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• W1984870795 abstract "Tumor necrosis factor-α (TNF-α) has been shown to reduce endothelial nitric-oxide synthase (eNOS) gene expression through post-transcriptional regulation of mRNA stability. The current study documented an independent effect of the cytokine on the eNOS gene promoter. TNF-α effected a time- and dose-dependent reduction in activity of a transiently transfected human –1197 eNOS-luciferase reporter. This reduction was inhibited by co-transfection of dominant negative IKKβ as well as a nonphosphorylatable constitutively suppressive mutant of IκB implying involvement of the NFκB cascade in the inhibitory effect. The locus of the TNF-α-dependent inhibition was traced to two Sp1-binding sites positioned between –109 and –95 and –81 and –67 relative to the transcription start site. Electrophoretic mobility shift analysis and immunoperturbation studies showed evidence for Sp1 and Sp3 binding to each element. TNF-α treatment had no effect on the binding pattern to the downstream (–81 to –67) site but did suppress association of Sp1 and Sp3 to the upstream (–109 to –95) site. Collectively, these data indicate that TNF-α exerts transcriptional, as well as post-transcriptional, effects on eNOS gene expression and suggest a potential mechanism to account for the endothelial dysfunction that accompanies disorders such as diabetes mellitus and heart failure. Tumor necrosis factor-α (TNF-α) has been shown to reduce endothelial nitric-oxide synthase (eNOS) gene expression through post-transcriptional regulation of mRNA stability. The current study documented an independent effect of the cytokine on the eNOS gene promoter. TNF-α effected a time- and dose-dependent reduction in activity of a transiently transfected human –1197 eNOS-luciferase reporter. This reduction was inhibited by co-transfection of dominant negative IKKβ as well as a nonphosphorylatable constitutively suppressive mutant of IκB implying involvement of the NFκB cascade in the inhibitory effect. The locus of the TNF-α-dependent inhibition was traced to two Sp1-binding sites positioned between –109 and –95 and –81 and –67 relative to the transcription start site. Electrophoretic mobility shift analysis and immunoperturbation studies showed evidence for Sp1 and Sp3 binding to each element. TNF-α treatment had no effect on the binding pattern to the downstream (–81 to –67) site but did suppress association of Sp1 and Sp3 to the upstream (–109 to –95) site. Collectively, these data indicate that TNF-α exerts transcriptional, as well as post-transcriptional, effects on eNOS gene expression and suggest a potential mechanism to account for the endothelial dysfunction that accompanies disorders such as diabetes mellitus and heart failure. Macrovascular disease is a major cause of morbidity and death in diabetes mellitus. Although a portion of this increased risk can be attributed to the dyslipidemia that occurs in diabetes, it has become clear that in a significant number of patients the presence of insulin resistance per se (a hallmark of type II diabetes) is a major contributor. The mechanism(s) linking insulin resistance to vascular disease remain(s) unknown. Similarly, the factor or factors responsible for the generation of insulin resistance remain(s) only incompletely understood. Tumor necrosis factor-α (TNF-α), 1The abbreviations used are: TNF-α, tumor necrosis factor-α; NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; iNOS, inducible isoform of nitric-oxide synthase; ROS, reactive oxygen species; BAEC, bovine aortic endothelial cells; l-NAME, N-nitro-l-arginine methyl ester hydrochloride; SSRE, shear stress response element; dn, dominant negative; IKK, IκB kinase; TRADD, TNF receptor-1-associated death domain protein; TRAF2, tumor necrosis factor receptor-associated factor 2; RIP, receptor-interacting protein; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-related kinase kinase kinase; NAK, NFκB activating kinase; NIK, NFκB inducing kinase. 