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COX-2-Derived Prostaglandin ProductionNitric Oxide Activates COX-1 But Inhibits Nitric Oxide Synthase/COX Cross-Talk:

AbramsonBallou, Mukundan Attur, Ashok R. Amin and Steven B. Robert Clancy, Branko Varenika, Weiqing Huang, Les

http://www.jimmunol.org/content/165/3/1582doi: 10.4049/jimmunol.165.3.1582

2000; 165:1582-1587; ;J Immunol 

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Nitric Oxide Synthase/COX Cross-Talk: Nitric Oxide ActivatesCOX-1 But Inhibits COX-2-Derived Prostaglandin Production1

Robert Clancy,2* Branko Varenika,* Weiqing Huang,* Les Ballou, † Mukundan Attur,*Ashok R. Amin,* and Steven B. Abramson*

It is recognized that there is molecular cross-talk between the inflammatory mediators NO and PGs that may regulate tissuehomeostasis and contribute to pathophysiological processes. However, the literature is divided with respect to whether NO acti-vates or inhibits PG production. In this study, we sought to determine whether conflicting observations could be accounted for bydivergent effects of NO on the two cyclooxygenase (COX) isoforms. Exposure of resting macrophages to NO (30mM) enhancedPGE2 release by 4.5-fold. This enhancement was inhibited by indomethacin but not by the COX-2 selective inhibitor NS398. Toseparate the activation of phospholipase A2 and COX, we performed experiments using fibroblasts derived from COX-1-deficientor COX-2-deficient mice. These cells exhibit increased basal PG production, which is due to a constitutively stimulated cytosolicphospholipase A2 and enhanced basal expression of the remaining COX isozyme. The exposure of COX- 2-deficient cells toexogenous NO (10mM) resulted in a 2.4-fold increase of PGE2 release above controls. Further studies indicated that NO stimulatedPGE2 release in COX-2-deficient cells, without altering COX-1 mRNA or protein expression. In contrast, NO inhibited COX-2-derived PGE2 production in both LPS-stimulated macrophages and COX-1 knockout cells. This inhibition was associated withboth decreased expression and nitration of COX-2. Thus, these studies demonstrate divergent effects of NO on the COX isoforms.The regulation of PGE production by NO is therefore complex and will depend on the local environment in which these pleiotropicmediators are produced. The Journal of Immunology,2000, 165: 1582–1587.

N itric oxide synthase (NOS)3 and cyclooxygenase (COX)produce important mediators of tissue homeostasis andpathophysiological processes. The regulation of these

enzymes and their capacity to participate in molecular cross-talk isan important focus of investigation. Both NOS and COX haveconstitutive and inducible isoforms which are encoded by twounique genes, located on different chromosomes (1, 2). COX-2 isthe inducible form of the COX enzyme, the synthesis of which istriggered by those cytokines that also induce NOS-2, or inducibleNOS (reviewed in Refs. 3 and 4). COX-1 is the constitutive iso-form that is expressed by most human cells and tissues (5). COX-1and COX-2 are 60% identical within a species (6) with the con-servation of a tyrosine (Tyr385) located in the active site. Tyr385

contributes to enzyme activity; in addition, the tyrosyl radical spe-cies is involved in suicide inactivation (7). In cell-free systems,NO has been reported to trap the tyrosyl radical of the COXs,which leads to tyrosine iminoxyl radical formation and inactiva-tion of the enzyme (8, 9).

The PG-biosynthetic pathway is initiated by activation of phos-pholipase A2 (PLA2), which are primarily responsible for agonist-

induced arachidonic acid release from membrane phospholipids(10). Conversion of arachidonic acid to PGH2, the committed stepin prostanoid biosynthesis, is mediated by both COX-1 andCOX-2. The PGH2 is subsequently converted to a variety of eico-sanoids depending on the downstream enzymatic machinerypresent in a particular cell type. The COX enzymes are thought tobe the primary target enzymes for nonsteroidal antiinflammatorydrugs, which block their ability to convert arachidonic acid toPGH2 (11).

