NO Inhibits Cytokine-Induced iNOS Expression and NF-κB Activation by Phosphorylation IκB-α

NO Inhibits Cytokine-Induced iNOS Expression and NF-␬B Activation by Interfering With Phosphorylation andDegradation of I␬B-␣Koichi Katsuyama,Masayoshi Shichiri,Fumiaki Marumo,Yukio HirataAbstract—Nitric oxide(NO)is known to have antiatherogenic and anti-inflammatory properties,but its effects on the cytokine-induced nuclear factor-kappa B(NF-␬B)activation pathway in relation to the regulation of inducible nitric oxide synthase(iNOS)gene in vascular smooth muscle cells(VSMCs)remain elusive.To elucidate the roles of NO in the regulation of cytokine-induced NF-␬B activation and consequent iNOS gene expression,we studied the effects of NO donors[(Ϯ)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide(NOR3)and sodium nitroprusside]on inter-leukin(IL)-1␤–induced NF-␬B activation and I␬B-␣degradation and subsequent iNOS expression in rat VSMCs.Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1␤–induced iNOS mRNA and protein expression.Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the down-stream NF-␬B site of rat iNOS promoter as a probe showed that NOR3inhibited IL-1␤–induced NF-␬B activation and its nuclear translocation,as demonstrated with immunocytochemical study.These effects were independent of guanylate cyclase activation;an inhibitor of soluble guanylate cyclase(1H-oxadiazolo-1,2,4-[4,3-␣]quinoxaline-1-one)had no effect on NOR3-induced inhibition of NF-␬B activation or iNOS mRNA expression by IL-1␤,and a cGMP derivative (8-bromo-cGMP)failed to mimic the effects of NO donors.Western blot analysis using anti–I␬B-␣and anti–phospho-I␬B-␣antibodies revealed that IL-1␤induced a transient degradation of I␬B-␣preceded by a rapid appearance of phosphorylated I␬B-␣,both of which were completely blocked by NOR3.A proteasome inhibitor(MG115)blocked IL-1␤–induced transient degradation of I␬B-␣and stabilized the appearance of phosphorylated I␬B-␣stimulated by IL-1␤.NOR3inhibited the appearance of IL-1␤–induced phosphorylated I␬B-␣even in the presence of MG115.Our results indicate that an inhibitory action by NO on cytokine-induced NF-␬B activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of I␬B-␣via the cGMP-independent pathway in rat VSMCs.(Arterioscler Thromb Vasc Biol.1998;18:1796-1802.)Key Words:NF-␬BⅢIL-1␤Ⅲinducible nitric oxide synthaseⅢI␬B-␣N itric oxide(NO)is synthesized from L-arginine by NO synthases(NOSs).Three distinct isozymes of NOS have been identified to date:2Ca2ϩ/calmodulin-dependent consti-tutive isozymes dominantly expressed in the brain and endo-thelium,and a Ca2ϩ-independent,cytokine-inducible isozyme (iNOS).1iNOS produces large amounts of NO in response to bacterial lipopolysaccharides(LPS)and certain cytokines in a variety of cells,including vascular smooth muscle cells (VSMCs).2NO possesses diverse physiological properties, such as vasodilation,neurotransmission,and mediation of immune responses.1High-output NO produced by iNOS in VSMCs not only causes inhibition of cell proliferation but apoptosis of VSMCs as well.3,4Therefore,regulation of iNOS gene expression has been implicated in the pathogenesis of vascular remodeling and atherosclerosis.5Many of the bio-logical effects of NO have been attributed to cGMP genera-tion via the stimulation of soluble guanylate cyclase,although a cGMP-independent mechanism is also involved in itsdiverse actions.The promoter region of the rat and mouse iNOS genecontains several potential cis-elements for the binding ofdifferent transcription factors,among which2putative bind-ing sites for nuclear factor-kappa B(NF-␬B)exist in the upstream(GGGGATTTTCC,nucleotidesϪ965toϪ955:NF-␬Bu)and downstream(GGGGACTCTCC,nucleotides Ϫ107toϪ97:NF-␬Bd)regions.6–8The sequence of NF-␬Bd is unique in that it is found only in murine and human iNOSgenes.It has been shown that a key region of the promoteractivity in mediation of LPS inducibility resides in theNF-␬Bd region in mouse macrophages.6,7However,its role in mediation of iNOS expression in response to cytokines in VSMCs remains largely unknown.NF-␬B complexes function as a pleiotropic regulator of many genes modulating immunologic and inflammatory pro-Received December11,1997;revision accepted June21,1998.From the Endocrine-Hypertension Division,Second Department of Internal Medicine,Tokyo Medical and Dental University,Tokyo,Japan. Correspondence to Yukio Hirata,MD,Endocrine-Hypertension Division,Second Department of Internal Medicine,Tokyo Medical and Dental University,1-5-45,Yushima,Bunkyo-ku,Tokyo113-8519,Japan.