1The abbreviations used are: TNF-α, tumor necrosis factor-α; NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; iNOS, inducible isoform of nitric-oxide synthase; ROS, reactive oxygen species; BAEC, bovine aortic endothelial cells; l-NAME, N-nitro-l-arginine methyl ester hydrochloride; SSRE, shear stress response element; dn, dominant negative; IKK, IκB kinase; TRADD, TNF receptor-1-associated death domain protein; TRAF2, tumor necrosis factor receptor-associated factor 2; RIP, receptor-interacting protein; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-related kinase kinase kinase; NAK, NFκB activating kinase; NIK, NFκB inducing kinase. free fatty acids, and a number of peptide hormones produced in the adipocyte (1.Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Crossref PubMed Scopus (691) Google Scholar, 2.Boden G. Exp. Clin. Endocrinol. Diabetes. 2003; 111: 121-124Crossref PubMed Scopus (270) Google Scholar, 3.Mohamed-Ali V. Pinkney J.H. Coppack S.W. Int. J. Obes. Relat. Metab. Disord. 1998; 22: 1145-1158Crossref PubMed Scopus (794) Google Scholar, 4.Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3913) Google Scholar) have each been suggested to play an important role in generating insulin resistance in humans and animal models.Nitric oxide produced in endothelial cells plays an important role as a regulator of vascular function. In addition to being a potent vasodilator (5.Griffith T.M. Edwards D.H. Davies R.L. Harrison T.J. Evans K.T. Nature. 1987; 329: 442-445Crossref PubMed Scopus (255) Google Scholar, 6.Palmer R.M. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9260) Google Scholar, 7.Furchgott R.F. Carvalho M.H. Khan M.T. Matsunaga K. Blood Vessels. 1987; 24: 145-149PubMed Google Scholar), it mediates antiproliferative effects on vascular smooth muscle cells (8.Garg U.C. Hassid A. J. Clin. Investig. 1989; 83: 1774-1777Crossref PubMed Scopus (1987) Google Scholar), inhibits platelet aggregation, and modulates leukocyte adhesion to the endothelium (9.Kubes P. Suzuki M. Granger D.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4651-4655Crossref PubMed Scopus (2805) Google Scholar). Disruption of endothelial function, including interference with NO production, is thought to contribute to the vasomotor dysregulation and the prothrombotic and hyperproliferative states that exist in the vasculature of patients with atherosclerosis, hypertension, and diabetes.Several studies have linked endothelial dysfunction and reduced NO bioactivity to insulin resistance (10.Rizzoni D. Porteri E. Guelfi D. Muiesan M.L. Valentini U. Cimino A. Girelli A. Rodella L. Bianchi R. Sleiman I. Rosei E.A. Circulation. 2001; 103: 1238-1244Crossref PubMed Scopus (239) Google Scholar, 11.Kuboki K. Jiang Z.Y. Takahara N. Ha S.W. Igarashi M. Yamauchi T. Feener E.P. Herbert T.P. Rhodes C.J. King G.L. Circulation. 2000; 101: 676-681Crossref PubMed Scopus (528) Google Scholar). This is thought to reflect a combination of decreased production and increased inactivation of NO, the latter through interaction with reactive oxygen species (ROS) such as superoxide (12.Harrison D.G. J. Clin. Investig. 1997; 100: 2153-2157Crossref PubMed Scopus (895) Google Scholar). There is compelling evidence to indicate that ROS play a major role in diminishing NO bioactivity in diabetics (13.Channon K.M. Guzik T.J. J. Physiol. Pharmacol. 2002; 53: 515-524PubMed Google Scholar). 5,6,7,8-Tetrahydrobiopterin is a required cofactor for eNOS activity. When 5,6,7,8-tetrahydrobiopterin is lacking, as is the case with diabetes mellitus (14.Meininger C.J. Marinos R.S. Hatakeyama K. Martinez-Zaguilan R. Rojas J.D. Kelly K.A. Wu G. Biochem. J. 2000; 349: 353-356Crossref PubMed Scopus (162) Google Scholar, 15.Shinozaki K. Nishio Y. Okamura T. Yoshida Y. Maegawa H. Kojima H. Masada M. Toda N. Kikkawa R. Kashiwagi A. Circ. Res. 2000; 87: 566-573Crossref PubMed Scopus (215) Google Scholar, 16.Hink U. Li H. Mollnau H. Oelze M. Matheis E. Hartmann M. Skatchkov M. Thaiss F. Stahl R.A. Warnholtz A. Meinertz T. Griendling K. Harrison D.G. Forstermann U. Munzel T. Circ. Res. 2001; 88: E14-E22Crossref PubMed Google Scholar), eNOS produces superoxide, a highly reactive free radical that interacts with NO (17.Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr, K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1215) Google Scholar, 18.Xia Y. Tsai A.L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 19.Landmesser U. Dikalov S. Price S.R. McCann L. Fukai T. Holland S.M. Mitch W.E. Harrison D.G. J. Clin. Investig. 2003; 111: 1201-1209Crossref PubMed Scopus (1348) Google Scholar) to form the potent oxidant, peroxynitrate (20.Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar). This results in depletion of bioactive NO and increased production of ROS. In addition, reduced generation of NO (21.Young M.E. Leighton B. Biochem. J. 1998; 329: 73-79Crossref PubMed Scopus (81) Google Scholar), presumably through decreased eNOS expression (11.Kuboki K. Jiang Z.Y. Takahara N. Ha S.W. Igarashi M. Yamauchi T. Feener E.P. Herbert T.P. Rhodes C.J. King G.L. Circulation. 2000; 101: 676-681Crossref PubMed Scopus (528) Google Scholar, 22.Chakravarthy U. Hayes R.G. Stitt A.W. McAuley E. Archer D.B. Diabetes. 1998; 47: 945-952Crossref PubMed Scopus (217) Google Scholar, 23.Ding Y. Vaziri N.D. Coulson R. Kamanna V.S. Roh D.D. Am. J. Physiol. 2000; 279: E11-E17Crossref PubMed Google Scholar, 24.Tickerhoof M.M. Farrell P.A. Korzick D.H. Am. J. Physiol. 2003; 285: H2694-H2703Crossref PubMed Scopus (24) Google Scholar), has also been proposed as a mechanism to account for endothelial dysfunction in diabetes.TNF-α, a cytokine that has been linked mechanistically to insulin resistance (1.Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Crossref PubMed Scopus (691) Google Scholar), is a purported contributor to endothelial dysfunction in type 2 diabetes (25.Pfeiffer A. Janott J. Mohlig M. Ristow M. Rochlitz H. Busch K. Schatz H. Schifferdecker E. Horm. Metab. Res. 1997; 29: 111-114Crossref PubMed Scopus (66) Google Scholar), android obesity (26.Winkler G. Lakatos P. Salamon F. Nagy Z. Speer G. Kovacs M. Harmos G. Dworak O. Cseh K. Diabet. Med. 1999; 16: 207-211Crossref PubMed Scopus (96) Google Scholar), heart failure (27.Fichtlscherer S. Rossig L. Breuer S. Vasa M. Dimmeler S. Zeiher A.M. Circulation. 2001; 104: 3023-3025Crossref PubMed Scopus (126) Google Scholar, 28.Agnoletti L. Curello S. Bachetti T. Malacarne F. Gaia G. Comini L. Volterrani M. Bonetti P. Parrinello G. Cadei M. Grigolato P.G. Ferrari R. Circulation. 1999; 100: 1983-1991Crossref PubMed Scopus (203) Google Scholar), and restenosis following coronary angioplasty (29.Krasinski K. Spyridopoulos I. Kearney M. Losordo D.W. Circulation. 2001; 104: 1754-1756Crossref PubMed Scopus (49) Google Scholar). Indeed, in vivo administration of TNF-α depresses endothelium-dependent relaxation (30.Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1994; 266: H2535-H2541Crossref PubMed Google Scholar) and reduces NO levels in endothelial cells (31.Johnson A. Phelps D.T. Ferro T.J. Am. J. Physiol. 1994; 267: L318-L325PubMed Google Scholar) particularly in response to vasodilators such as bradykinin (32.Zhang D.X. Yi F.X. Zou A.P. Li P.L. Am. J. Physiol. 2002; 283: H1785-H1794Crossref PubMed Scopus (61) Google Scholar). TNF-α has the ability to increase arterial ROS generation likely accounting for some of the reduction in NO levels (33.Corda S. Laplace C. Vicaut E. Duranteau J. Am. J. Respir. Cell Mol. Biol. 2001; 24: 762-768Crossref PubMed Scopus (290) Google Scholar, 34.Muzaffar S. Jeremy J.Y. Angelini G.D. Stuart-Smith K. Shukla N. Thorax. 2003; 58: 598-604Crossref PubMed Scopus (78) Google Scholar). In addition, a number of groups have shown that TNF-α suppresses expression of eNOS, although predominantly through destabilization of eNOS mRNA (35.Michel T. Lamas S. J. Cardiovasc. Pharmacol. 