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Experimental Cell Research 266, 203–212 (2001)doi:10.1006/excr.2001.5218, available online at http://www.idealibrary.com on

Nitric Oxide Suppresses IL-8 Transcription by InhibitingC-Jun N-Terminal Kinase-Induced AP-1 Activation

Ramesh Natarajan,* Seema Gupta,† Bernard J. Fisher,* Shobha Ghosh,* and Alpha A. Fowler, III*,1

*Division of Pulmonary and Critical Care Medicine and †Division of Gastroenterology, Department of Internal Medicine,
Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298
The role of activator protein-1 (AP-1) in tumor ne-crosis factor-a (TNF-a)-induced interleukin-8 (IL-8)gene expression was evaluated. We showed that TNF-aactivates AP-1 in the transformed endothelial cell lineECV304 by transient transfections of IL-8 promoterconstruct pGL-3BF2. Mutation of either the AP-1 site

r the NF-IL-6 site on the IL-8 promoter suppressedhe TNF-a-induced activation, suggesting cooperation

between these transcription factors and transcriptionfactor NF-kB. Overexpression of dominant negative

utants of c-Jun suppressed AP-1-driven transcrip-ion of the IL-8 promoter following stimulation byNF-a, suggesting that cooperative interaction be-

ween AP-1 and NF-kB is essential for IL-8 transcrip-ion in the presence of TNF-a. We also showed that

nitric oxide (NO), in the form of an exogenous NOdonor, suppressed the level of activation of the AP-1subunit, c-Jun, by down-regulation of c-Jun NH2 ter-minal kinase. This down-regulation could be the puta-tive mechanism of action for NO-mediated inhibitionof IL-8 secretion in activated endothelium. These ob-servations suggest for the first time that NO has broadsuppressive activities on various proinflammatory ef-fectors in activated endothelium. © 2001 Academic Press

Key Words: interleukin-8; activator protein-1; nu-clear factor kappa B; c-Jun N terminal kinase; nitricoxide.

INTRODUCTION

Interleukin-8 (IL-8)2, a member of the C-X-C class ofhemokines, mediates adhesion, activation, and migra-ion of neutrophils into sites of inflammation [1, 2].dministration of neutralizing antibodies against IL-8

1 To whom reprint requests should be addressed at the Division ofPulmonary and Critical Care Medicine, Department of Internal Med-icine, Virginia Commonwealth University, Box 980050, Richmond,VA 23298. Fax: (804) 828-3559. E-mail: [email protected].

2 Abbreviations used: IL-8, interleukin-8; TNF-a, tumor necrosisactor-a; NF-kB, nuclear factor kappa B; AP-1, activator protein-1;

JNK, c-Jun NH2 terminal kinase; GSNO, S-nitroso-L-glutathione;
LU, relative light units.
203

blocks neutrophil infiltration and tissue damage inanimal models of acute inflammation, suggesting thatoverproduction of IL-8 by endothelium is a key featureof acute vascular injury [3–5]. Exposure to proinflam-matory peptides such as tumor necrosis factor-a(TNF-a) causes endothelial cells to rapidly producelarge quantities of IL-8 with little or no IL-8 produced“constitutively” [6].

Current research shows that IL-8 expression is me-diated by differential activation and binding of induc-ible transcription factors to the 59-flanking DNA of theIL-8 gene. The proximal 130 nucleotides upstreamfrom the TATA box in the IL-8 promoter are sufficientfor producing maximal transcriptional responses toproinflammatory mediators such as TNF-a [7–9]. Bind-ing sites for nuclear factor-kappa B (NF-kB), nuclearfactor-IL-6 (NF-IL-6), and activator protein-1 (AP-1),among others, are present within this region. Mukaidaet al. demonstrated that TNF-a and lipopolysaccharideinduce IL-8 gene transcription by promoting binding ofNF-kB and possibly NF-IL-6 to their cognate DNAbinding sites on the IL-8 promoter [10]. NF-IL-6 is abZip transcription factor that belongs to the C/EBPfamily. While NF-IL-6 alone binds weakly to the IL-8promoter, its binding activity increases in the presenceof NF-kB. Moreover, the rel homology domain of NF-kBand the bZip domain of NF-IL-6 enable these transcrip-tion factors to physically interact independent of DNAbinding [11]. AP-1, an inducible transcription factor, isalso a member of the bZip family and is also essentialfor many genes involved in the inflammatory response[11]. Binding of TNF-a to its cell surface receptor acti-vates AP-1 through a cascade of kinases that include aMAP kinase kinase kinase (MEKK1), a MAP kinasekinase (MKK4 and MKK7), and a MAP kinase [JNK(c-Jun NH2 terminal kinase)] [12, 13]. Once activated,JNK translocates to the nucleus where it phosphory-lates and activates c-Jun, a component of the transcrip-tion factor AP-1.