©1998American Heart Association,Inc.Arterioscler Thromb Vasc Biol.is available at cesses.NF-␬B contains heterodimeric complexes,usually consisting of p50and p65(Rel-A)subunits and p50/p50 homodimers in human VSMCs.9p50/p65heterodimer asso-ciates with its cytoplasmic inhibitor,I␬B-␣,to form an inactive cytoplasmic ternary complex.p65subunit may also complex with p105,a precursor protein of p50,as an inactive form.Activation of NF-␬B by LPS or cytokines requires either degradation of I␬B-␣10or proteolytic cleavage of p105 through a common ubiquitin-proteasome pathway after phos-phorylation.11After degradation of I␬B-␣,an active NF-␬B translocates to the nucleus and activates the expression of a plethora of genes.Recently,a cytokine-responsive I␬B-␣kinase(IKK)that activates NF-␬B by phosphorylation of Ser32and Ser36residue in I␬B-␣has been identified.12–16It has been reported that NO inhibits NF-␬B by induction and stabilization of I␬B-␣in human endothelial cells.17However, it remains unknown whether NO inhibits cytokine-induced NF-␬B activation via inhibition of phosphorylation and deg-radation of I␬B-␣,thereby blocking iNOS gene expression in VSMCs.These observations led us to examine(1)whether interleu-kin(IL)-1␤–induced iNOS gene expression is mediated by the NF-␬B activation pathway involving phosphorylation and subsequent degradation of I␬B-␣in cultured rat VSMCs and (2)whether NO inhibits IL-1␤–induced iNOS gene expres-sion by interfering with phosphorylation and subsequent degradation of I␬B-␣.MethodsMaterialsHuman recombinant IL-1␤was kindly provided by Otsuka Pharma-ceutical.Murine recombinant tumor necrosis factor(TNF)-␣was purchased from Gibco BRL;(Ϯ)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide(NOR3),SDS,and EDTA were purchased from Wako Pure Chemical;8-bromo-cGMP,N-acetyl-L-cysteine,1H-oxadiazolo-1,2,4-[4,3,-␣]quinoxaline-1-one(ODQ),PMSF,DTT, poly(dI-dC),and EGTA were purchased from Sigma Chemical; pyrrolidinedithiocarbamate was purchased from Katayama Chemi-cal;sodium nitroprusside(SNP)was purchased from Calbiochem Novabiochem;MG115was purchased from Peptide Institute; [␣-32P]dCTP was purchased from Amersham International;and dNTP and a Klenow fragment of DNA polymerase I were purchased from Takara Shuzo.Cell CultureVSMCs from the thoracic aorta of15-week-old male Wistar rats were prepared by the explant method and cultured in Dulbecco’s modified Eagle’s medium containing10%FBS at37°C in a humidified atmosphere of95%air and5%CO2as described previously.18Subcultured VSMCs(15th to20th passages)from2 independent isolates were used in the experiments. Electrophoretic Mobility Shift Assay(EMSA) Confluent VSMCs(5ϫ106cells per dish)pretreated with or without NO donors(NOR3,SNP)for60minutes were stimulated with IL-1␤for2hours,washed with ice-cold PBS,and harvested in0.4mL ice-cold hypotonic lysis buffer(10mmol/L HEPES,pH7.8; 10mmol/L KCl;2mmol/L MgCl2;1mmol/L DTT;0.1mmol/L EDTA;0.1mmol/L PMSF;and5␮g/mL leupeptin).After15 minutes of incubation,25␮L10%Nonidet P-40was added.The nuclei pellets were collected,resuspended in30␮L hypertonic extraction buffer(50mmol/L HEPES,pH7.8;50mmol/L KCl; 300mmol/L NaCl;0.1mmol/L EDTA;1mmol/L DTT;10% glycerol;and0.1mmol/L PMSF),and centrifuged,and the superna-tant was subjected to EMSA.The single-stranded oligonucleotides (forward:5Ј-TGGGGACTCTCC-3Ј,complement:5Ј-AAGGGAG-AGTCC-3Ј)corresponding to the NF-␬B