Although it is recognized that there is “cross-talk” betweenproducts of the NOS and COX pathways, the literature is dividedwith respect to whether NO activates or inhibits PG production.For example, nitroglycerin (NO surrogate) is reported to inhibitplatelet activation in vivo, which occurs via the stimulation of PGsynthesis by endothelial cells (12). Salvemini and Masferrer (13)reported that NO stimulates COX activity in RAW 264.7 murinemacrophages, possibly via reaction with the heme componentwhich binds to the active site of the COX enzyme. In contrast, weand others have reported that endogenous NO inhibits PG synthe-sis in chondrocytes and LPS-stimulated macrophages (14, 15). Inthis study, we sought to determine whether these conflicting ob-servations could be accounted for by divergent effects of NO onthe two COX isoforms. In addition, the capacity of NO to promoteNO modifications, such as tyrosine nitration, was investigated. Ourstudies indicate that NO exerts divergent effects on the constitutiveand inducible COX isoforms, activating COX-1 but inactivatingCOX-2. Mechanisms by which NO exerts these effects on COXare explored.

Materials and MethodsCulture of murine macrophages

Mouse monocyte/macrophage cell line J774.A1 (ATCC TIB 67) was cul-tured in DMEM medium plus 10% FBS, 2 mM glutamine, 50 U/ml pen-icillin, and 50mg/ml streptomycin. The cells were activated by IFN-g(100U/ml) and LPS (5mg/ml) in the presence and absence ofN-L-methylarginine.

*Department of Rheumatology, Hospital for Joint Diseases and Division of Rheu-matology, New York University School of Medicine, New York, NY 10003; and†Department of Veterans Affairs Medical Center and Departments of Medicine andBiochemistry, University of Tennessee, Memphis, TN 38163

Received for publication January 27, 2000. Accepted for publication May 11, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a Biomedical Science Grant from the National ArthritisFoundation to S.B.A. and by the Joseph and Sophia Abeles Foundation.2 Address correspondence and reprint requests to Dr. Robert Clancy, Department ofRheumatology, Hospital for Joint Diseases, 301 E. 17the Street, New York,NY 10003.3 Abbreviations used in this paper: NOS, NO synthase; COX, cyclooxygenase; DEA,diethylamine; PLA29 phospholipase A2.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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Culture of COX-deficient pulmonary fibroblasts

We utilized COX-12/2 (COX-1) or COX-22/2 (COX-2) lung fibroblasts,which express an immortalized phenotype and a hygromycin resistancegene as described by Kirtikara et al. (16). Cells were seeded in DMEMmedium plus 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50mg/mlstreptomycin, and 250mg/ml hygromycin.

Viability measurements

Cell viability was determined using a lactic dehydrogenase kit (Sigma,St. Louis, MO), and measurements were determined following the recom-mendations of the manufacturer. Results are expressed as a percentagecompared with values obtained after treatment with PBS plus 0.1% TritonX-100.

RNA extraction

RNA was extracted using a Promega kit (Promega, Madison, WI). North-ern blots used a hybridization probe prepared from full length cDNA forCOX-1, and the procedure was performed as previously described (23).

NO treatment

NO solutions were prepared by dissolving diethylamine (DEA)/NO in 10mM NaOH. NO was quantitated by measuring the absorbance at 250 nmas described by the manufacturer.

PGE2 measurement

In the macrophage studies, PGE2 biosynthesis was measured in cell fluidrecovered after treatment by NO or LPS (24 h). In studies using COX-1/COX-2 cells, NO treatment was varied. The treatment medium was thenremoved, and cells were washed twice with fresh medium. Cells wereplaced in fresh medium, and the interval to harvest PGE2 release was 10 or3 min (as described in text). PGE2 in the medium was measured by ELISAusing a commercial kit (Cayman Chemicals, Ann Arbor, MI) following theinstructions of the manufacturer.