Mastronarde and colleagues used the alveolar epi-thelial cell line, A549, and found a role for AP-1 inNF-kB-mediated IL-8 expression, suggesting that AP-1
may be the preferred transcription factor over NF-IL-6
0014-4827/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

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204 NATARAJAN ET AL.

for cooperative interaction with NF-kB [14]. Mori et al.also suggested that cooperation between NF-kB andAP-1 is essential for transactivation of the IL-8 gene byhuman T-cell leukemia virus type I [15]. Molecularcooperation between AP-1 and NF-kB appears essen-tial for transcriptional activation of other genes. Cyclo-oxygenase-2 induction by nitric oxide requires activa-tion of both AP-1 and NF-kB [16]. The transcriptionfactors AP-1 and NF-kB are also essential for inductionof the human inducible nitric oxide synthase gene [17].We and others have conclusively demonstrated tran-scriptional regulation of IL-8 by NF-kB in endothelialcells [18, 19]. The AP-1 site has been proposed asinfluencing both the basal and the inducible activitiesof the IL-8 promoter and playing a critical role in celltype-specific expression of IL-8. Moreover, the differ-ential regulation of IL-8 in endothelial and epithelialcells is proposed as modulating the different roles di-verse cell types play in mediating acute inflammatoryresponses. Therefore, it is essential to study the role ofAP-1 in the regulation of cytokine-induced IL-8 tran-scription in endothelial cells.

Nitric oxide (NO), a key signaling molecule in vas-cular regulation, neuronal communication, and hostdefense, exerts potent anti-inflammatory properties indiseases characterized by excessive endothelial activa-tion and IL-8 secretion [20]. NO, administered as a gas,into the lungs of patients with acute respiratory dis-tress syndrome, resulted in dramatic improvements inlung function and oxygenation [21]. In prior reports, weshowed that inhaled NO gas dramatically attenuatedacute pulmonary vascular inflammation and subse-quent lung injury resulting from bacterial sepsis inanesthetized swine [22]. More recently, in the endothe-lial cell line ECV304, we demonstrated that NO atten-uated IL-8 secretion by preventing binding of NF-kB toits regulatory sequence on the IL-8 promoter [19].Work done by various groups suggests that NO has abroad effect on the pathways involved in the activationof AP-1 and NF-kB [23–26]. However, the in vivo effectof NO on AP-1-driven IL-8 transcription in endothelialcells remains undefined.

In this report, we show the importance of AP-1 inTNF-a mediated up-regulation of IL-8 transcription inan endothelial cell line (ECV304). We also show thatnitric oxide strongly down-regulates AP-1-driven tran-scription by inhibiting the activation of the c-Jun com-ponent of AP-1 by the upstream kinase JNK.

MATERIALS AND METHODS

Reagents and kits. The nitric oxide donor S-nitroso-L-glutathione(GSNO) used in these studies was obtained from Cayman ChemicalCo. (Ann Arbor, MI). Recombinant human TNF-a was purchasedfrom Collaborative Biomedical Products (Bedford, MA). The AP-1and NF-kB luciferase reporter constructs pAP1Luc and pNFkBLuc
were obtained from Stratagene (La Jolla, CA). The dual luciferase i
assay system and pRL-TK vector were purchased from Promega(Madison, WI). The Quik-Change site-directed mutagenesis kit wasobtained from Stratagene. Effectene transfection reagent was pur-chased from Qiagen, Inc. (Valencia, CA). DNA purification kits wereobtained from Bio-Rad (Hercules, CA). Oligonucleotides for site-directed mutagenesis were synthesized by Genosys BiotechnologiesInc. (The Woodlands, TX). The ABI Prism Dye Terminator CycleSequencing kit was purchased from Perkin Elmer (Foster City, CA).Rabbit anti-c-Jun NH2 kinase antibody was obtained from SantaCruz Biotechnology (sc-571; Santa Cruz, CA). Cell culture media andreagents were purchased from Mediatech (Herndon, VA). [g-32P]ATPwas obtained from New England Nuclear (Boston, MA). All otherreagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture. The transformed human umbilical vein endothelialcell line, ECV304, was obtained from the American Type CultureCollection (Rockville, MD). Cells were maintained in M-199 plus 10%fetal bovine serum, penicillin–streptomycin (100 m/ml), and ampho-tericin-B (250 ng/ml) and incubated under 37°C, 5% CO2 conditions.