1992; 20: S45-S49Crossref PubMed Scopus (21) Google Scholar, 36.Yoshizumi M. Perrella M.A. Burnett Jr., J.C. Lee M.E. Circ. Res. 1993; 7: 205-209Crossref Scopus (702) Google Scholar, 37.Mohamed F. Monge J.C. Gordon A. Cernacek P. Blais D. Stewart D.J. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 52-57Crossref PubMed Scopus (76) Google Scholar, 38.Alonso J. Sanchez de Miguel L. Monton M. Casado S. Lopez-Farre A. Mol. Cell. Biol. 1997; 17: 5719-5726Crossref PubMed Scopus (129) Google Scholar) without a major effect on transcription of the eNOS gene.In the present study, we investigated the effect of TNF-α on human eNOS gene promoter activity in bovine aortic endothelial cells (BAEC). We have established that at physiological concentrations, TNF-α inhibits eNOS promoter activity. This repressive effect of TNF-α is dependent on activation of the transcription factor NFκB and subsequent reduction in the association of Sp1/Sp3-containing complexes with the eNOS promoter.EXPERIMENTAL PROCEDURESMaterials—Bovine aortic endothelial cells were obtained from Bio-Whittaker, Inc. (Walkersville, MD). Recombinant human TNF-α was from R&D Systems (Minneapolis, MN). [3H]Arginine was from Amersham Biosciences. Monoclonal antibody against eNOS was from BD Biosciences. N-Nitro-l-arginine methyl ester hydrochloride (l-NAME) was from Sigma. Polyclonal antibodies against IκBα, Sp1, Sp3, and the p50 subunit of NFκB were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibody against the p65 subunit of NFκB was from Active Motif (Carlsbad, CA). Oligonucleotides were synthesized by Qiagen Operon (Valencia, CA).Wild-type and Mutant Plasmids—The –1197 human eNOS-luciferase was generated as described by Ye et al. (39.Ye Q. Chen S. Gardner D.G. Hypertension. 2003; 41: 675-681Crossref PubMed Scopus (41) Google Scholar) using the genomic clone provided by Nadaud et al. (40.Nadaud S. Bonnardeaux A. Lathrop M. Soubrier F. Biochem. Biophys. Res. Commun. 1994; 198: 1027-1033Crossref PubMed Scopus (198) Google Scholar). Dominant negative NAK mutant was provided by M. Nakanishi (41.Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (312) Google Scholar). Dominant negative NIK, IKKα, and IKKβ mutants were provided by D. Goeddel (42.Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1062) Google Scholar). Wild-type IκBα and IκBα mutants 2N, 3C, and 2N+3C were provided by Dr. J. Hiscott (McGill University, Montreal, Quebec, Canada).Site-directed Mutagenesis—Site-directed mutagenesis was carried out with the QuikChange kit (Stratagene) according to the manufacturer's protocol. The sequence of each mutagenic primer (sense strand) was as follows (mutagenized bases are identified by lowercase letters): NFκB-like –84/–69, 5′-CGGGGCGAGGGCCAGCACTttAGAGaaaaCTCCCACTGCCCCCTC-3′; shear stress response element (SSRE)-like –643/–637, 5′-GAGTCATGGGGGTGTttGGGggaaAGGAAATTGGGGCTGGGAGGG-3′; Sp1-like –146/–141, 5′-GAGGCTTTAGAGaagaaaAGCCGGGCTTGTTCCTGTCCCATTGTG-3′; Ets –129/–126, 5′-GCTTTAGAGCCTCCCAGCCGGGCTTGggaaTGTCCCATTGTG-3′; SSRE-like –259/–254, 5′-CCCAGCCCCCATGCTGCAGCCCCAttGagaTGCTGGACACCTGGG-3′; SSRE-like –248/–243, 5′-GCAGCCCCAGGGCTCTGCTGtcacaaTGGGCTCCCACTTATCAGC-3′; Myc-associated zinc finger protein-related transcription factor (MAZF)-like –194/–181, 5′-GCGGAACCCAGGCGTCCGGaaaaCacCCCTTCAGGCGAGCGGGCG-3′; Ying Yang (YY1)-like –122/–117, 5′-CAGCCGGGCTTGTTCCTGTaaacggGTGTATGGGATAGGGGCG GG-3′; Sp1-like –81/–67, 5′-CGGGGCGAGGGCCAGCACTaagaAGCCCCCTtttACTGCCCC CTC-3′, Sp1 –109/–95, 5′-CCCATTGTGTATGGtAgAGtGtCtGtGCGAGGGCCAGCACTGGAG-3′; GATA –231/–225, 5′-GGACACCTGGGCTCCCACggcgacGCCTCAGTCCTCACAGCGG-3′.Cell Culture—BAEC were cultured in Dulbecco's modified Eagle's medium (DME-H16) containing 5% fetal bovine serum, 2 mm glutamine, 10 units/ml penicillin, and 100 units/ml streptomycin. Cell viability was assessed using the CellTiter-Glo™ Cell Viability assay (Promega) according to the manufacturer's instructions.Western Blotting—Following treatment with TNF-α, as indicated in the figure legends, cells were harvested in 1 ml of phosphate-buffered saline, centrifuged briefly, lysed under denaturing