binding sequence of the downstream region(Ϫ107toϪ97)of rat iNOS gene promoter8were annealed at65°C for15minutes and filled with[␣-32P]dCTP(111 TBq/mmol),dNTP,and a Klenow fragment of DNA polymerase I. Nuclear proteins(10␮g)were incubated with20000cpm32P-labeled NF-␬B double-stranded oligonucleotide and1␮g poly(dI-dC)in EMSA buffer(10mmol/L Tris-HCl,pH7.5;2%glycerol;0.2mmol/L EDTA;0.5mmol/L DTT;50mmol/L NaCl)for30 minutes and subjected to polyacrylamide gel electrophoresis.To examine the specificity of the NF-␬B binding protein,the gel shift assay was performed in parallel with the same samples in the presence of a100-fold excess unlabeled oligonucleotide as a com-petitor.For gel supershift assay,nuclear protein was preincubated for 30minutes with goat polyclonal antibodies against human NF-␬B p50or p65subunit(Santa Cruz Biotechnology).Northern Blot AnalysisConfluent VSMCs(5ϫ106cells per dish)pretreated with or without NO donors(NOR3,SNP)for60minutes were stimulated with IL-1␤for6hours,and total RNAs were extracted by the acid guanidinium thiocyanate-phenol-chloroform methods.19Total RNAs(20␮g), separated by formaldehyde/1.1%agarose gel electrophoresis,were transferred to a Magna Graph nylon membrane(Micron Separations Inc).The cDNA probe for rat iNOS recently cloned from rat endothelial cells20was labeled with[␣-32P]dCTP(111TBq/mmol)by random-primed labeling method.RNA immobilized on the mem-brane was hybridized with the labeled probes,washed in0.1ϫSSPE/ 0.5%SDS,and autoradiographed.Western Blot AnalysisWestern blot analyses were performed essentially as described.21 Confluent cells(5ϫ106cells per dish),pretreated with or without NO donors(NOR3,SNP)or MG115for60minutes,were stimulated with IL-1␤for the indicated times for I␬B-␣and phospho-I␬B-␣,or for15hours for iNOS.Cells were lysed in50mmol/L Tris-HCl,pH 6.8(10%glycerol,1%SDS,1␮g/mL pepstatin,2␮g/mL leupeptin, 2␮g/mL aprotinin,and1mmol/L PMSF).Whole-cell lysates were boiled,and extracted proteins were separated on12%(for I␬B-␣and phospho-I␬B-␣)or7.5%(for iNOS)SDS-polyacrylamide gel and transferred to Hybond ECL nitrocellulose membranes(Amersham), which were incubated overnight with rabbit polyclonal antibody for human I␬B-␣(1:500;Santa Cruz Biotechnology),rabbit polyclonal antibody for human phospho-I␬B-␣(Ser32)(1:1000;New England Biolabs),or mouse monoclonal antibody for murine iNOS(1:1000; Transduction Laboratories)at4°C.Anti–phospho-I␬B-␣antibody detects I␬B-␣only when phosphorylated at Ser32and has no cross-reactivities with the corresponding phosphorylated Ser of I␬B-␤or I␬B-⑀.After extensive washing,the secondary antibody (donkey anti-rabbit IgG or sheep anti-mouse IgG horseradish perox-idase;1:500,Amersham)was applied for1hour,and exposure was performed by using an ECL kit(Amersham). Immunocytochemical StainingSubconfluent cells grown on LAB-TEK Chamber Slide(Nalge Nunc Int)were treated with IL-1␤in the absence or presence of NOR3for 2hours,fixed with70%acetone for20minutes at room temperature, and then washed with PBS for10minutes.Goat polyclonal antibody specific for NF-␬B p50subunit(Santa Cruz Biotechnology)was used;the antibody did not show any cross-reactivities with p105, p52,or p100.Immunostaining was visualized with the indirect immunoperoxidase avidin-biotin-peroxidase kit(Vector Laboratory).ResultsNF-␬B Activation by IL-1␤To determine whether NF-␬B activation was induced with IL-1␤in rat VSMCs,EMSA was performed using synthetic oligonucleotides corresponding to the NF-␬Bd site(Ϫ107to Katsuyama et al November19981797Ϫ97)of the rat iNOS promoter as a probe (Figure 1).IL-1␤(10ng/mL)caused a distinct shifted band,whereas there was no distinct band in the control cells.The specificity of the NF-␬B binding gel shift assay was examined using coincubation with excess unlabeled probe as a competitor.The band was com-pletely eliminated in the presence of a 100-fold molar excess of unlabeled oligomers (Figure 1A).For characterization of NF-␬B subunits,specific antibodies for