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Inducible Nitric Oxide Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2
Sangwon F. Kim,1Daniel A. Huri,1Solomon H. Snyder1,2,3*
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase(iNOS) are two major inflammatory mediators. Here we show thatiNOS specifically binds to COX-2 and S-nitrosylates it, enhancingCOX-2 catalytic activity. Selectively disrupting iNOS–COX-2binding prevented NO-mediated activation of COX-2. This synergisticmolecular interaction between two inflammatory systems may informthe development of anti-inflammatory drugs.
1 Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. 2 Department of pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. 3 Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
* To whom correspondence should be addressed. E-mail: ssnyder{at}jhmi.edu
Inflammatory processes are mediated by multiple molecular mechanisms.Two of the most prominent are the production of nitric oxide(NO) by inducible NO synthase (iNOS) and the formation of prostaglandinsby cyclooxygenase-2 (COX-2; prostaglandin H2 synthase) (1, 2).COX-2 inhibitors have attained widespread use as anti-inflammatoryagents, although they elicit potentially adverse side effects(1, 3, 4), whereas iNOS inhibitors are not presently employedtherapeutically. Inflammatory stimuli elicit the synthesis ofiNOS and COX-2 proteins with similar time courses, which suggeststhat the two systems may interact (5, 6). Stimulants of iNOSsuch as bradykinin (7) and lipopolysaccharide (LPS) plus interferon-(IFN-), two components of endotoxin, enhance prostaglandin formation(8). NOS inhibitors prevent the formation of prostaglandins(9).
To determine whether iNOS and COX-2 interact, we used a murinemacrophage cell line (RAW264.7) in which LPS and IFN- massivelyactivate both iNOS and COX-2. INOS immunoprecipitated with COX-2–specificantibodies from lysates of cells treated with LPS–IFN-(Fig. 1A). This was also observed in transfected human embryonickidney cells (HEK293T) overexpressing both proteins (fig. S1A).The two enzymes also coimmunoprecipitated from peritoneal macrophagesobtained from mice injected with thioglycollate, an inflammatorystimulus that induces peritonitis or pleuritis (fig. S1B). Todetermine whether catalytic activity of the enzymes influencestheir interactions, cells that were induced by LPS–IFN-were also treated with the iNOS-selective inhibitor 1400W (Fig. 1B)or the COX-2–selective inhibitor SC58125 (Fig. 1C).Coimmunoprecipitation of iNOS and COX-2 by antibodies specificto either protein was unaffected by either inhibitor. The bindingof iNOS and COX-2 was selective, because COX-1 did not immunoprecipitatewith iNOS. To map the binding sites on both proteins, we generatedselective deletions of iNOS (Fig. 1, D to F) and COX-2 (Fig. 1, G and H)sequences. The amino acid segment 1 to 144 of iNOS,which is within the oxygenase domain, is required, whereas theC terminus of COX-2 mediates binding and includes amino acids484 to 604, which do not exist in COX-1.
Fig. 1. COX-2 and iNOS bind selectively in vitro and in intact cells. (A) RAW264.7 cells were treated with LPS (2 µg/ml) and IFN- (100 U/ml). COX-2 was immunoprecipitated by COX-2–specific antibody and analyzed by Western blot with antibodies against COX-2 and iNOS. Control indicates untreated cells. (B and C) RAW264.7 cells were treated with LPS–IFN- with or without an iNOS inhibitor 1400W (100 µM) or COX-2 inhibitor SC58125 (100 µM). Cell lysates were subjected to immunoprecipitation (IP) and Western blot analysis with antibodies against COX-2 and iNOS. (D) The fragments of iNOS denoted in red bind to full-length COX-2, whereas fragments labeled purple do not, as determined by coimmunoprecipitation of full-length COX-2 by iNOS fragment fused to glutathione S-transferase (GST). The numbers represent the number of the amino acid sequence. (E) Transfected HEK293T cells expressing COX-2 and iNOS fragments expressed as fusion proteins with GST were precipitated with glutathione-conjugated beads. Proteins were detected by Western blot with antibodies against GST or COX-2. (F) Transfected HEK293T cells expressing COX-2 and epitope-tagged (Myc) iNOS fragments were immunoprecipitated with Myc-specific antibody and then analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.) (G) Generated fragments of COX-2 that bind to full-length iNOS are labeled in red; those that do not bind are labeled in yellow. (MBD, membrane-binding domain). (H) Transfected HEK293T cells expressing iNOS and Myc-tagged COX-2 fragments were immunoprecipitated with Myc-specific antibody and analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.)