conditions (50 mm Hepes at pH 7.4, 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, 1 mm EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride), and centrifuged at 12,800 × g for 10 min. 20 μg of supernatant protein was electrophoresed on 7.5% SDS-polyacrylamide gels and transferred electrophoretically onto polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences). Membranes were incubated with monoclonal anti-eNOS antibody (1:2500 dilution) or polyclonal anti-IκBα (1:250 dilution) in Tris-buffered saline/Tween buffer (20 mm Tris-HCl at pH 7.5, 150 mm NaCl, 0.05% Tween 20, and 5% nonfat milk) overnight at 4 °C and then washed with Tris-buffered saline/Tween buffer. Membranes were later incubated with horseradish peroxidase-conjugated goat anti-mouse antibody or donkey anti-rabbit antibody, respectively, for 1 h at room temperature and washed with Tris-buffered saline/Tween. Blots were immersed for 1 min in ECL Detection reagent (Amersham Biosciences) and then exposed to film. Signals were quantified using a Kodak Image Station (440CF).NOS Activity Assay—NOS activity was measured by monitoring the conversion of [3H]arginine to [3H]citrulline (43.Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar). Equal amounts of cell extracts were added to 100 μl of reaction buffer containing 25 mm Tris (pH 7.4), 0.1 μCi of [3H]arginine, 3 μm tetrahydrobiopterin, 1 μm FAD, 1 μm FMN, 100 nm calmodulin, and 1 mm NADPH. After incubation for 20 min at room temperature, reactions were terminated with 4 ml of 50 mm Hepes, pH 5.5, and 5 mm EDTA and applied to Dowex 50-X8-400 1-ml columns (Sigma-Aldrich). [3H]Citrulline was quantified by liquid scintillation of the flow-through.Transfection and Luciferase Assay—BAEC were cultured as described above and then transiently co-transfected with 0.5 μg of –1197 human eNOS-luciferase (wild-type or mutant) and 0.05 μg of actin-β-galactosidase using Lipofectin reagent (Invitrogen) under conditions recommended by the manufacturer. 18–24 h post-transfection, cells were treated as described in the individual figure legends, after which cells were washed twice with phosphate-buffered saline and lysed with lysis buffer (Promega). Luciferase activity was measured using the luciferase assay system (Promega). β-Galactosidase activity was assayed using the Galactolight Plus chemiluminescence assay (Tropix, Bedford, MA). For each well, luciferase levels were normalized for β-galactosidase activity.Preparation of Nuclear Extracts—Endothelial cells were cultured and treated with vehicle or TNF-α as described above. Cells were harvested and lysed with buffer A (10 mm Hepes at pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5% Nonidet P-40, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin) on ice for 10 min. Lysates were centrifuged for 5 min at 4 °C. Particulates were resuspended in buffer C (20 mm Hepes at pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol and the above protease inhibitors) and kept on ice for 30 min. Nuclear suspensions were centrifuged at 12,800 × g for 10 min, and the supernatants (nuclear extracts) were stored at –80 °C until use.Electrophoretic Mobility Shift Assay—Oligonucleotides used for electrophoretic mobility shift assay were as follows (sequence represents sense strand with mutagenized bases in lowercase): Sp1-like –81/–67, wild-type 5′-GCCAGCACTGGAGAGCCCCCTCCCACTG-3′, mutant 5′-GCCAGCACTaagaAGCCCCCTtttACTG-3′; Sp1 –109/–95, wild-type 5′-CATTGTGTATGGGATAGGGGCGGGGCGAGGGCCAG-3′, mutant 5′-CATTGTGTATGGtAgAGtGtCtGtGCGAGGGCCAG-3′. Nuclear extracts (5–10 μg) were incubated in binding reaction buffer (10 mm Hepes at pH 7.9, 50 mm KCl, 0.2 mm EDTA, 2.5 mm dithiothreitol, 10% glycerol, and 0.05% Nonidet P-40) containing 0.5 μg of poly(dI-dC) and 32P-end-labeled double-stranded wild-type oligonucleotide at room temperature for 30 min. For immunoperturbation experiments, nuclear extracts were preincubated on ice for 2 h with 1 μg of polyclonal antibody directed against Sp1, Sp3, p50, or