Nitric oxide donor. The NO donor S-nitroso-L-glutathione waschosen for these studies because of its frequent use in the literature,its prolonged half-life in biological systems, and its low cytotoxicity[18]. The times displayed for the experiments shown below, as wellas the concentrations of GSNO utilized, were optimized in prelimi-nary experiments. Viability was assessed by lactate dehydrogenaserelease, and there was no evidence of cytotoxicity.

Site-directed mutagenesis. Mutant oligonucleotide primers (senseand antisense) containing transversion point mutations were de-signed at the AP-1 (2126 to 2120) and NF-IL-6 (294 to 281) sites onthe IL-8 promoter. The Quik-Change site-directed mutagenesis kitwas used for the mutagenesis. The construct used for the generationof the replacement mutants was a wild-type luciferase reporter vec-tor, pGL-3BF2, engineered in this laboratory and previously reported19]. pGL-3BF2 contains the base pairs from 2421 to 155 of the

human IL-8 promoter. The mutant AP-1 primer sequences designedwere 59-GAACAAATAGGAAGTGTGATGCAGAAGGTTTGCCCTG-AGGGGATGG-39 and 59-CCATCCCCTCAGGGCAAACCTTCTG-CATCACACTTCCTATTTGTTC-39. The mutant NF-IL-6 primersequences were 59-GCCCTGAGGGGATGGGCCATAGCTTGCAA-ATCGTGGAATTTCC-39 and 59-GGAAATTCCACGATTTGCAAGC-TATGGCCCATCCCCTCAGGGC-39, where the shaded bases showthe transverse mutations introduced into the AP-1 and NF-IL-6sites, respectively. Mutant primers have a high melting point (Tm)nd anneal to the identical sequences on opposite strands of theonstruct. Temperature cycling was performed per the manufactur-r’s instructions using Pfu DNA polymerase, which replicates bothtrands with high fidelity without displacing the mutant primers.he resulting thermocycling product was treated with DpnI endonu-lease, which specifically digests methylated and hemimethylatedNA, selecting for the “mutation-containing” synthesized DNA.NA isolated from almost all Escherichia coli strains is dam-meth-lated and therefore susceptible to DpnI digestion. The nicked vectorNA incorporating the desired mutation was transformed into Epi-

urian Coli XL1-Blue supercompetent cells and sequenced. The re-ulting mutated luciferase reporter vectors were designatedAP1MT and pNFIL-6MT.Transient transfections and dual luciferase reporter assay. TheP-1 and NF-kB luciferase reporter constructs pAP1Luc and pNFkBuc, the pGL-3BF2 reporter, and the mutant luciferase reporters

pAP1MT and pNFIL-6MT were used in transient transfection stud-ies to examine the role of AP-1 in TNF-a-stimulated IL-8 transcrip-tion. For these studies, ECV304 cells were seeded into 24-well plates(80% confluence) in growth medium and incubated overnight. Cellswere transfected using the proprietary nonliposomal lipid Effecteneoptimized according to the manufacturer’s instructions. Cells weretreated with TNF-a (10 ng/ml) for 4 h prior to harvesting. Fireflyuciferase activity generated from the above reporters was normal-
zed to Renilla luciferase activity by cotransfecting the pRL-TK con-

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205NITRIC OXIDE SUPPRESSES c-JUN N-TERMINAL KINASE

struct that contains a herpes simplex thymidine kinase promoterupstream from the Renilla luciferase gene. Dual luciferase outputDual-Luciferase Reporter Assay System) was quantified with a lu-inometer (Lumat LB9501, Berthold) and the results were ex-

ressed as an index of relative light units (RLU). The dominantegative AP-1 mutants were designated DBMT, for the c-Jun mu-ant lacking its DNA binding domain, and LZMT, for the c-Junutant lacking the leucine zipper domain, and were driven by the

ytomegalovirus (CMV) promoter. The above vector, with the CMVromoter alone, was used in the control transfections. The cotrans-ection assays were performed with pGL-3BF2 in the 24-well format