human p50and p65subunits were examined.The IL-1␤–induced NF-␬B protein-DNA com-plexes were supershifted by anti-p50antibody,whereas anti-p65antibody caused a supershifted band in addition to the reduced shifted band (Figure 1B).NO Inhibits IL-1␤–Induced NF-␬B Activation and iNOS mRNA and Protein ExpressionWe studied whether NO donors affect NF-␬B activation and iNOS expression induced by IL-1␤in rat VSMCs (Figure 2).Northern blot analysis using rat iNOS cDNA as a probe revealed that IL-1␤(10ng/mL)induced iNOS mRNA ex-pression (4.5kb),the effects of which were dose-dependently (10Ϫ5to 10Ϫ3mol/L)suppressed by NOR3(Figure 2A;top panel).EMSA showed that NOR3similarly blocked the IL-1␤–induced NF-␬B activation in a dose-dependent man-ner (10Ϫ5to 10Ϫ3mol/L)(Figure 2A;bottom panel).NOR3(10Ϫ3mol/L)completely blocked both NF-␬B activation and iNOS mRNA expression stimulated by IL-1␤.SNP (10Ϫ3mol/L)also attenuated the IL-1␤–induced iNOS mRNA expression and NF-␬B activation (Figure 2B).To determine whether the inhibitory effect of NO donors is mediated via a cGMP-dependent mechanism,the effects of a selective inhibitor of soluble guanylate cyclase (ODQ)and a cell-permeable cGMP analogue (8-bromo-cGMP)were tested (Figure 3).ODQ (10Ϫ5mol/L)did not affect the inhibitory effect of NOR3(10Ϫ4mol/L)on IL-1␤–induced iNOS mRNA expression or NF-␬B activation (Figure 3A).8-Bromo-cGMP (10Ϫ3mol/L)did not inhibit the IL-1␤–induced iNOS mRNA expression or NF-␬B activation (Figure 3B).Western blot analysis using specific anti-murine iNOS antibody demon-strated a distinct band of 130-kDa iNOS protein after stimu-lation with IL-1␤(10ng/mL),which was completely abol-ished in the presence of NOR3(10Ϫ4mol/L)(Figure 4).These data indicate that NO blocks IL-1␤–induced NF-␬B activa-tion as well as iNOS mRNA and protein expression via a cGMP-independent mechanism.NO Prevents IL-1␤–Induced NF-␬B Nuclear TranslocationTo confirm that NO donor prevents nuclear translocation of active NF-␬B in rat VSMCs after cytokine stimulation,immunohistochemical staining using anti-p50antibody was performed.Nonstimulated cells revealed a diffuse but faint distribution of immunoreactive p50within the cytoplasm (Figure 5A).In contrast,exposure of rat VSMCs to IL-1␤(10Figure 1.NF-␬B activation by IL-1␤in rat VSMCs.Confluent cells were incubated with or without IL-1␤(10ng/mL)for 2hours.Nuclear proteins (10␮g)were extracted and subjected to EMSA using synthetic NF-␬B oligomer as a probe (A)without (Ϫ)or with (ϩ)100-fold excess unlabeled probe as a competi-tor,and (B)without (Ϫ)or with pretreatment of antibody against human NF-␬B p50or p65subunit.Figure 2.Effect of NO donors on IL-1␤–induced iNOS mRNA expression and NF-␬B activation in rat VSMCs.Confluent cells pretreated with or without (A)NOR3(10Ϫ5to 10Ϫ3mol/L)or (B)SNP (10Ϫ3mol/L)were stimulated with IL-1␤(10ng/mL)for 6hours for Northern blot analysis of iNOS mRNA (top panel),28S ribosomal RNA (middle panel),and 2hours for EMSA of NF-␬B activity (bottom panel),respectively.Figure 3.Effects of NOR3,ODQ,and 8-bromo-cGMP on IL-1␤–induced iNOS mRNA expression and NF-␬B activation in rat VSMCs.Confluent cells pretreated without (Ϫ)or with (ϩ)(A)NOR3(10Ϫ4mol/L)and ODQ (10Ϫ5mol/L)or (B)8-bromo-cGMP (10Ϫ3mol/L)were stimulated with IL-1␤(10ng/mL)for 6hours for Northern blot analysis of iNOS mRNA (top panel),28S ribo-somal RNA (middle panel),and 2hours for EMSA of NF-␬B activity (bottom panel),respectively.1798NO Inhibits IL-1␤–Induced I ␬B-␣Phosphorylationng/mL)resulted in dense accumulations of immunoreactive p50within the nucleus (Figure 5B),the effect of which was prevented by pretreatment with NOR3(10Ϫ3mol/L)(Figure 5D);NOR3added alone was without effect (Figure 5C).NO Prevents IL-1␤–Induced I ␬B-␣DegradationTo determine whether IL-1␤causes I ␬B-␣degradation in rat VSMCs,Western blot analysis using anti–I ␬B-␣antibody was performed.Addition of IL-1␤(10ng/mL)resulted in a rapid (within 15to 30minutes)decrease in I ␬B-␣protein,which then