transformed ECs, it is unlikely to have a role in the cytokine-mediated pathways to NF-κB in transformed or primary ECs. In transformed cells, TNF but not IL-1 was sensitive to PDTC, while in primary cells neither stimulus was inhibited. Further, we show that the ability of PDTC to inhibit NF-κB activation by TNF in transformed ECs involves inhibition of iron-catalyzed lipid peroxidation that is not important for activation of NF-κB by IL-1. The immortalized human endothelial cell line ECV304 (27Takahashi K. Sawasaki Y. Hata J.-I. Mukai K. Goto T In Vitro Cell. Dev. Biol. 1990; 25: 265-274Crossref Scopus (418) Google Scholar) and human Jurkat T cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, United Kingdom). Pooled human umbilical vein endothelial cells (HUVECs) were obtained at first passage from Clonetics Corporation (San Diego, CA). RPMI 1640 medium, heat-inactivated fetal bovine serum (FBS), trypsin-EDTA, and penicillin-streptomycin-glutamine were from Life Technologies, Inc. (Paisley, Scotland). Human recombinant IL-1α was a gift from NCI, National Institutes of Health (Frederick, MD), while human recombinant TNFα was a gift from Dr. Steve Foster (Zeneca Pharmaceuticals, Macclesfield, UK). The 22-base pair oligonucleotide, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, containing the NF-κB consensus sequence (underlined), T4 polynucleotide kinase, and the Cytotox 96TM nonradioactive cytotoxicity assay were from Promega Corp. (Madison, WI). The 22-base pair oligonucleotide, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′, containing the mutated NF-κB consensus sequence (underlined), the rabbit polyclonal antibody to human IκBα, and the antisera to the NF-κB subunits p50 and c-Rel were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antiserum to the NF-κB subunit RelA (p65) was a gift from Dr. Jean Imbert (INSERM, Marseille, France). [γ-32P]ATP (3000 Ci/mmol) and enhanced chemiluminescence (ECL) reagent were from Amersham International (Aylesbury, UK). Poly(dI·dC) was from Pharmacia Biosystems (Milton Keynes, UK). All other reagents, including heparin (sodium salt), human recombinant acidic fibroblast growth factor, medium 199 (HEPES modification), PDTC (ammonium salt), diethyldithiocarbamate (DDTC), pyrrolidine, pyroglutamic acid, NAC, desferrioxamine, allopurinol, the spin traps 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), FeCl3, H2O2, PMA, butylated hydroxyanisole (BHA),tert-butylhydroperoxide (t-BHP), thiobarbituric acid, anti-mouse IgG peroxidase conjugate, scopoletin, horseradish peroxidase (type II), and catalase were from Sigma (Poole, UK). ECV304 cells were grown in medium 199 (HEPES modification) containing 10% (v/v) FBS, and passaged when confluent using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA. HUVECs were grown in medium 199 containing 20% FBS, 10 ng/ml acidic fibroblast growth factor, and 90 μg/ml heparin. The medium was changed every 48 h, and cells were passaged when 80–90% confluent using trypsin-EDTA. Jurkat T cells were grown in RPMI 1640 medium containing 10% FBS. All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine, and cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cells were pretreated with the test compounds or left untreated before the addition of IL-1, TNF, PMA, or H2O2 as described in the figure legends. All experiments were carried out in complete medium at 37 °C. PDTC, DDTC, and pyrrolidine were dissolved in PBS; pyroglutamic acid (PGA), desferrioxamine (DFO), and FeCl3were dissolved in H2O; NAC was dissolved in 25 mm Tris-HCl, pH 7.5; BHA was dissolved in ethanol; and PMA, allopurinol, TEMPO, and DMPO were dissolved in Me2SO. NAC and PGA were adjusted to pH 7.4 with 1 n NaOH. None of the vehicles had any effect on NF-κB alone at the concentrations used. No metal spatulas were used. The effect of the compounds on cell viability was assessed using the Cytotox 96TM nonradioactive cytotoxicity assay, as described by the manufacturers. This system uses lactate dehydrogenase release as an index of cell toxicity. At the concentrations used here, NAC interfered with this assay, and thus the magnitude of release of lactate dehydrogenase activity from NAC-treated cells was determined directly by monitoring spectrophotometrically the decrease in absorbance at 340 nm in the presence of 75 mmTris-HCl, pH 7.2, containing 150 mm KCl, 0.2 mmNADH, and 4.8 mm sodium pyruvate. Measurement of LDH release from intact cells and examination of monolayer morphology revealed that none of the compounds used were toxic to the cells at the exposure times and concentrations used here. Nuclear extracts were prepared using a modified version of the method of Osborn et al. (28Osborn L. Kunkel S. Nable G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1370) Google