described above.Overexpression of dominant negative c-Jun. ECV304 cells were

plated in 48-well plates at 2000 cells/well. They were transientlytransfected with pAP1Luc (Stratagene) or pGL-3BF2 using the pro-prietary nonliposomal lipid Effectene optimized according to themanufacturer’s instructions as described above. Cells were theninfected either with null (empty) virus or with a recombinant ade-novirus encoding for the dominant negative c-Jun mutant (TAM67)[27] at multiplicities of infection (m.o.i.) of 1, 2, and 3. TAM67 is adominant negative mutant of c-Jun and results in expression of thec-Jun protein lacking the transactivation domain. Cells were incu-bated for 24 h to permit infection and overexpression. Appropriatelyinfected cells were then treated with TNF-a (10 ng/ml) or medium for4 h prior to harvesting and measurement of luciferase activity asdescribed above.

Electrophoretic mobility shift assay. Appropriately treatedECV304 cells in 60-mm dishes were placed on ice, washed once(PBS), and then exposed to buffer A (10 mM Hepes, 10 mM KCl, 100mM EDTA, 100 mM EGTA, 2 mM NaF, 2 mM Na3VO4, 100 mMPMSF) for 15 min at 4°C. Cells were then policed into 1.5 ml tubes,lysed (Nonidet P-40), and centrifuged (13,000g). Nuclear pellets wereresuspended in buffer B (20 mM Hepes, 390 mM NaCl, 1 mM EDTA,1 mM EGTA, 2 mM NaF, 2 mM Na3VO4, 2 mM PMSF), proteinoncentration assessed (Bicinchoninic acid solution, Sigma), andtored at 270°C. An electrophoretic mobility shift assay (EMSA) waserformed as described previously [19]. Briefly, double-stranded con-ensus AP-1 oligonucleotide was end-labeled with [g-32P]ATP using

T4 polynucleotide kinase according to the manufacturer’s specifica-tions (Promega). DNA binding reactions were performed at roomtemperature for 20 min in 50 mM Tris (pH 7.5), 50 mM NaCl, 1 mMdithiothreitol, 2 mg poly[d(I–C)], 0.05% Nonidet P-40 with 10,000–0,000 cpm end-labeled oligonucleotide and 5 mg nuclear protein.

Specificity was determined by preincubation with excess unlabeledAP-1 oligonucleotide. Samples were subjected to EMSA on native 5%polyacrylamide gels at 100 V and visualized by autoradiography.

c-Jun NH2 terminal kinase assay. The JNK assay was performeds described previously [28]. Briefly, following appropriate treat-ents, ECV304 in 60-mm dishes were washed with ice-cold PBS,

ollowed by homogenization in cold lysis buffer (25 mM Hepes, pH.4, 5 mM EDTA, 5 mM EGTA, 50 mM NaCl, 1 mM Na3VO4, 1 mMa4P2O7, 0.05% SDS, 0.05% (w/v) sodium deoxycholate, 1% Triton-100, 5 mM NaF, 0.1% (v/v) 2-mercaptoethanol, 1 mM PMSF, 1 mM

Microcystin-LR, and 40 mg/ml each of pepstatin A, aprotinin, andeupeptin). Cell lysates were centrifuged (13,700g, 10 min) and su-ernatants incubated with 1 mg rabbit anti-JNK antibody at 4°C for

3 h (antibody specific for JNK isoforms, JNK1, JNK2, and JNK3).Antigen–antibody complexes were immunoprecipitated overnight(4°C) by adding protein A–agarose beads. Immunoprecipitates wererecovered by centrifugation and washed sequentially for 10 minwith: (1) lysis buffer, (2) PBS, and (3) kinase assay buffer (2 mMHepes, pH 7.4, 15 mM MgCl2, 0.1 mM Na3VO4, and 0.1% (v/v)2-mercaptoethanol). JNK activity was determined by incubating im-munoprecipitates in a reaction mixture containing kinase assaybuffer, 10 mg glutathione S-transferase–c-Jun (GST–c-Jun) protein,.1 mM ATP, 1 mM Microcystin-LR, and 10 mCi [g-32P]ATP for 20 min
t 37°C. Reactions were terminated by boiling in 53 SDS–PAGE
sample buffer for 5 min. Phosphorylated GST–c-Jun was resolved on10% SDS–PAGE. The gels were dried and autoradiographed and theradioactivity was incorporated in GST–c-Jun, determined by densi-tometry (Kodak Digital Sciences EDAS 120 system using 1D Imageanalysis software).

