Artigo Acesso aberto Revisado por pares

Suppression of Nuclear Factor-κB Activity by Nitric Oxide and Hyperoxia in Oxygen-resistant Cells

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m202623200

ISSN

1083-351X

Autores

William R. Franek, Yalamanchali C Chowdary, Xinchun Lin, Maowen Hu, Edmund J. Miller, Jeffrey A. Kazzaz, Pasquale Razzano, John Romashko, Jonathan M. Davis, Pramod Narula, Stuart Horowitz, William Scott, Lin L. Mantell,

Tópico(s)

Immune Response and Inflammation

Resumo

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO ± O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-κB activity. Cells lacking functional NF-κB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-κB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-κB activity in cells exposed to NO ± O2, IκBα was degraded, suggesting that pathways regulating the steady-state levels of IκB were not involved. However, exposure to NO/O2caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-κB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-κB activity. Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO ± O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-κB activity. Cells lacking functional NF-κB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-κB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-κB activity in cells exposed to NO ± O2, IκBα was degraded, suggesting that pathways regulating the steady-state levels of IκB were not involved. However, exposure to NO/O2caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-κB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-κB activity. inhaled nitric oxide reactive oxygen species phosphatidylserine fluorescein isothiocyanate propidium iodide phosphate-buffered saline 4′,6-diamidine-2-phenylindole dihydrochloride cytomagalovirus (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate reactive nitrogen species tumor necrosis factor Inhaled nitric oxide (iNO)1 is used clinically as a therapeutic modality to selectively manipulate the pulmonary vasculature for the treatment of persistent pulmonary hypertension of the newborn. In most cases, these patients receive simultaneous oxygen therapy with supraphysiological concentrations of oxygen. However, studies have shown that prolonged exposure to the combination of NO and hyperoxia (NO/O2) causes significantly more lung injury than hyperoxia alone in several animal models, including piglets (1Robbins C.G. Davis J.M. Merritt T.A. Amirkhanian J.D. Sahgal N. Morin F.C. Horowitz S. Am. J. Physiol. 1995; 269: L545-L550PubMed Google Scholar) and rats (2Nader N.D. Knight P.R. Bobela I. Davidson B.A. Johnson K.J. Morin F. Anesthesiology. 1999; 91: 741-749Crossref PubMed Scopus (25) Google Scholar). This increased lung injury is associated with increased apoptosis of lung cells (3Ekekezie I.I. Thibeault D.W. Zwick D.L. Rezaiekhaligh M.H. Mabry S.M. Morgan R.E. Norberg M. Truog W.E. Biol. Neonate. 2000; 77: 37-44Crossref PubMed Scopus (27) Google Scholar). We have previously shown that exposure of cultured pulmonary cells to NO and hyperoxia causes a synergistic cytotoxicity (4Narula P., Xu, J. Kazzaz J.A. Robbins C.G. Davis J.M. Horowitz S. Am. J. Physiol. 1998; 274: L411-L416PubMed Google Scholar), similar to that observed in lungs of exposed animals. In addition, significantly more apoptosis has been found when isolated human neutrophils and primary cultures of normal human lung fibroblasts were exposed to NO/O2 than those exposed to O2alone (5Fortenberry J.D. Owens M.L. Brown M.R. Atkinson D. Brown L.A. Am. J. Respir. Cell Mol. Biol. 1998; 18: 421-428Crossref PubMed Scopus (61) Google Scholar, 6Raghuram N. Fortenberry J.D. Owens M.L. Brown L.A. Biochem. Biophys. Res. Commun. 