Western blot analysis

Laemmli buffer was directly added to the cells at the termination of thereaction. Proteins were separated by SDS-PAGE (10%), and proteins weretransferred to nitrocellulose and analyzed using rabbit anti-nitrotyrosine(Upstate Biotechnology, Lake Placid, NY), rabbit anti-COX-2 (Transduc-tion Laboratories, Lexington, KY) rabbit anti-actin (Sigma), rabbit anti-GAPDH (Sigma), rabbit anti-glucose 6 phosphate dehydrogenase (giftfrom Dr. E. Beutler), and rabbit anti-cytosolic PLA2 (Upstate Biotechnol-ogy). Images were evaluated after scanning of autoradiographs using thestorage phosphor technique performed with the Molecular Dynamics 400APhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Two-dimensional gel electrophoresis and trypsin digestion

Two-dimensional electrophoresis was performed as previously described(17–19). Detection of anti-COX-2 was by ECL, and then the same blot wasreprobed with anti-nitrotyrosine which was reported by an alkaline phos-phatase method. Lysate (20 mg/ml) was treated with bead-conjugated tryp-sin (Pierce, Rockford, IL, Immobilized tosylphenylchloromethyl ketonetrypsin, 1 volume sample to 1 volume resin) at 4°C or 37°C (10 min). Thebeads were removed at 10,0003 g (1 min), and SDS sample buffer wasadded to the supernatant fraction. Proteins were separated by SDS-PAGE(10%), and proteins were transferred to nitrocellulose and analyzed usingrabbit anti-nitrotyrosine, rabbit anti-COX-2 (Transduction), or rabbitanti-actin.

Data variability

Data were analyzed as the mean and the SEM. The levels of significancewere calculated using Student’st test.

ResultsNO increases PGE2 production in resting murine macrophages

Exposure of resting macrophages to NO enhanced PGE2 release(Fig. 1). The enhancement of PGE2 production was inhibited byindomethacin (nonselective COX inhibitor) but not by the COX-2selective inhibitor NS398. The dose-response relationship betweenNO and PGE2 production by macrophages was obtained by com-bining the data from four separate experiments. The amount of

PGE2 produced was significantly greater than control (p , 0.01)at NO concentrations of 30mM and above. NO-dependent PGE2

release by unstimulated macrophages was not due to increase inexpression of PlA2 or COX-1 as assessed by Western blot analysis(Fig. 1). These findings suggest that activation of PLA2 or COX-1is necessary for NO-dependent PGE2 production. To further sep-arate the activation of PLA2 and COX-1, we performed experi-ments using immortalized, nontransformed cells derived fromCOX-1-deficient or COX-2-deficient mice (16). These cells exhibit

FIGURE 1. Effect of exogenous NO on PGE2 production by murinemacrophages.A andB, PGE2 release by resting macrophages with or with-out NO (30mM, 24 h) in the presence or absence of indomethacin (INDO,5 mM); NS 398 (10mM) was analyzed by ELISA.C, Western blot analysisof COX-1 and cytosolic PLA2 in resting and macrophages with or withoutNO treatment.

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increased basal PG production, which is due to a constitutivelystimulated PLA2 and enhanced basal expression of the remainingCOX isozyme.

NO increases PGE2 production in COX-2-deficient (but notCOX-1-deficient) cells

The above studies, which showed enhancement of PGE2 produc-tion in unstimulated macrophages (not inhibited by NS-398), in-dicated that NO activates COX-1-dependent PGE2 production. Wenext examined the effect of exogenous NO on PGE2 release byCOX-1 and COX-2-deficient cells. As shown in Fig. 2, treatmentof COX-2-deficient cells with as low as 1mM NO increased PGE2release to 155% control and NO stimulated PGE2 synthesis in adose-dependent manner. As expected, in COX-2-deficient cells,the NO-stimulated PGE2 release was inhibited by indomethacinbut not by the COX-2 selective agent NS-398. Exposure of COX-2-deficient cells to NO (1mM) in the presence of exogenouslyadded arachidonic acid (10mM) also enhanced PGE2 release by180% (not shown). In contrast, and consistent with the conclusionsdrawn from studies of macrophages, exogenous NO failed to stim-ulate PGE2 release in COX-1-deficient cells; NO treatment, in fact,inhibited PGE2 release from these cells (Fig. 2).