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The two major mechanisms whereby NO influences its intracellulartargets are stimulation of guanylyl cyclase by direct bindingof NO to iron in heme at the active site of guanylyl cyclase(10) or S-nitrosylation of protein targets on appropriate cysteines(11, 12). Because COX-2 has heme at its active site (13), thiswould be a potential target. However, NO binding to heme inCOX-1 does not alter its activity (14). COX-2 also contains13 cysteines whose roles are not fully understood (15). To explorethe possibility of S-nitrosylation of COX-2 by NO, we examinedmultiple NO donors including nitroso-S-glutathione (GSNO) (Fig. 2A),sodium nitroprusside (SNP), spermine-NO, and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) (fig. S3A). Usingthe biotin switch method in which all the S-nitrosylated cysteinesare selectively biotinylated (16), we observed that all fourNO donors elicited S-nitrosylation of COX-2 in transfected HEK293Tcells expressing COX-2–Myc (Fig. 2A). S-Nitrosylationof COX-2 was also observed in RAW264.7 cells treated with LPS–IFN-.This was prevented when cells were treated with iNOS inhibitor1400W (Fig. 2B and fig. S3B). The biotin switch method was specific,as H2O2 did not elicit S-nitrosylation (fig. S4). We also ruledout the possibility that sulfenic acid modification was detectedby the biotin switch assay by demonstrating that arsenite, whichreverses sulfenic acid modifications but not S-nitrosylation,failed to provide the biotin switch signal afforded by ascorbateusing GSNO with purified COX-2 or LPS–IFN- treatment ofRAW 264.7 cells (fig. S4B). In some instances there may be noneed to deliver NO directly to targets, as some actions of NOare prevented by hemoglobin, which sequesters freely diffusibleNO (17). We examined the effects of hemoglobin on S-nitrosylationof COX-2 under varying conditions. In transfected HEK293T cellsexpressing COX-2, hemoglobin prevented the S-nitrosylation elicitedby GSNO (fig. S5A), whereas it failed to alter S-nitrosylationof COX-2 in RAW264.7 cells activated by LPS–IFN- (fig.S5B). Thus, in the more physiologic macrophage cell line, theS-nitrosylation of COX-2 induced by an inflammatory stimulusdoes not appear to be elicited by freely diffusible NO.
Fig. 2. S-Nitrosylation of COX-2 enhances enzyme activity. (A) COX-2 expressed in transfected HEK293T cells is S-nitrosylated in the presence of GSNO (100 µM) or glutathione (reduced form) (100 µM) as determined by biotin-switch assay. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. COX-2 was selectively S-nitrosylated by GSNO. (B) LPS–IFN- treatment of RAW264.7 cells elicits S-nitrosylation of COX-2, which is prevented by the iNOS inhibitor 1400W (100 µM). COX-2 was selectively S-nitrosylated by endogenously generated NO. (C) COX-2enzyme activity was measured from the cell lysate of transfected HEK293T cells expressing COX-2–Myc in the presence or absence of SNP and ascorbate. Bars represent the mean ± SEM of three independent cell cultures performed in triplicate (*statistically significant by Student's t test). (D) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated by various concentrations of GSNO. The dose-dependence of GSNO-mediated activation of PGE2 was measured. Data were pooled from at least three independent determinations, each in triplicate. (E) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated in the presence of SNP and reversed by the addition of ASC. All the S-nitrosylated proteins were precipitated, and COX-2 was detected by Western blot with COX-2–specific antibody. (F) Recombinant human COX-2 was treated with SNP, and COX-2 activity was measured (n = 3, control: Vmax = 81.3 ± 4.8 nmol/min per mg, Km = 16.2 ± 2.2 µM; SNP: Vmax = 132 ± 6.5 nmol/min per mg, Km = 17.0 ± 2.0 µM). (G) Recombinant human COX-2 was treated with SNP, and its turnover rate (kcat) was measured in the presence of various concentrations of sucrose. Data were expressed as kcat-control over kcat in each viscosity versus viscosity ratio.