returned to baseline levels within 1to 2hours (Figure 6A).Pretreatment with NOR3(10Ϫ3mol/L)com-pletely prevented the IL-1␤–induced transient decrease in I ␬B-␣levels (Figure 6B);NOR3added alone was without effect.A proteasome inhibitor,MG115(10Ϫ5mol/L),also prevented the transient decrease in I ␬B-␣levels induced by IL-1␤(Figure 6B).These data suggest that both NO andMG115interfere with the transient degradation of I ␬B-␣induced by IL-1␤in rat VSMCs.NO,but Not Proteasome Inhibitor,Prevents IL-1␤–Induced I ␬B-␣PhosphorylationTo determine whether IL-1␤causes I ␬B-␣phosphorylation in rat VSMCs,Western blot analysis using anti–phospho-Ser 32of I ␬B-␣antibody was performed.Addition of IL-1␤(10ng/mL)resulted in a rapid (within 3to 5minutes)appearance of phosphorylated I ␬B-␣,which peaked at 5minutes and then decreased by 30minutes (Figure 7A;top panel).Pretreatment with MG115(10Ϫ5mol/L)stabilizedtheFigure 4.Effect of NO donor on IL-1␤–induced iNOS protein expression in rat VSMCs.Confluent cells pretreated without (Ϫ)or with (ϩ)NOR3(10Ϫ4mol/L)were stimulated with IL-1␤(10ng/mL)for 15hours and subjected to Western blot analysis using murine iNOS antibody.An arrow denotes 130-kDa iNOSprotein.Figure 5.Effect of NO donor on IL-1␤–induced nuclear translo-cation of NF-␬B by immunohistochemical staining.Cells pre-treated with or without NOR3(10Ϫ3mol/L)were stimulated with IL-1␤(10ng/mL)for 2hours,fixed with 70%acetone,and stained with anti-human p50subunit antibody.A,Control;B,IL-1␤;C,NOR3;D,IL-1␤ϩNOR3.Figure 6.Effects of NO and proteasome inhibitor on degrada-tion of I ␬B-␣by IL-1␤in rat VSMCs.A,Confluent cells were incubated with IL-1␤(10ng/mL)for the indicated times.B,Cells were pretreated with (ϩ)or without (Ϫ)NOR3(10Ϫ3mol/L)and/or MG115(10Ϫ5mol/L)for 60minutes and stimulated with or without IL-1␤(10ng/mL)for 15minutes.The extracted cell lysates were subjected to Western blot analysis using anti–I ␬B-␣antibody.Figure 7.Effects of NO donor and proteasome inhibitor on phosphorylation of I ␬B-␣by IL-1␤in rat VSMCs.A,Confluent cells were incubated with IL-1␤(10ng/mL)pretreated without (top panel)or with (bottom panel)MG115(10Ϫ5mol/L)for the indicated times.B,Cells were pretreated with (ϩ)or without (Ϫ)NOR3(10Ϫ3mol/L)and/or MG115(10Ϫ5mol/L)for 60minutes and stimulated with or without IL-1␤(10ng/mL)for 5minutes.The cell lysates were subjected to Western blot analysis using anti–phospho-I ␬B-␣antibody.Katsuyama et al November 19981799IL-1␤–induced phosphorylated I␬B-␣during a30-minute incubation period(Figure7A;bottom panel).NOR3(10Ϫ3 mol/L)prevented the IL-1␤-stimulated transient increase in phosphorylated I␬B-␣with or without pretreatment with MG115(Figure7B);NOR3or MG115added alone was without effect.These data suggest that NO inhibits rapid phosphorylation of Ser32of I␬B-␣induced by IL-1␤before its degradation.DiscussionThe present study clearly demonstrates that IL-1␤–induced iNOS mRNA and protein expression is associated with NF-␬B activation in rat VSMCs.By EMSA using a synthetic oligomer corresponding to the unique downstream NF-␬B site of rat iNOS promoter as a probe,we have shown that the IL-1␤specifically activated NF-␬B composed of p50/p65 heterodimer that is immunologically similar,if not identical, to its human counterpart.These results are in agreement with a recent report demonstrating that NF-␬B complexes were mainly composed of p50/p65heterodimers in human cultured VSMCs.9Transcriptional activity of p50/p65heterodimer of NF-␬B can be regulated by at least2pathways.11The p50/p65 heterodimer constitutes an inactive cytoplasmic ternary com-plex with the inhibitor protein,I␬B-␣,which masks the nuclear localization sequences of p50/p65heterodimer. I␬B-␣can be rapidly phosphorylated and degraded after stimulation with LPS or cytokines,allowing transcriptionally active p50/p65heterodimers to translocate to the nucleus to activate a set of genes related to inflammation and prolifer-ation.An alternative pathway to regulate p65(Rel-A)is derived