Further studies were undertaken to define the manner in whichNO stimulates PGE2 production. COX-2-deficient cells were in-cubated with 10mM NO for 20 and 60 min and protein and RNAwere extracted to examine COX-1 expression. As shown in Fig. 2,NO treatment did not affect COX-1 expression of protein ormRNA. We next examined the kinetics of NO-dependent PGE2

production in COX-2-deficient cells (Fig. 3). Exposure of COX-2-deficient cells to 10mM NO resulted in a rapid PGE2 releasedetectable at 1 min. After exposure to NO, PGE2 release reachedits peak at 10 min and returned to baseline by 60 min. NO treat-ment at 100mM resulted in a similar profile with a more gradualdecrease to baseline. A23187 exposure resulted in a rapid increasein PGE2 release that was first measurable at 1 min and that, unlikethe response to NO, was sustained during the 60-min observationperiod. Taken together, these results suggest that NO stimulatesPGE2 release due to COX-1 activation and not by altering COX-1expression.

NO inhibits PGE2 biosynthesis by COX-1-deficient cells or byLPS-stimulated macrophages in association with decreasedCOX-2 expression

Additional experiments were performed to further explore the ef-fects of NO on COX-2 activity and expression. As shown in Fig.4, the exposure of COX-1 deficient cells to exogenous NO for 1 hdecreased basal PGE2 release by.70%. In addition, we observeda 50% decrease in COX-2 protein expression compared with con-trols at 1 h. NO treatment did not affect cellular viability, nor didit reduce the expression of actin and GAPDH (control protein notshown). Similarly, in LPS-stimulated macrophages, NO exposureresulted in a decrease in PGE2 release and total COX-2 protein (at24 h, Western blot (Fig. 4)). In experiments using varying doses ofNO, we observed a correlation between PGE2 release and COX-2expression (not shown).

NO inhibits PGE2 biosynthesis by COX-1-deficient cells or byLPS-stimulated macrophages in association with COX-2nitration

The inhibition of PGE2 production in LPS-treated macrophageswas accompanied by the formation of a 72-kDa nitrated protein(nitrotyrosine immunoblot (Fig. 5)). Coincubation of 3-nitroty-rosine (10 mM) prevented Ab binding to the 72-kDa protein(Western blot assay, not shown), confirming recognition by the Ab

of a nitrated protein. To assess whether the 72-kDa protein couldbe identified as COX-2, we utilized two-dimensional gel electro-phoresis and trypsin digestion. In lysates of LPS-stimulated mac-rophages exposed to 30mM NO, the nitrated protein was separatedby isoelectric focusing and by SDS-PAGE followed by transfer tonitrocellulose. As shown (Fig. 6), autoradiographic analysis re-vealed that COX-2 eluted at 72 kDa with an apparent pI of 7.5. Thesame blot was reanalyzed by Western blot using a rabbit anti-nitrotyrosine Ab. The analysis revealed that one of two nitratedproteins migrated with an identical molecular mass and apparent pIas COX-2. COX-2 is trypsin sensitive, and we next focused on thecapacity of trypsin to digest the 72-kDa nitrated protein. The 72-kDa nitrated protein digestion was separated by SDS gels, trans-ferred to nitrocellulose, and sequentially probed with anti-COX-2and anti-nitrotyrosine. Treatment resulted in a loss of both immu-nodetectable COX-2 and the nitrated 72-kDa protein (Fig. 6); tryp-sin digestion did not affect G6PDH examined as a control (notshown).