1999; 262: 685-691Crossref PubMed Scopus (27) Google Scholar). These studies suggest that prolonged exposure to the combination of NO and hyperoxia has direct toxic effects on lung cells and the resulting injury and death of pulmonary cells may lead to impaired pulmonary function and lung injury. NF-κB is a key redox-sensitive transcription factor in inflammatory diseases, where it regulates the inflammatory response by modulating the gene expression of cytokines, chemokines, and adhesion molecules (7Yamamoto Y. Gaynor R.B. J. Clin. Invest. 2001; 107: 135-142Crossref PubMed Scopus (1352) Google Scholar, 8Tak P.P. Firestein G.S. J. Clin. Invest. 2001; 107: 7-11Crossref PubMed Scopus (3287) Google Scholar). In addition, NF-κB has been shown to play a pivotal role in mediating cell proliferation and survival against a variety of cell death stimuli, including oxidative and nitrosative stress (9Mattson M.P. Culmsee C., Yu, Z. Camandola S. J. Neurochem. 2000; 74: 443-456Crossref PubMed Scopus (434) Google Scholar, 10Mattson M.P. Goodman Y. Luo H., Fu, W. Furukawa K. J. Neurosci. Res. 1997; 49: 681-697Crossref PubMed Scopus (523) Google Scholar, 11Ibe W. Bartels W. Lindemann S. Grosser T. Buerke M. Boissel J.P. Meyer J. Darius H. Cell Physiol. Biochem. 2001; 11: 231-240Crossref PubMed Scopus (15) Google Scholar, 12D'Acquisto F. de Cristofaro F. Maiuri M.C. Tajana G. Carnuccio R. Cell Death Differ. 2001; 8: 144-151Crossref PubMed Scopus (27) Google Scholar, 13Nakshatri H. Bhat-Nakshatri P. Martin D.A. Goulet Jr., R.J. Sledge Jr., G.W. Mol. Cell. Biol. 1997; 17: 3629-3639Crossref PubMed Google Scholar, 14Raziuddin A. Court D. Sarkar F.H. Liu Y.L. Kung H. Raziuddin R. J. Biol. Chem. 1997; 272: 15715-15720Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 15Shattuck-Brandt R.L. Richmond A. Cancer Res. 1997; 57: 3032-3039PubMed Google Scholar). NO has also been shown to be closely associated with NF-κB. NF-κB is an essential transcription factor for the production of endogenous NO and acts by mediating the gene expression of iNOS (16Xie Q.W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar, 17Marshall H.E. Stamler J.S. Am. J. Respir. Cell Mol. Biol. 1999; 21: 296-297Crossref PubMed Scopus (32) Google Scholar). Exogenous NO can also affect NF-κB transcriptional activity by directly interacting with or modulating upstream pathways of its activation (18Bogdan C. Trends Cell Biol. 2001; 11: 66-75Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). The role of exogenous NO in modulating NF-κB activity in vitro is cell line-, NO donor-, and dose-dependent (18Bogdan C. Trends Cell Biol. 2001; 11: 66-75Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 19Connelly L. Palacios-Callender M. Ameixa C. Moncada S. Hobbs A.J. J. Immunol. 2001; 166: 3873-3881Crossref PubMed Scopus (289) Google Scholar). In human peripheral blood mononuclear cells, NF-κB is activated by NO (20Deora A.A. Win T. Vanhaesebroeck B. Lander H.M. J. Biol. Chem. 1998; 273: 29923-29928Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), whereas, in human and bovine vascular endothelial cells, exogenous NO inhibits tumor necrosis factor-mediated NF-κB activation (21Matthews J.R. Botting C.H. Panico M. Morris H.R. Hay R.T. Nucleic Acids Res. 1996; 24: 2236-2242Crossref PubMed Scopus (461) Google Scholar, 22Spiecker M. Peng H.B. Liao J.K. J. Biol. Chem. 1997; 272: 30969-30974Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). All normal cells are sensitive to hyperoxia and suffer oxygen toxicity. Reactive oxygen species (ROS), especially superoxide, are thought to have a pivotal role in oxygen toxicity. In the presence of ROS, NO can form highly reactive species, including peroxynitrite, resulting in enhanced cytotoxicity (18Bogdan C. Trends Cell Biol. 2001; 11: 66-75Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). To delineate the role of the pathways induced by hyperoxia in the absence of oxygen toxicity, we utilized HeLa-80 cells, a mutant oxygen-resistant cell line. HeLa-80 cells, derived from HeLa cells, a cervical epithelial cell line, are capable of stable proliferation in 80% O2, a lethal dose to many other cell types (23Joenje H. Gille J.J. Oostra A.B. Van der Valk P. Lab. Invest. 