RESULTS

Effects of TNF-a on AP-1-induced transcription inECV304 cells. The AP-1 binding site contributes toIL-8 promoter activity in several epithelial cells lines[11]. However, the differential regulation of IL-8 inendothelial cells has not been extensively studied. Todetermine the importance of AP-1 in transcription ofIL-8 in cytokine-activated endothelium, we first soughtto demonstrate that TNF-a activated AP-1 in thetransformed endothelial cell line ECV304. This wasachieved by using the firefly luciferase reporter con-struct pAP1Luc, which possesses only a TATA box and7 AP-1 sites. Since TNF-a is known to activate NF-kBn ECV304 cells, another firefly luciferase reporter vec-or, pNFkBLuc, possessing only a TATA box and 5

NF-kB sites, was used as a positive control for thesestudies. For the assay, separate sets of triplicate wellscontaining confluent ECV304 were transiently trans-fected with either pAP1Luc or pNFkBluc. pRL-TK, avector that expresses Renilla luciferase, was cotrans-fected into all cells and then incubated in the presenceor absence of TNF-a (10 ng/ml) for 4 h. Firefly lucif-erase values obtained were normalized to lumines-cence produced by the cotransfected Renilla luciferaseector and expressed as RLU. Figure 1 shows that theuciferase activity of pAP1Luc increased approxi-

ately 12-fold in the presence of TNF-a, suggestingthat this cytokine stimulates transcription by AP-1 in

FIG. 1. AP-1 activation in ECV304 cells by TNF-a. ECV304 cellswere transiently transfected with pAP1Luc or pNFkBluc as de-scribed under Materials and Methods. Cells were activated withTNF-a (10 ng/ml) for 4 h prior to harvest. Luciferase activities weremeasured using the Dual Luciferase System and reported as indexedrelative light units (RLU). Values are means 6 SEM of three inde-pendent transfections.

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Effects of TNF-a on IL-8 promoter activity once theAP-1 and NF-IL-6 sites are mutated. In these studies,we compared TNF-a-induced firefly luciferase expres-sion obtained from the construct pGL-3BF2, a wild-type IL-8 reporter, to the luminescence obtained withthe AP-1 mutated plasmid, pAP1MT, and the NF-IL-6mutated plasmid, pNFIL-6MT. Another pGL-3BF2 mu-ant construct, possessing a mutated NF-kB site

(pNFkBMT), described previously [19], was also usedalong with the promoterless luciferase reporter pGL3-Basic (Promega) as a negative control. Triplicate wellsof ECV304 cells were transiently transfected withthese vectors and pRL-TK. Cells were exposed to eithermedium alone or medium containing TNF-a (10 ng/ml)or 4 h. Firefly luciferase values obtained were normal-zed to Renilla luciferase luminescence and expresseds RLU. As shown in Fig. 2, pGL-3BF2, which pos-esses intact AP-1, NF-IL-6, and NF-kB sites, exhib-

ited an approximate 650% increase in RLU followingTNF-a exposure. However, neither pAP1MT nor pN-

IL-6MT was activated to any appreciable extent byNF-a. Expectedly, the pNFkBMT was also not acti-

vated. Therefore, intact binding sites for NF-kB, NF-IL-6, and AP-1 appear to be essential for TNF-a-medi-ted activation of the IL-8 promoter in endothelialells. The data also suggest that a high level of “inter-ependence” occurs between these transcription fac-ors, as the absence of even one of these factors resultsn loss of cytokine activation of the IL-8 promoter.

AP-1 is essential for IL-8 transcription. We showed,s described above, that the DNA binding site for AP-1n the IL-8 promoter was required for activation in theresence of TNF-a. The aim of these studies was to

FIG. 2. IL-8 promoter activity in the presence of mutated AP-1,NF-IL-6, and NF-kB sites. ECV304 cells in triplicate wells weretransiently transfected with one of the following: (1) the wild-typeIL-8 promoter construct pGL-3BF2, (2) the above construct with amutated NF-kB site (pNFkBMT), (3) the above construct with amutated AP-1 site (pAP1MT), or (4) the above construct with amutated NF-IL-6 site. Cells were stimulated with TNF-a (10 ng/ml)or 4 h prior to harvest. Luciferase activities were measured usinghe Dual Luciferase System and reported as percentage activation byNF-a. Values are means 6 SEM of three independent transfections.