p65. All samples were resolved on 4% nondenaturing polyacrylamide gels. Gels were dried and exposed to x-ray film.Statistical Analysis—Data were analyzed by one-way analysis of variance using Student-Newman-Keuls or Bonferroni Multiple Comparison post hoc tests as applicable.RESULTSTreatment of cultured BAEC with increasing concentrations of TNF-α resulted in a dose-dependent inhibition of eNOS protein levels after 24 h of incubation (Fig. 1A). Maximal reduction in eNOS levels (∼40%) was seen at 1,000 pg/ml. This was accompanied by a reduction in NOS activity, assessed using a conventional enzymatic assay (43.Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar). As shown in Fig. 1B, TNF-α effected a dose-dependent reduction in NOS activity, which was maximal (∼50% inhibition) at 3,000 pg/ml. To assess the potential of TNF-α to reduce transcription of the eNOS gene, we examined the effects of the cytokine on a transiently transfected human eNOS promoter. As shown in Fig. 1C, TNF-α treatment led to a dose-dependent reduction in eNOS promoter-dependent luciferase activity that was statistically significant at 100 pg/ml (∼40% reduction) and maximal (60%) at 1,000 pg/ml cytokine.TNF-α had no significant effect on readily detectable (∼15,000–20,000 arbitrary light units) luciferase activity (control = 100 ± 0% versus TNF-α = 101 ± 12%, p > 0.05) associated with the background vector.The suppression by TNF-α of eNOS expression was time-dependent with reductions in both eNOS protein levels (Fig. 2A) and eNOS promoter activity (Fig. 2B) seen after 8 h of TNF-α exposure. Interestingly, although the effect on protein levels appeared to plateau between 8 and 48 h, the reduction in promoter activity required the full 48 h to achieve maximal inhibition.Fig. 2Time course of TNF-α-dependent reduction of eNOS protein levels and human eNOS promoter activity. A, BAEC were treated with TNF-α (100 pg/ml) for 0–48 h. Cells were harvested, lysed, and assayed for eNOS protein using Western blot analysis; n = 6. B, BAEC were co-transfected with 0.5 μg of –1197 human eNOS-luciferase and 0.05 μg of actin β-galactosidase. 18 –24 h post-transfection, the cells were treated with TNF-α (100 pg/ml) for 0–48 h and then processed and assayed for luciferase activity (n = 4). Data are expressed as means ± S.E. *, p < 0.05; ***, p < 0.001 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TNF-α is known to activate expression of the inducible isoform of NOS (iNOS) in the vasculature, primarily in smooth muscle cells (44.Wen J.K. Han M. Biochemistry (Mosc.). 2000; 65: 1376-1379Crossref PubMed Scopus (7) Google Scholar). The effect of TNF-α on iNOS expression in endothelial cells and, in particular, bovine endothelial cells is reportedly negligible (44.Wen J.K. Han M. Biochemistry (Mosc.). 2000; 65: 1376-1379Crossref PubMed Scopus (7) Google Scholar, 45.Wagner A.H. Schwabe O. Hecker M. Br. J. Pharmacol. 2002; 136: 143-149Crossref PubMed Scopus (84) Google Scholar, 46.Zhang J. Patel J.M. Li Y.D. Block E.R. Res. Commun. Mol. Pathol. Pharmacol. 1997; 96: 71-87PubMed Google Scholar), but because NO has been shown to feed back and suppress eNOS gene expression (47.Vaziri N.D. Wang X.Q. Hypertension. 1999; 34: 1237-1241Crossref PubMed Google Scholar) in these cells, we questioned whether increased NO in our BAEC cultures (through enhanced activity of iNOS or other NOS isoforms) might account for the TNF-α-dependent repression of the eNOS promoter. As shown in Fig. 3, inhibiting the activity of all NOS isoforms using the nonselective NOS inhibitor l-NAME (1 mm) had no effect on the TNF-α-dependent suppression of eNOS promoter activity. This suggests that NO generation does not mediate the inhibitory effect of TNF-α.Fig. 3Effect of NOS inhibition on TNF-α-dependent reduction of eNOS promoter activity. BAEC were co-transfected with 0.5 μg of –1197 human eNOS-luciferase and 0.05 μg of actin β-galactosidase. 