1985; 52: 420-428PubMed Google Scholar, 24Joenje H. Gille J. Horowitz S., Li, Z. Whyzmuzis C. Massaro D. Oxygen Regulation of Genes and Metabolism. Marcel Dekker, New York1997: 67-73Google Scholar). In this study, we examined the cytotoxicity resulting from the exposure to NO ± O2 in HeLa-80 cells, determined the mode of cell death, and examined the role of NF-κB regulation in the cytotoxicity of NO/O2. HeLa-80 cells and human lung adenocarcinoma A549 cells were grown and maintained at 37 °C as described previously (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). NIH 3T3 wild type and RelA−/− cells (26Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 95% room air and 5% CO2. Cells were exposed to 0.5–7.5 mm DETA NONOate, a NO donor from Cayman Chemical, Ann Arbor, MI, in the presence or absence of 80% O2 for up to 6 days as previously described (4Narula P., Xu, J. Kazzaz J.A. Robbins C.G. Davis J.M. Horowitz S. Am. J. Physiol. 1998; 274: L411-L416PubMed Google Scholar, 25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Media and gases were refreshed daily. Cell death/viability was determined by trypan blue exclusion or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (27Carmichael J. de Graff W.G. Gazdar A.F. Minna J.D. Mitchell J.B. Cancer Res. 1987; 47: 943-946PubMed Google Scholar). All experiments were performed independently and at least twice. The data were expressed as mean ± S.E. and analyzed for statistical significance using the unpaired Student's ttest and analysis of variance with p < 0.05 considered significant. The Annexin V incorporation assay, which detects the flipping of phosphatidylserine (PS) from the cytosolic surface to the extracellular surface, was performed as previously described (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, cells were trypsinized and combined with cells detached during the exposure. After centrifugation, cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the vendor's instructions (R&D Systems, Minneapolis, MN). Ten thousand cells from each sample were analyzed on a BD Biosciences flow cytometer. Both Annexin V binding and cell shrinkage were used to characterize apoptosis (Cell Quest software, BD Biosciences, Franklin Lakes, NJ). Relative fluorescence units (FITC) higher than 210 or cell sizes smaller than 502 relative units (the Forward Scatter) were considered positive for apoptosis, whereas cells with relative fluorescence units (PI) higher than 250 were deemed necrotic. To assess caspase activation, cells were trypsinized and centrifuged at 200 × g for 5 min. The cell pellet was resuspended in cold cell lysis buffer at 25 μl per 1 × 106 cells according to the vendor's instructions (R&D Systems, Minneapolis, MN). At least 5 × 106cells were collected for the caspase-3-like activity assay for each sample. Equal volumes of cell lysate and 2× reaction buffer were mixed with DEVD-pNA for 2 h at 37 °C. Colorimetric reactions were analyzed at 405 nm. Results were normalized to total protein content of each sample. After cell lysates were collected, protein concentrations were determined, and Western blot analyses were performed as previously described (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, 5 μg of protein of each sample were loaded onto 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) for electrophoresis. Polyclonal antibodies directed against IκBα, NF-κB p65 subunit, actin (Santa Cruz Biotechnology, Santa Cruz, CA), and nitrotyrosine (Cayman Chemical) were used for Western blot analysis. Modification of NF-κB p65 subunit was assessed by determining the presence of carbonyl