FIGURE 2. NO increases PGE2 production by COX-2-, but not COX-1-, deficient cells. COX-1-deficient cells and COX-2-deficient cells weretreated with NO (10mM, 20 min, 37°C) with or without indomethacin (5mM), NS 398 (10mM), and then PGE2 in the media was assessed asdescribed inMaterials and Methods. Protein and RNA were isolated fromthe cells, and COX-1 expression (GAPDH as RNA loading control) wasdetermined.

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Thus, these findings indicate that NO induces the nitration ofCOX-2 in LPS-stimulated macrophages. As has been shown incell-free systems by Gunther et al. (9), it is likely that such nitra-tion inhibits the catalytic activity of the enzyme, as those authorshave reported (8, 9). Whether COX-2 nitration decreases the sta-bility of the enzyme and accounts for decreased COX-2 expressionin NO-treated cells remains to be determined.

As shown in Fig. 5, exposure of resting macrophages to NO didnot lead to protein nitration. We next examined the effect of NO onnitration of protein in COX-1-deficient cells. The exposure of thesecells to NO lead to the formation of a 72-kDa nitrated protein (Fig.5). In contrast to observations made with COX-1-deficient cells,NO exposure did not lead to the formation of a 72-kDa nitratedprotein in COX-2-deficient cells (Fig. 5). These data indicate thatCOX-2 (but not COX-1) undergoes protein nitration after exposureto NO. The percentage of the protein that is nitrated is not known;thus, it is impossible to equate nitration with enzyme inhibition orinstability of COX-2 expression.

DiscussionIn this study, the effect of NO on PG biosynthesis by murine mac-rophages and COX-1 or -2-deficient murine fibroblasts was exam-ined. We found that exposure of resting macrophages to NO en-hanced PGE2 release. This enhancement was inhibited byindomethacin (nonselective COX inhibitor) but not by the COX-2selective inhibitor NS398. In contrast, exposure of LPS-stimulatedmacrophages to NO inhibited PGE2 release. To test whether thesedivergent effects of NO depend on the COX isoform, we studiedPGE2 production in immortalized, nontransformed cells derivedfrom COX-1-deficient or COX-2-deficient mice. These cells re-lease PGE2 due to increased basal expression of the remaining

FIGURE 3. Kinetics of PGE2 release by COX-2-deficient cells exposedto NO or the calcium ionophore A23187. COX-2-deficient cells weretreated with NO (10 or 100mM, time varied) or A23187 (5mM, timevaried). After exposure to NO or A23187, the medium was replaced, andeach sample was incubated for 3 min. Thex-axis refers to the treatmentinterval with NO or A23187, and they-axis indicates PGE2 detected in thereplacement media.

FIGURE 4. NO inhibits PGE2 pro-duction and decreases COX-2 expres-sion in COX-1-deficient cells andLPS-treated murine macrophage.A,PGE2 release by COX-1-deficient fi-broblasts with or without NO (1 mM,time varied).B, PGE2 release by LPS-stimulated macrophages which weregiven a pretreatment with LPS (5mg/ml) plus IFN-g (100 U/ml) (LPS-stimulated macrophages, 5 h) and thena second treatment with NO (30mM,18 h).C andD, Western blot analysisof COX-2 (actin as protein loadingcontrol).

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COX isozyme as well as the elevated expression of PLA2 (16).Using COX-deficient cell lines, we confirmed that NO activatedCOX-1- but inhibited COX-2- derived PG production. In addition,our studies demonstrate that the capacity of NO to inhibit PGE2

release is associated with a decrease in COX-2 expression as wellas nitration of the enzyme. The NO dependent decrease in COX-2expression has been previously reported and may be, in part, dueto the attenuation of cytokine elicited transcription factors whichregulate COX-2 expression such as NFk B (4, 15, 20–22). Re-cently, we have reported that NO also inhibits the translocation ofCOX-2 to a cytosolic compartment which favors enzyme activity(23).