Scholar). Confluent ECV304 cells or HUVECs in six-well plates (3-ml volume) were treated as described in the figure legends. Stimulation was terminated by removal of medium followed by washing twice with 3 ml of ice-cold PBS (0.145m NaCl, 0.027 m phosphate buffer, pH 7.6). Washed cells were then scraped into 1 ml of hypotonic buffer (10 mm Hepes buffer, pH 7.9, containing 1.5 mmMgCl2, 10 mm KCl, 0.5 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride). Stimulation of Jurkat T cells (5 × 106 in 1 ml of medium) was terminated by the removal of cells into 5 ml of ice-cold PBS, followed by centrifugation (170 g, 10 min). Cell pellets were resuspended in 1 ml of hypotonic buffer. Nuclear extracts were subsequently prepared as described previously (17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar). Protein concentrations were determined using the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). Extracts were then stored at −20 °C and assayed for NF-κB activity the next day. Nuclear extracts (2 μg of protein) were incubated with 10,000 cpm of a 22-base pair oligonucleotide containing the NF-κB consensus sequence that had previously been labeled with [γ-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. Incubations were performed for 30 min at room temperature, in the presence of 2 μg of poly(dI·dC) as nonspecific competitor and 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 4% glycerol, and 100 μg/ml nuclease-free bovine serum albumin. For competition studies, unlabeled wild type or mutant NF-κB oligonucleotides were added to the binding reaction 30 min before the addition of the radiolabeled probe. In experiments involving antisera to NF-κB subunits, 0.5 μl of a specific antiserum to p50, RelA, or c-Rel was incubated with nuclear extracts for 20 min on ice prior to the binding reaction. All incubation mixtures were subjected to electrophoresis on native 5% (w/v) polyacrylamide gels, which were subsequently dried and autoradiographed. Confluent ECV304 cells in six-well plates (3-ml volume) were treated as described in the figure legends. Treatment was terminated by washing monolayers twice with ice-cold PBS. Cells were then scraped into 1 ml of ice-cold radioimmune precipitation buffer (1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS in PBS) containing 10 μg/ml phenylmethylsulfonyl fluoride, 7 μg/ml aprotinin, and 1 mm Na3VO4. Following further disruption of cells by passage through a 21-gauge needle (5 strokes), an additional 0.1 mg/ml phenylmethylsulfonyl fluoride was added to samples, which were then incubated on ice for 45 min. Samples were then centrifuged at 14,000 × g for 20 min at 4 °C, and the supernatant was removed as cell lysate. Supernatants were assayed for protein (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). Equal amounts of protein (2–4 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose, and IκBα immunoblot analysis was performed as described previously (30Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). H2O2released from ECV304 cells was measured by the horseradish peroxidase-mediated oxidation of the fluorescent probe scopoletin to its nonfluorescent oxidized form (31Harpe J.D.L. Nathan C.F. J. Immunol. Methods. 1985; 78: 323-336Crossref PubMed Scopus (137) Google Scholar). Since hydrogen peroxide freely diffuses across the cell membrane, measuring extracellular release is an indication of intracellular levels. Confluent monolayers of ECV304 cells in six-well plates (3-ml volume) were washed twice with Hanks' balanced salt solution to remove phenol red and serum. Monolayers were incubated with 3 ml of assay solution containing stimulant (IL-1 (10 ng/ml), TNF (10 ng/ml), or PMA (100 ng/ml)), sodium azide (1 mm), scopoletin (1 μm), and horseradish peroxidase (0.2 units/ml) in Hanks' balanced salt solution, for 30 min at 37 °C. The supernatant was then transferred to a test tube and cooled to room temperature, and the fluorescence was measured on a Perkin-Elmer LS 50B fluorimeter using an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Controls included cell-free plates with full assay mix and cells incubated in the absence of peroxidase. The specific decrease in fluorescence due to H2O2 was assessed by measuring scopoletin oxidation for each sample in the presence and absence of catalase (50 units/ml). H2O2 concentration was then determined against a standard curve, using concentrations of 0.05–0.5 μm H2O2, as determined spectrophotometrically (ε240 = 43.6m−1 cm−1). Protein concentrations of samples were determined (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar) following alkali digestion of monolayers. H2O2 release was then expressed as pmol/min/mg of protein. Lipid peroxidation was assessed by the TBARS assay, which detects mainly malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters. TBARS were measured by a modification of the method of Ohkawa et al. (32Ohkawa H. Ohishi N. Yagi K. Anal. Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23298) Google Scholar). Confluent monolayers of ECV304 cells in 100-mm dishes were treated as described in the figure legends and washed in PBS before undergoing three cycles of freeze-thawing in 200 μl of water. A 20-μl aliquot was subsequently removed for Bradford protein determination (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar), and 800 μl of assay mix (0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, 9.4% (v/v) acetic acid, pH 3.5) was added to the remaining sample. Samples were incubated for 60 min at 95 °C, cooled to room temperature, and centrifuged at 14,000 ×g for 10 min, and the absorbance of the supernatants was read at 532 nm against a standard curve prepared using the MDA standard (10 mm 1,1,3,3-tetramethoxypropane in 20 mmTris-HCl, pH 7.4). Results were calculated as nmol of MDA equivalents/mg of protein and expressed as a percentage of matched control values. Significance was evaluated by Student's t test. Fig.1 A shows that nuclear extracts from untreated ECV304 cells contained trace amounts of NF-κB, the levels of which varied slightly between experiments (lanes 1, 9, 17, and 25). Following treatment with 10 ng/ml IL-1 (lanes 2–8) or 10 ng/ml TNF (lanes 10–16), NF-κB was activated, as evidenced by the increased retardation of the DNA probe containing the κB motif. This activation was evident from 5 min (lanes 2 and 10) and peaked at 30 min (lanes 4 and 12). NF-κB activity was strong for at least 4 h (lanes 6 and 14) and still detectable at 24 h (lanes 8 and 16). Treatment of cells with 100 ng/ml PMA (lanes 18–24) gave a somewhat different time course of activation in that active NF-κB only became detectable at 30 min (lane 20) and was greatly reduced by 24 h (lane 24). Prolonged treatment of ECV304 cells with H2O2 led to activation of NF-κB. When cells were treated with 0.2 mm H2O2 for 4 h, a strong activation was observed (compare lanes 28 and 25). In contrast to the rapid response seen for IL-1 and TNF, the H2O2-stimulated activation was not detectable after 1 h (compare lanes 26 and 25) and did not peak until 4 h (lane 29). H2O2-mediated activation was also more transient than that seen for IL-1 and TNF, with activity greatly decreased by 8 h (compare lanes 29 and 28) and identical to control levels at 24 h (compare lanes 30 and 25). A concentration of 0.2 mmH2O2 was optimal in this effect. Fig.1 B confirms that the protein-DNA complexes activated by IL-1, TNF, PMA, and H2O2 were all specific for NF-κB, since 18 or 180 fmol of unlabeled NF-κB wild type consensus sequence effectively competed with each binding activity, while the same concentrations of a mutant NF-κB oligonucleotide containing a single base pair change in the consensus sequence failed to compete with binding. Since this is the first study reporting NF-κB activation in ECV304 cells, we also characterized the NF-κB subunits present in the complexes activated by the four stimuli. Fig. 1 Cdemonstrates that IL-1, TNF, PMA, and H2O2activated similar NF-κB complexes. Using specific antisera to p50, RelA, and c-Rel, the same pattern of supershifting was seen for IL-1-, TNF-, PMA-, and H2O2-activated NF-κB (lanes 1–4, 5–8, 9–12, and 13–16, respectively). This revealed the presence of two main NF-κB complexes. Antiserum to p50 affected both the lower and upper complex (lanes 2, 6, 10, and 14), while RelA antiserum only reacted with the upper complex (lanes 3, 7, 11, and 15). There was no detectable reaction with c-Rel antiserum (lanes 4, 8, 12, and 16). Hence, it was likely that IL-1, TNF, PMA, and H2O2 were activating two NF-κB complexes, tentatively identified as p50/p50 homodimers and p50/RelA heterodimers. Fig. 1 D shows that IL-1, TNF, and PMA also activated NF-κB in HUVECs as has been well characterized by others (2Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1572) Google Scholar). IL-1 (10 ng/ml) gave a similar time course of activation of NF-κB to that seen in ECV304 cells, with activity detectable after 5 min (compare lanes 2 and 1), maximal at 1 h (lane 5) and still apparent at 24 h (lane 8). Activation of NF-κB by TNF (10 ng/ml) and PMA (100 ng/ml) at a single time point (1 h) is also shown (compare lanes 10 and 11, respectively, with lane 9). In contrast to ECV304 cells, H2O2 failed to activate NF-κB in HUVECs. No activation was apparent upon a 2- or 4-h incubation of HUVECs with either 0.2 or 0.4 mm H2O2 (comparelanes 12–15 with lane 9). Higher doses of H2O2 also failed to activate NF-κB (not shown). These results highlight an important dif