groups using a standard kit (Oxy-blot, Intergen Co., Purchase, NY) following derivatization with 2,4-dinitrophenylhydrazine in the presence of trifluoroacetic acid according to the vendor's protocol. Five micrograms of each derivatized protein sample were loaded onto 10% SDS-polyacrylamide gels, and blots were probed with the primary and secondary antibodies supplied in the kit and developed as Western blots. To determine whether some protein carbonyl bands contain NF-κB p65 subunit, blots were stripped and reprobed with a 1:1000 dilution of anti-p65 polyclonal antibody. To further confirm protein carbonyl modification of NF-κB p65 subunit, total p65 protein from cell lysates was immunoprecipitated by agarose conjugated with anti-p65 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) according to the vendor's protocol. Protein carbonyl formation was then determined as described above in the resulting immunopurified p65. Cells grown on chamber slides were washed twice with PBS and fixed for 10 min in 10% buffered formalin as previously described (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, slides were rinsed with PBS and incubated in a 1% bovine serum albumin in PBS solution (Panvera Corp., Madison, WI) for 30 min. Cells were then incubated with anti-p65 NF-κB polyclonal IgG (Santa Cruz Biotechnology) for 1 h. Subsequently, the slides were washed and incubated for 1 h with anti-rabbit IgG-rhodamine antibody (Roche Molecular Biochemicals, Indianapolis, IN). The cells were then stained with 5 μg/ml 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI, Roche Molecular Biochemicals, Indianapolis, IN) for 10 min. Nuclear extracts from HeLa-80, and A549 cells were prepared as described previously (28Dignam J.R. Lebovitz M. Roeder R. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) from at least 4 × 107 cells per sample. To prepare mitochondrial extracts, cells were washed with PBS, trypsinized, and pelleted at 1500 ×g. Cells were then washed and homogenized in cold fractionation buffer (0.25 m sucrose, 10 mmTris-HCl, pH 7.4, 0.1 mm EDTA, 2 mm sodium citrate, 1 mm sodium succinate) using a Dounce homogenizer. Disruption of the plasma membrane was monitored by trypan blue staining. Lysates were centrifuged twice for 5 min at 2,000 ×g to pellet nuclei and other debris. The supernatant was then centrifuged at 10,000 × g for 10 min. The remaining pellet was resuspended in a buffer containing 50 mm HEPES, pH 7.0, 500 mm NaCl, and 1% Nonidet P-40, supplemented with a mixture of protease inhibitors, and collected as the mitochondrial extracts. To determine the levels of NF-κB transactivation activity, a reporter gene expression assay was performed as described (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, HeLa-80 cells were transiently cotransfected with pCMV-SPORT-β-galactosidase, pBlue, and a reporter plasmid containing 5 NF-κB binding sites inserted upstream of the luciferase reporter (Stratagene, La Jolla, CA). Cells were exposed to NO and 80% O2 24 h post-transfection. NF-κB activity was determined by luciferase activity (luminescence) with a Lumat LB9501 luminometer and normalized to β-galactosidase expression. 3T3 RelA−/− cells were transiently transfected with a plasmid containing the gene encoding NF-κB p65 subunit. Transient transfections were performed using the LipofectAMINE reagent kit (Invitrogen, Carlsbad, CA) according to the vendor's protocol. Double-stranded NF-κB oligonucleotide (Promega, Madison, WI) was end-labeled with [γ-32P]ATP (PerkinElmer Life Sciences, Boston, MA) using T4 polynucleotide kinase in a Invitrogen labeling buffer at 37 °C for 60 min. Binding reactions were performed by mixing 100,000 cpm of the above 32P-labeled probe with 5 μg of nuclear proteins, 1 μg of poly(dI·dC), 1 μg of poly-l-lysine, 20 mm HEPES, pH 7.6, 1 mm EDTA, 10 mm NaCl, 1 mm dithiothreitol, 