As noted, our studies indicate that the inhibition of PGE syn-thesis by NO is accompanied by nitration of COX-2. Although ithas been previously demonstrated that NO converts tyrosine tonitrotyrosine in cell-free cyclooxygenases (8, 9), this is the firstreport to show that the reaction occurs in cells and that COX-2 ismore sensitive to nitration than COX-1. In these studies, we foundthat micromolar NO stimulated COX-2 nitration in LPS-stimulatedmacrophages and in COX-1-deficient cells. COX-2 nitration hasbeen shown to inhibit the catalytic activity of the enzyme (8, 9).Whether nitration also decreases COX-2 protein stability remainsto be determined.

In contrast to these effects of NO on COX-2, we did not findevidence for nitration of COX-1. However, the activation ofCOX-1 by NO may occur via an allosteric effect subsequent toS-nitrosylation that stimulates enzymatic activity as reported in acell-free system by Hajjar et al. (24). Alternatively, the activationof COX-1 by NO could be linked to the effect of NO on glutathi-one metabolism. Our laboratory has demonstrated that neutrophilsexposed to NO convert intracellularglutathione to a nitrosylatedadduct (25). This has been termed “nitrosative stress” and had beenimplicated in the activation of signaling proteins such as p21ras andthe transcription factor OxyR (26, 27). NO may therefore serve a roleas a potent peroxide activator of COX-1 due to the depletion ofreduced glutathione as suggested by Goodwin et al. (28).

In summary, NO activates COX-1 but inhibits COX-2-derivedPG production. Because a variety of cells (e.g., endothelium, mac-

rophages, chondrocytes) produce NO and PGs simultaneously inresponse to cytokines and other activators (3, 29), we speculatethat enhanced PG biosynthesis by COX-1 in the presence of ele-vated levels of NO may contribute to inflammatory mitogenic andangiogenic processes. The studies here reported help resolve a lin-gering controversy in the literature regarding NOS/COX cross-talkby demonstrating divergent effects of NO on the COX isoforms.The regulation of PGE production by NO is therefore complex andwill depend on the local environment in which these pleiotropicmediators are produced.

AcknowledgmentsWe thank Maddy Rios for assistance in the preparation of the manuscript.

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FIGURE 5. COX-2 undergoes protein nitration after exposure to NO.A, control macrophages (as in Fig. 1) and LPS-stimulated macrophages (asin Fig. 4) were incubated with or without NO (NO varied, 1 h). B, COX-1-deficient cells were exposed to NO (1 mM, time varied). After treat-ments, proteins were separated on SDS-PAGE, transferred to nitrocellu-lose, and probed with an anti-nitrotyrosine Ab. As shown, a 72-kDanitrated protein was detected in LPS-stimulated and COX-1-deficient cells,but not in controlled macrophages or COX-2-deficient cells.

FIGURE 6. Evidence that the 72-kDa nitrated protein reported in LPSmacrophage exposed to NO is COX-2 as indicated by two-dimensional gelelectrophoresis and trypsin digestion. LPS-stimulated macrophages weretreated with DEA/NO (as in Fig. 5A, 1 mM).A, Lysates were separated byisoelectric focusing and by SDS-PAGE followed by transfer to nitrocel-lulose, and the blots were probed with an anti-nitrotyrosine Ab or with ananti-COX-2 Ab. Arrow indicates region of two-dimensional gel contain-ing colocalization of Ag after probing for COX-2 and nitrotyrosine.B,Evidence that the 72-kDa nitrated protein as indicated by trypsin diges-tion. Lysate was incubated with or without trypsin: untreated (Lane 1);trypsin 4°C (5 min) (Lane 2); trypsin treatment 37°C (5 min) (Lane 3).Digest was separated by SDS-PAGE, and nitrocellulose blots were probedwith an anti-nitrotyrosine Ab or with an anti-COX-2 Ab.

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