18–24 h post-transfection, cells were pre-treated with l-NAME (1 mm for 1 h) and then treated with TNF-α (100 pg/ml) for 48 h. The cells were processed and assayed for luciferase activity. Data are expressed as means ± S.E., n = 3. *, p < 0.05 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TNF-α has been shown to exert at least a portion of its pro-inflammatory effects through activation of the NFκB pathway (48.Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Stimulation of the NFκB pathway involves activation of one or more upstream kinases called IκB kinases or IKKs. The IKKs phosphorylate IκB, an inhibitory protein that complexes with NFκB in the cytoplasm, thereby blocking its entry into the nuclear compartment. This phosphorylation targets IκB for degradation through the ubiquitin-sensitive proteasome pathway (49.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1160) Google Scholar), resulting in increased nuclear localization of NFκB and activation or suppression of NFκB-regulated genes. Treatment of BAEC with TNF-α resulted in a time-dependent reduction in levels of IκBα in these cells between 15 and 30 min (Fig. 4), reflecting increased degradation of the inhibitor and, inferentially, activation of the NFκB signaling pathway. By 60 min, levels of the protein began to increase toward control levels, a finding that presumably results from increased synthesis of IκB (50.Rothwarf, D. M., and Karin, M. (1999) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;1999/5/re1Google Scholar).Fig. 4TNF-α causes degradation of IκBα. BAEC were treated with TNF-α (100 pg/ml) for 0–60 min and then harvested, lysed, and assayed for IκBα using Western blot analysis. Data are expressed as means ± S.E., n = 3–5. **, p < 0.01; ***, p < 0.001 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that NFκB is involved in signaling the TNF-α-dependent inhibition, we transfected a nonphosphorylatable, and therefore constitutively suppressive, mutant of IκB (2N) into BAEC along with the eNOS luciferase reporter and treated them with TNF-α. As shown in Fig. 5A, the 2N mutant completely reversed the TNF-α-dependent inhibition. Co-transfection of wild-type IκB led to partial reversal of the inhibition (Fig. 5B), whereas an IκB mutant (3C) that was altered at phosphorylation sites that are not involved in IκB turnover (51.Beauparlant P. Kwon H. Clarke M. Lin R. Sonenberg N. Wainberg M. Hiscott J. J. Virol. 1996; 70: 5777-5785Crossref PubMed Google Scholar) was devoid of activity. A third mutant that combined mutations at both types of phosphorylation sites (2N + 3C) also reversed the inhibition.Fig. 5Nondegradable IκBα mutants prevent TNF-α-dependent reduction of eNOS promoter activity. BAEC were co-transfected with 0.5 μg of –1197 human eNOS-luciferase, 0.05 μg of actin β-galactosidase, and 0.01 μg of wild-type (WT), nondegradable mutant (panels A and B, 2N or 2N+3C), degradable mutant (panel B, 3C) IκBα or control plasmid (—). 18–24 h post-transfection, the cells were treated with TNF-α (100 pg/ml) for 48 h and then processed and assayed for luciferase activity. Data are expressed as means ± S.E., n = 3. *, p < 0.05; **, p < 0.01 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IKK is a heterotrimeric complex harboring α, β, and γ subunits. Both the α and β subunits harbor kinase catalytic activity (52.DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1896) Google Scholar, 53.Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1839) Google Scholar), whereas the γ subunit appears to subserve a regulatory role in the complex (54.Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (841) Google Scholar, 55.Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). IKK can be activated through a number of different kinase pathways (56.Karin M. Delhase M. Semin. Immunol. 2000; 12" @default.
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• W1984870795 title "Tumor Necrosis Factor-α Inhibits Endothelial Nitric-oxide Synthase Gene Promoter Activity in Bovine Aortic Endothelial Cells" @default.
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