0.2% Tween 20 (w/v), 30 mm KCl in a volume of 20 μl. Reactions were incubated for 30 min at room temperature before electrophoretic analysis on 6% non-denaturing polyacrylamide gels in 0.5× TBE buffer (0.045 m Tris borate, 1 mm EDTA). Supershift analyses were performed by incubating nuclear proteins with polyclonal antibodies against NF-κB p65 (Santa Cruz Biotechnology) prior to adding 32P-labeled probe. The DNA-protein complexes were visualized by autoradiography at −80 °C for 12–24 h. To determine if the synergistic cytotoxicity induced by the exposure to NO/O2 is primarily due to oxygen toxicity, HeLa-80 cells were exposed to 80% O2 and 0.5 mm DETA NONOate either separately or in combination. These cells were viable and proliferated for 6 days when cultured either in room air or 80% O2 (Fig.1 A). However, a decrease of cell viability was observed in cells exposed to NO/O2 after 5 days, whereas cells exposed to NO alone were alive after 6 days (Fig.1 A). To determine whether there is an increase in cell death in cells exposed to NO/O2 than to NO, we determined the cytotoxicity of DETA NONOate in HeLa-80 cells. Exposure to up to 2.5 mm DETA NONOate for 24 h caused little or no significant cell death, whereas 5–7.5 mm DETA NONOate induced marked cell death (data not shown). We therefore used 5 mm DETA NONOate in the subsequent experiments. After 24-h exposure to 5 mm NO, the amount of cell death was increased to 24 ± 3.6%, whereas 81 ± 8.1% (p < 0.05) cells exposed to both 80% O2 and 5 mmDETA NONOate were dead (Fig. 1 B). Results derived from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis further validated these results (data not shown). Because HeLa-80 cells tolerate 80% O2, these data demonstrate that synergistic cytotoxicity of NO and hyperoxia is not due to pleiotropic oxygen toxicity. Stress from both ROS and reactive nitrogen species (RNS) have been shown to induce either apoptosis or necrosis in a dose- and cell type-dependent manner (29Bonfoco E. Krainc D. Ankarcrona M. Nicotera P. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7162-7166Crossref PubMed Scopus (1863) Google Scholar,30Davies K. IUBMB Life. 1999; 48: 41-47Crossref PubMed Google Scholar). To determine the mode of cell death in HeLa-80 cells exposed to 5 mm DETA NONOate, either alone or in combination with 80% O2, Annexin V incorporation, cell shrinkage, nuclear condensation, and caspase activation were measured as indicators of apoptosis. Externalization of PS from the cytosolic surface to the extracellular surface is an early event in apoptosis (31Martin S. Reutelingsperger C. McGahon A. Rader J. van Schie R. LaFace D. Green D. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar). Using FITC-conjugated Annexin V to detect PS translocation and PI exclusion to detect membrane permeability, we analyzed the mode of cell death. More than 90% of the cells cultured in room air or hyperoxia were alive based on PI exclusion and Annexin V incorporation analyses (TableI). Exposure to NO, either alone or in combination with hyperoxia, led to increased apoptosis (Table I). At 24 h, NO induced apoptosis in 18 ± 3.1% of the exposed cells, while NO/O2 increased apoptosis to 79 ± 3.8%. After 2 days, 68 ± 0.5% and 95 ± 1.4% of cells exposed to NO alone, or with O2, respectively, were apoptotic. To demonstrate that apoptosis can be distinguished from necrosis, cells were exposed to 0.01% Nonidet P-40 for 0.5 min, and 73% were found to die by necrosis (Table I).Table IFlow cytometry analysis of HeLa-80 cells exposed to NO±O2RAO2NONO/O2NP4024 h16 h24 h48 h16 h24 h48 h%%%%%%%%%Normal95 ± 0.290 ± 0.282 ± 2.374 ± 5.028 ± 1.169 ± 1.412 ± 3.41.6 ± 0.626 ± 8Apoptotic3 ± 0.16 ± 0.114 ± 1.718 ± 3.168 ± 0.525 ± 0.579 ± 3.895 ± 1.411 ± 1.0Necrotic2 ± 0.24 ± 0.24 ± 0.78 ± 1.94 ± 0.96 ± 0.99 ± 0.53.4 ± 0.763 ± 5.9Annexin V incorporation and PI