from its association with the unprocessed p105,the C-terminal portion of which bears a striking resemblance to I␬B-␣to form an inactive p105/p65cytoplasmic complex. Processing of p105results in a rapid degradation of the I␬B-␣homologous sequence and formation of transcriptionally active p50/p65heterodimer.In both pathways,phosphoryla-tion of I␬B-␣and p105by a protein kinase(s)is essential for the subsequent degradation of I␬B-␣and the processing of p105,respectively.Resynthesis of I␬B-␣after its rapid degradation depends on the induction of I␬B-␣expression after NF-␬B activation.22Cellular responses to cytokine signaling,such as activation of several protein kinases,including ceramide-activated pro-tein kinase,p42/p44mitogen-activated protein kinase,p38 mitogen-activated protein kinase,and Jun N-terminal kinase, have been suggested to exert their diverse biological conse-quences.23However,little information is yet available as to how immediate signals by cytokines cause NF-␬B activation in VSMCs.The present experiments using cultured rat VSMCs clearly demonstrated that stimulation with IL-1␤caused a rapid phosphorylation of Ser32of I␬B-␣and subse-quent transient degradation of I␬B-␣,followed by NF-␬B activation and its nuclear translocation.A cytokine-responsive I␬B-␣kinase recently identified phosphorylates two serine residues(Ser32and Ser36)of I␬B-␣.24Phosphory-lation of these residues is a prerequisite for polyubiquitination and subsequent degradation of I␬B-␣by20S proteasome.25 TNF-␣rapidly activates I␬B-␣kinase,peaking at5to10minutes in HeLa cells.24In the present study,IL-1␤induced a rapid(3to5minutes)phosphorylation of Ser32residue of I␬B-␣,followed by a transient(15minutes)degradation and subsequent resynthesis of I␬B-␣in rat VSMCs.In the present study,a proteasome inhibitor,MG115,completely prevented the IL-1␤–induced transient degradation of I␬B-␣and also stabilized the phosphorylated I␬B-␣induced by IL-1␤in rat VSMCs.These data suggest that IL-1␤rapidly stimulates I␬B-␣kinase to phosphorylate Ser32and possibly Ser36 residues of I␬B-␣,which in turn is degraded via the ubiquitin/ proteasome pathway,thereby leading to NF-␬B activation in rat VSMCs.In fact,we have shown that the IL-1␤–induced NF-␬B activation and its nuclear translocation in rat VSMCs were completely prevented by pretreatment with MG115 (K.K.et al,unpublished data,1998).Our present study has clearly shown that NO donors (NOR3,SNP)inhibited the IL-1␤–induced NF-␬B activation and its nuclear translocation,accompanied by inhibition of both iNOS mRNA and protein expression.The present study has further shown that NO donor blocked both the transient decrease in I␬B-␣levels as well as its rapid phosphorylation induced by IL-1␤,whereas NO donor added alone had no effect on basal I␬B-␣levels.Furthermore,NO donor abol-ished the appearance of IL-1␤–induced phosphorylated I␬B-␣,even in the presence of a proteasome inhibitor,which stabilized I␬B-␣.These results suggest that the inhibitory effect of NO on the IL-1␤–induced iNOS expression is mediated via inhibition of NF-␬B activation primarily due to its inhibition of I␬B-␣phosphorylation rather than the proteasome-mediated I␬B-␣degradation.Our data are in agreement with those of previous reports showing the inhib-itory effects of NO on TNF-␣–induced NF-␬B activation in endothelial and neuronal cells.26,27However,our results ap-pear to be in contrast to2recent(though contradictory) reports from the same laboratory.17,28Liao and his associates have shown that NO donors inhibited TNF-␣–induced NF-␬B activation by induction and stabilization of I␬B-␣in human endothelial cells;S-nitrosoglutathione(GSNO)prevented I␬B-␣degradation30minutes after stimulation with TNF-␣.17The same group has subsequently reported that GSNO did not prevent phosphorylation and degradation of I␬B-␣15 minutes after stimulation with TNF-␣in the same cells, suggesting that NO inhibits NF-␬B activation by the late(2 hours)induction and nuclear