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To determine whether S-nitrosylation of COX-2 alters enzymeactivity, we examined transfected HEK293T cells expressing COX-2–Myc.The NO donor SNP, added to cell lysates, elicited a twofoldincrease in COX-2 activity, reflecting S-nitrosylation. Ascorbicacid reversed S-nitrosylation (16, 18) and prevented the increase(Fig. 2, C and D). The reversal by ascorbate of COX-2 activationby NO donors is not merely a reflection of ascorbate influenceson enzyme substrates or intermediate products, as ascorbatefailed to affect COX-2 activity in preparations not treatedwith SNP. Further evidence that S-nitrosylation and COX-2 activationare related is the closely similar concentration-response relationbetween the effects of the NO donor GSNO on S-nitrosylationand on COX-2 activity (Fig. 2E).
NO activates COX-2 by increasing its apparent Vmax without changingits Km (Fig. 2F). The higher concentration of SNP required toactivate COX-2 in vitro compared with intact cells accords withearlier studies showing greater potency of NO donors in intactcells (19). To ascertain the kinetic basis for NO activationof COX-2, we conducted enzyme assays with increasing concentrationsof sucrose to augment viscosity and slow down enzyme kinetics(Fig. 2G). As expected, with increasing viscosity, the ratioof control enzyme activity to the activity in more viscous solutionsincreased. This increase was diminished in SNP samples, consistentwith SNP's accelerating the release of product from the enzyme.
To determine which of the 13 cysteines of COX-2 are criticalfor the augmentation of COX-2 activity elicited by S-nitrosylation,RAW 264.7 cells were transfected to express the N-terminal 483amino acids or the C-terminal 120 amino acids of COX-2. LPS–IFN-treatment induced S-nitrosylation of the C-terminal fragment(which contains three cysteines) but not the N-terminal fragment(fig. S6). To ascertain which of these three cysteines is responsiblefor augmented COX-2 activity, each was mutated to serine. Themutation in which Ser is substituted for Cys526 (C526S) preventedactivation of COX-2 by the NO donor SNP, whereas the C561S mutationdid not (fig. S6). The C555S mutation abolished enzyme activity,so the effects of NO stimulation could not be assessed. Individualmutation of the 13 cysteines in COX-2 did not detectably diminishtotal S-nitrosylation of the enzyme, which suggests that multiplecysteines can be S-nitrosylated, but only C526 is responsiblefor enzyme activation by NO.
To clarify the influence of NO on prostaglandin E2 (PGE2), aprostaglandin synthesized by COX-2 formation in a physiologiccontext, we examined RAW264.7 cells. The formation of PGE2 inresponse to LPS–IFN- was inhibited by the iNOS inhibitor1400W, with 50% reduction of PGE2 formation at drug concentrationsthat provide 50% inhibition of iNOS activity (Fig. 3A). Specificityof the NO association was evident by inhibition of PGE2 formationwith the active L-isomer of the NOS inhibitor N-nitro-L-argininemethyl ester (L-NAME) but not by D-NAME; the effects of L-NAMEwere reversed by added L-arginine (Fig. 3B). Thus, about 50%of induced COX-2 activity is determined by S-nitrosylation.
Fig. 3. Endogenously generated NO enhances COX-2 activity. (A) RAW264.7 cells were activated by LPS–IFN- and treated with various concentrations of iNOS inhibitor 1400W for 18 hours. The dose dependence of 1400W-mediated suppression of PGE2 and nitrite was then measured. Data were pooled from at least three independent determinations, each in triplicate (*statistically significant by Student's t test). (B) Combinations of L-NAME (500 µM), L-NAME + L-Arg (1 mM) or D-Arg (1 mM), and D-NAME (500 µM) were added to RAW264.7 cells treated with LPS–IFN-. PGE2 was measured. The data were pooled from three independent experiments performed, each in triplicate. (C) PGE2 and nitrite were measured from primary peritoneal macrophages isolated from wild-type (WT) or iNOS knockout (KO) mice. Macrophages were treated with LPS–IFN- or untreated (*statistically significant by Student's t test). (D) S-Nitrosylation of COX-2 of WT primary peritoneal macrophages treated with LPS–IFN- is abolished in iNOS KO macrophages. All the S-nitrosylated proteins were precipitated, and COX-2 was detected by Western blot with COX-2–specific antibody.