incorporation was measured by flow cytometry to assess the mode of cell death. Relative fluorescence units represents the intensity of Annexin V incorporation or PI incorporation. The percent cells undergoing apoptosis or necrosis was determined as described under “Experimental Procedures.” Cells treated with NP-40 were used as controls for necrosis. Values are means ± S.E. (n = 6). Open table in a new tab Annexin V incorporation and PI incorporation was measured by flow cytometry to assess the mode of cell death. Relative fluorescence units represents the intensity of Annexin V incorporation or PI incorporation. The percent cells undergoing apoptosis or necrosis was determined as described under “Experimental Procedures.” Cells treated with NP-40 were used as controls for necrosis. Values are means ± S.E. (n = 6). To further characterize the mode of cell death, we examined cell size and nuclear morphology. Fig.2 A shows the nuclear morphology of exposed cells visualized with DAPI staining. Condensed chromatin was evident in the nuclei of cells exposed to NO ± O2 (Fig. 2 A). In addition, apoptotic bodies were apparent in cells exposed to NO/O2 for 24 h (Fig.2 A). As expected, cells cultured in room air or in hyperoxia alone showed no signs of nuclear condensation (Fig. 2 A). Cell shrinkage was quantified by flow cytometry analysis as illustrated in Fig. 2 B. Cell shrinkage was apparent in cells exposed to NO/O2 (79% at 24 h, 97% at 48 h) compared with those exposed to NO alone (25% at 24 h, 71% at 48 h). Fig.2 C demonstrates the activation of caspase-3-like caspases using synthetic substrates. Caspase activity was induced in cells exposed to NO alone for 24 h, whereas such an increase in caspase activity was detected much earlier in NO/O2-exposed cells (6 h). To test whether caspase activation plays a causal role in cell death induced by these exposures, cells were pretreated for 60 min with 50 μm Z-DEVD-FMK (Calbiochem), a cell-permeable caspase-3-like inhibitor, prior to the exposure to NO ± O2. 99% (±1.0%) or 90 ± 1.6% of cell death, induced by either exposure to NO alone or to NO/O2, respectively, was prevented or delayed with this pre-treatment, suggesting that caspase activation is necessary for NO ± O2-induced cell death. These data demonstrate that 5 mm DETA NONOate induces significant amounts of apoptosis in HeLa-80 cells, which is significantly enhanced with hyperoxia, even though hyperoxia alone is not toxic to these cells. NF-κB has been shown to play a crucial role in mediating cell survival under nitrosative stress (11Ibe W. Bartels W. Lindemann S. Grosser T. Buerke M. Boissel J.P. Meyer J. Darius H. Cell Physiol. Biochem. 2001; 11: 231-240Crossref PubMed Scopus (15) Google Scholar, 12D'Acquisto F. de Cristofaro F. Maiuri M.C. Tajana G. Carnuccio R. Cell Death Differ. 2001; 8: 144-151Crossref PubMed Scopus (27) Google Scholar). To determine whether NF-κB activity is regulated upon exposure to NO ± O2, we assayed for the transcriptional activity of NF-κB using a luciferase reporter assay as described previously (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Constitutive NF-κB activity was detected in HeLa-80 cells (Fig.3). There was a moderate decrease in NF-κB activity when cells were grown in 80% O2 compared with basal levels in room air (Fig. 3 A). However, NF-κB activity was significantly suppressed in cells exposed to NO. This NO-induced suppression of NF-κB activity was further pronounced by the addition of hyperoxia. Fig. 3 A shows that the synergistic suppression of NF-κB activity was apparent by 6 h (p < 0.05). By 24 h, most of the NF-κB activity was inhibited in cells exposed to NO either alone or in combination with hyperoxia. In addition, NF-κB binding activity in nuclei of HeLa-80 cells was determined by EMSA. As a positive control, A549 cells were treated with TNFα for 