translocation of I␬B-␣.28In the present study,however,NOR3itself had no effect on basal I␬B-␣expression during a75-minute incubation period.The exact reasons for the apparent discrepancy between their reports and ours are unknown.This may be accounted for by the different cell types,species,cytokines,and NO donors used in the experiments.Among these,caution must be paid to the physicochemical nature of NO donors used,because they release NO with different kinetics and generate various metabolites that may affect cell functions.NOR3,(Ϯ)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (FK409),is a novel and potent NO donor that spontaneously releases NO under neutral aqueous conditions with a half-life of46minutes.29Although the possible involvement of the metabolite cannot be excluded,concentration-dependent in-hibition of NF-␬B activation by NOR3,with its greater1800NO Inhibits IL-1␤–Induced I␬B-␣Phosphorylationpotency compared with other NO donors(such as SNP and GSNO)as demonstrated in this study,strongly suggests that the effect of NOR3is due to the biologically active NO molecule.The exact intracellular signaling mechanism by which NO blocks cytokine-induced NF-␬B activation remains unknown. To address the question of whether the effect of NO is mediated by guanylate cyclase activation and subsequent cGMP generation,a cell-permeable cGMP analogue(8-bromo-cGMP)and a soluble guanylate cyclase inhibitor (ODQ)were tested.8-Bromo-cGMP did not inhibit IL-1␤–induced NF-␬B activation or iNOS mRNA expression, whereas ODQ did not affect the NO-induced inhibition of either NF-␬B activation or iNOS mRNA expression.These results argue against an intermediate role of cGMP and the involvement of cGMP-dependent protein kinase in the mech-anism of NO inhibition of both NF-␬B activation and iNOS expression.NO modifies activities of several heme-and nonheme-containing enzymes by direct nitrosylation.For example,NO interacts with the heme moiety of soluble guanylate cyclase to stimulate its enzyme activity,whereas NO inhibits enzyme activity of heme-containing iNOS.30NO also binds to nonheme-containing enzymes,particularly to iron-sulfur clusters(cis-aconitase,mitochondrial complexes I and II, ribonucleotide reductase),to inhibit their enzymatic activi-ties.31–33NO can also modify proteins by nitrosylation of Cys residue to form S-nitrosothiols.34Therefore,it is possible to speculate that the inhibitory effect of NO on cytokine-in-duced NF-␬B activation may be due to its direct nitrosylation of Cys residue(s)of I␬B-␣kinase to decrease its enzymatic activity.It is also possible that NO may inhibit NF-␬B by dephosphorylation of I␬B-␣,because NO has been shown to activate protein phosphatases in monocytes.35In fact,okadaic acid,an inhibitor of protein phosphatase2A,has been shown to activate NF-␬B and induce I␬B-␣phosphorylation.36NO may function as an antioxidant to scavenge pro-oxidants, such as superoxide anion and hydrogen peroxide,which in turn stimulate redox-sensitive protein kinase(s)to activate NF-␬B.37However,antioxidants,such as N-acetyl-L-cysteine and pyrrolidinedithiocarbamate,failed to block IL-1␤–in-duced NF-␬B activation and I␬B-␣degradation in our cell culture(K.K.et al,unpublished data,1998).The NO-induced suppression of cytokine-induced I␬B-␣phosphorylation and degradation in rat VSMCs as demon-strated in the present study,along with the direct inhibition by NO on the enzyme activity of iNOS as recently reported,30 may constitute an auto-inhibitory mechanism to lessen the magnitude of NO-induced deleterious effects by high-output iNOS.Excessive NO production by augmented iNOS expres-sion in the blood vessel,such as in inflammation,atheroscle-rosis,and septic shock,could be terminated not only via inhibition of iNOS enzyme activity,but also via transcrip-tional inhibition of iNOS gene by NO per se.Because NO inhibits cytokine-induced endothelial expression of adhesion molecules(vascular cell adhesion molecule-1,E-selectin, intercellular