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As RAW264.7 cells are a continuous macrophage cell line thatmay not behave the same as macrophages in intact organisms,we used peritoneal macrophages from mice lacking iNOS. PGE2formation from macrophages of mice treated with LPS–IFN-was reduced in the iNOS knockout mice by 70%, in parallel witha similar reduction in nitrite formation by the macrophages(Fig. 3C) and a decrease in S-nitrosylated COX-2 (Fig. 3D).These observations concur with findings of decreased urinaryPGE2 in iNOS knockout mice (20).
We hypothesized that the increase in PGE2 formation by iNOSactivation reflects binding of iNOS to COX-2 to deliver NO inappropriate proximity for S-nitrosylation. To explore this possibilitywe blocked iNOS–COX-2 binding with the fragment of COX-2(amino acids 484 to 604), which binds iNOS (Fig. 4A). Expressionof COX-2(484–604) in transfected RAW264.7 cells abolishedthe coprecipitation of iNOS and COX-2. Instead, COX-2(484–604)associated with iNOS (Fig. 4A). Moreover, this interferenceof binding between COX-2 and iNOS by COX-2(484–604) decreasedS-nitrosylation of COX-2 in RAW264.7 cells (Fig. 4B). The dominant-negativeeffect of COX-2(484–604) reduced PGE2 formation by morethan 50%, whereas expression of a COX-2 fragment of amino acids1 to 483, which does not bind iNOS, failed to influence PGE2formation (Fig. 4, C and D).
Fig. 4. COX-2–Myc fragment attenuates iNOS binding to COX-2– and NO-mediated activation of PGE2 production. Transfected RAW264.7 cells expressing COX-2–Myc fragments 1 to 483 or 484 to 604 were treated with LPS–IFN-. (A) Cell lysates were immunoprecipitated with rabbit iNOS-specific antibody and analyzed by Western blot with antibodies against mouse iNOS, goat COX-2, and mouse Myc. (B) COX-2–Myc fragment (484 to 604) decreases S-nitrosylation of COX-2 in RAW264.7 cells. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. (C) Transfected RAW264.7 cells expressing the indicated COX-2 fragments were treated with LPS–IFN-. PGE2 levels were measured and the data were pooled from three independent experiments performed, each in triplicate (*statistically significant by Student's t test). (D) PGE2 and the indicated COX-2 fragments were visualized with confocal microscopy using antibodies against mouse Myc and rabbit PGE2. Images of COX-2 (red) and PGE2 (green) were superimposed to show colocalization. Nuclei were visualized with Hoechst staining (blue). In D1, arrows point to two RAW264.7 cells, only one of which is expressing the COX-2 fragment 484 to 604 (red). In D2, the same two cells are analyzed for presence of endogenous PGE2 after activation of RAW264.7 cells by LPS–IFN- treatment. Immunofluorescent staining shows a reduction in the PGE2 expression in cells expressing COX-2(484–604) compared with the nontransfected cell (D2). This observation contrasts with D4, where the arrows point to a nontransfected cell and a transfected cell expressing COX-2(1–483). D5 does not show a reduction of PGE2 in the transfected cell as compared with the nontransfected cell.
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In summary, our study establishes a physiologic binding interactionof iNOS and COX-2 bringing NO in proximity to COX-2, facilitatingits S-nitrosylation and activation, and fitting with earlierfindings that NOS inhibition decreases prostaglandin formation(9, 21). Our findings accord with recent evidence that manyphysiologic actions of NO require its delivery to moleculartargets (12, 22, 23). Whereas scaffolding proteins such as CAPON(22) or PSD95 (23) link neuronal NOS, respectively, to Dexras1(22) and N-methyl-D-aspartate receptors (23), iNOS and COX-2bind directly. The molecular synergism between iNOS and COX-2may represent a major mechanism of inflammatory responses. Drugsthat block the iNOS–COX-2 interaction may be anti-inflammatory,synergizing with COX-2 inhibitors and permitting lower doses.As the binding site on iNOS is in the catalytic domain, derivativesof iNOS inhibitors that also prevent binding to COX-2 may decreaseboth NO and prostaglandin formation.
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24. This work was supported by U.S. Public Health Service grant DA000266, Research Scientist Award DA00074 (to S.H.S.), and a Canadian Institute of Health Research fellowship (to S.F.K).
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(IFN-
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