30 min to induce NF-κB activation (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A similar NF-κB DNA binding complex was detected in nuclear extracts prepared from cells cultured in room air. Exogenous addition of anti-p65 antibodies supershifted this band, suggesting that p65 is one of the components of this complex. Exposure to NO/O2 for 16 h significantly reduced this binding activity (Fig.3 B). We have previously demonstrated that NF-κB provides a protective role in hydrogen peroxide-induced apoptosis in epithelial cells (25Franek W.R. Horowitz S. Stansberry L. Kazzaz J.A. Koo H.C., Li, Y. Arita Y. Davis J.M. Mantell A.S. Scott W. Mantell L.L. J. Biol. Chem. 2001; 276: 569-575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To investigate any causal effects of NF-κB suppression in NO/O2-induced cell death, we exposed fibroblasts lacking a functional NF-κB p65 subunit, RelA(p65)−/−, to 95% O2 and 0.5 mm DETA NONOate. Similar to the observations in HeLa-80 cells and lung epithelial A549 cells, exposure of fibroblasts to the combination of 0.5 mm DETA NONOate and 95% O2induces a synergistic cytotoxicity (data not shown). Fig.4 shows that mutant cells lacking functional NF-κB were hypersensitive to NO/O2 exposure relative to their wild type counterparts. After 2 days of exposure to NO/O2, 46 ± 2.9% of wild type cells died compared with 96 ± 1.2% of the mutant cells (Fig. 4). This difference in cytotoxicity in response to the exposure to NO/O2 is not due to a difference in their growth rate, because mutant cells proliferate at a similar rate as the wild type fibroblasts (data not shown). To test whether the addition of functional p65 can rescue such injury, RelA−/− cells were transfected with a p65 expression construct. This resulted in the rescue of (36 ± 5% after 1 day and 37 ± 4% after 2 days, p < 0.05) from injury caused by exposure to NO/O2. These data indicate that repression of NF-κB increases susceptibility to the cytotoxicity induced by NO/O2. Inactive NF-κB is sequestered in the cytoplasm by IκB proteins, the inhibitors of NF-κB. Upon activation, IκB is phosphorylated and degraded (32Baldwin A.J. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5579) Google Scholar). NF-κB is then released and translocates to the nucleus, regulating gene expression. It has been shown that NO regulates NF-κB activity by increasing the steady-state levels of IκB by enhancing its mRNA stability or reducing its phosphorylation (21Matthews J.R. Botting C.H. Panico M. Morris H.R. Hay R.T. Nucleic Acids Res. 1996; 24: 2236-2242Crossref PubMed Scopus (461) Google Scholar, 22Spiecker M. Peng H.B. Liao J.K. J. Biol. Chem. 1997; 272: 30969-30974Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). To determine the status of IκB in cells exposed to NO ± O2, we examined the steady-state levels of IκBα by Western blots. As illustrated in Fig.5, IκBα levels were decreased in cells exposed to NO for 24 h. This reduction was more pronounced in cells exposed to NO/O2 for 24 h. Therefore, a lack of degradation of IκB is clearly not responsible for the reduction of NF-κB activity. We then examined if the reduced activity is due to decreased NF-κB expression. While the protein level of NF-κB p65 subunit was maintained upon exposure to NO/O2 for up to 16 h, a decrease was detected in cells exposed to NO/O2 for 24 h (Fig. 5). However, the decrease was only moderate, compared with the 85 ± 2% reduction of NF-κB activity observed after only 6-h exposure (Fig. 3). As a loading control, the steady-state levels of actin were examined. Fig. 5 shows that there was no substantial changes in the levels of actin upon exposure to NO ± O2, suggesting that the effects of NO ± O2 on IκBα and p65 are specific. These data suggest that the reduction of NF-κB activity upon exposure

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