adhesion molecule-1)and proinflammatory cy-tokines(IL-1␤,IL-8)via inhibition of NF-␬B,38the ability of NO to inhibit NF-␬B to decrease iNOS gene expression in VSMCs may also contribute to the prevention of atherogen-esis and inflammation in the vessel walls.AcknowledgmentsThis work was supported in part by grants-in-aid(Y.H.)from the Ministry of Education,Science and Culture,Japan,and from the Ministry of Health and Welfare,Japan.References1.Moncada S,Palmer R,Higgs E.Nitric oxide:physiology,pathophys-iology,and pharmacology.Pharmacol Rev.1991;43:109–142.2.Farivar RS,Chobanian AV,Brecher P.Salicylate or aspirin inhibits theinduction of the inducible nitric oxide synthase in rat cardiac fibroblasts.Circ Res.1996;78:759–768.3.Cornwell TL,Arnold E,Boerth NJ,Lincoln TM.Inhibition of smoothmuscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP.Am J Physiol.1994;267:C1405–C1413.4.Fukuo K,Hata S,Suhara T,Nakahashi T,Shinto Y,Tsujimoto Y,Morimoto S,Ogihara T.Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle.Hypertension.1996;27:823–826.5.Collins T.Endothelial nuclear factor-␬B and the initiation of the athero-sclerotic b Invest.1993;68:499–508.6.Lowenstein C,Alley E,Raval P,Snowman A,Snyder S,Russell S,Murphy W.Macrophage nitric oxide synthase gene:two upstream regions mediate induction by interferon␥and lipopolysaccharide.Proc Natl Acad Sci U S A.1993;90:9730–9734.7.Xie QW,Whisnant R,Nathan C.Promoter of the mouse gene encodingcalcium-independent nitric oxide synthase confers inducibility by interferon ␥and bacterial lipopolysaccharide.J Exp Med.1993;177:1779–1784.8.Eberhardt W,Kunz D,Hummel R,Pfeilschifter J.Molecular cloning ofthe rat inducible nitric oxide synthase gene promoter.Biochem Biophys Res Commun.1996;223:752–756.9.Bourcier T,Sukhova G,Libby P.The nuclear factor␬-␤signalingpathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis.J Biol Chem.1997;272:15817–15824.10.Beg AA,Finco TS,Nantermet PV,Baldwin AJ.Tumor necrosis factorand interleukin-1lead to phosphorylation and loss of I␬B␣:a mechanism for NF-␬B activation.Mol Cell Biol.1993;13:3301–3310.11.Palombella VJ,Rando OJ,Goldberg AL,Maniatis T.The ubiquitin-proteasome pathway is required for processing the NF-␬B1precursor protein and the activation of NF-␬B.Cell.1994;78:773–785.12.DiDonato JA,Hayakawa M,Rothwarf DM,Zandi E,Karin M.A cyto-kine-responsive I␬B kinase that activates the transcription factor NF-␬B.Nature.1997;388:548–554.13.Mercurio F,Zhu H,Murray BW,Shevchenko A,Bennett BL,Li J,YoungDB,Barbosa M,Mann M.IKK-1and IKK-2:cytokine-activated I␬B kinases essential for NF-␬B activation.Science.1997;278:860–866. 14.Woronicz JD,Gao X,Cao Z,Rothe M,Goeddel DV.I␬B kinase-␤:NF-␬B activation and complex formation with I␬B kinase-␣and NIK.Science.1997;278:866–869.15.Zandi E,Rothwarf DM,Delhase M,Hayakawa M,Karin M.The I␬Bkinase complex(IKK)contains two kinase subunits,IKK␣and IKK␤, necessary for I␬B phosphorylation and NF-␬B activation.Cell.1997;91: 243–252.16.Regnier CH,Song HY,Gao X,Goeddel DV,Cao ZD,Rothe M.Identi-fication and characterization of an I-␬-B kinase.Cell.1997;90:373–383.17.Peng HB,Libby P,Liao JK.Induction and stabilization of I␬B␣by nitricoxide mediates inhibition of NF-␬B.J Biol Chem.1995;270: 14214–14219.18.Hirata Y,Yoshimi H,Takaichi S,Yanagisawa M,Masaki T.Binding andreceptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells.FEBS Lett.1988;239:13–17.19.Chomczynski P,Sacchi N.Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem.1987;162:156–159.20.Iwashina M,Hirata Y,Imai T,Sato K,Marumo F.Molecular cloning ofendothelial,inducible nitric oxide synthase gene from rat aortic endothe-lial cell.Eur J Biochem.1996;237:668–673.21.Hanson KD,Shichiri M,Follansbee MR,Sedivy JM.Effects of c-mycexpression on cell cycle progression.Mol Cell Biol.1994;14:5748–5755.22.Thompson JE,Phillips RJ,Erdjument BH,Tempst P,Ghosh S.I␬B-␤regulates the persistent response in a biphasic activation of NF-␬B.Cell.1995;80:573–582.Katsuyama et al November19981801。