determine whether c-Jun, with all three of its func-

tional domains intact, namely the DNA binding do-main, the transactivation domain, and the leucine zip-per dimerization domain, was required for IL-8transcription following TNF-a activation in ECV304cells. NF-kB and NF-IL-6 physically interact throughthe rel homology domain and bZip domain of NF-kBand NF-IL-6, respectively. Stein et al. showed thatAP-1 physically interacts with NF-kB [29]. This inter-action leads to synergistic activation of IL-8 [30, 31]. Todetermine whether AP-1 could synergistically activateIL-8 in a similar manner in endothelial cells, we used arecombinant adenovirus encoding for a dominant neg-ative c-Jun mutant (TAM67), a mutant having intactc-Jun DNA binding and leucine zipper domains, butlacking the transactivation domain. To test the activityof this dominant negative mutant, confluent ECV304in 48-well plates were transfected with pAP1Luc orpGL-3BF2, followed by infection with either a null(empty) virus or TAM67. Following infection, cellswere treated with TNF-a (10 ng/ml) for 4 h and har-ested and luciferase activity was measured. Figure 3Ahows that TNF-a activates AP-1-driven transcription

of pAP1Luc even when infected with the null virus.Although the extent of increased transcription is sig-nificant in null virus-infected cells, the level of activa-tion was lower than that observed in the absence ofnull virus. We feel that this decrease in activationprobably arises as a consequence of the infectionscheme. Following infection with TAM67, this AP-1-driven transcription of pAP1Luc was suppressed withincreasing multiplicities of infection. Thus, the domi-nant negative c-Jun mutant was effective in down-regulating AP-1-driven transcription from pAP1Luc inECV304 cells. Proportional overexpression of domi-nant negative c-Jun in these cells was verified by West-ern blot analysis with an antibody recognizing theC-terminal portion of c-Jun (sc-44, data not shown).

In contrast, overexpression of TAM67 (Fig. 3B) failedto suppress cytokine-activated transcription of the IL-8promoter, as assessed by transient transfection assays.IL-8 promoter activity was significantly increased be-yond control values (null) at the highest m.o.i. of 3,suggesting that this dominant negative mutant withintact DNA binding and dimerization domains couldstimulate IL-8 transcription even in the absence of itstransactivation domain. Therefore, this c-Jun mutantmust bind to its cognate site on the IL-8 promoterthrough its DNA binding domain, while the leucinezipper domain could interact with either C/EBP orNF-kB subunits to facilitate transcription.

To test the importance of the DNA binding domainand the leucine zipper domain of c-Jun, we cotrans-fected dominant negative mutants of c-Jun lacking ei-ther the DNA binding domain (DBMT) or the leucinezipper domain (LZMT) with the IL-8 promoter con-

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transiently transfected with the above vectors andpRL-TK. The identical vector, possessing the CMV pro-

FIG. 3. Effect of overexpression of dominant negative c-Jun onL-8 promoter activity. ECV304 cells were grown to confluence in8-well plates. Cells were transfected with either pAP1Luc (A) orGL-3BF2 (B) using Effectene followed by infection with either a nullempty) virus or a recombinant adenovirus encoding for a dominantegative c-Jun mutant (TAM67) at various multiplicities of infectionMOI) for 24 h. AP-1-driven transcription was stimulated by theddition of TNF-a (10 ng/ml) for 4 h. Luciferase activities wereeasured using the Dual Luciferase assay system and reported as

ercentage stimulation by TNF-a over untreated controls. (C)CV304 cells in 48-well plates were cotransfected with pGL-3BF2

and pCMV, a vector possessing only a CMV promoter, or DBMT, avector which expresses, through the same CMV promoter, a domi-nant negative c-Jun mutant lacking the DNA binding domain, orLZMT, a vector also expressing through the same CMV promoter adominant negative c-Jun mutant lacking the leucine zipper domain.AP-1-driven transcription was stimulated by the addition of TNF-a(10 ng/ml) for 4 h. Luciferase activities were measured using theDual Luciferase System and reported as indexed luminescence(RLU). All reported values are means 6 SEM of three independentobservations.

moter but lacking the c-Jun coding regions, was co-

transfected with pGL-3BF2 in control wells. Cells wereexposed to medium alone or medium containing TNF-a(10 ng/ml) for 4 h. Firefly luciferase values obtainedwere normalized to Renilla luciferase luminescenceand expressed as RLU. As seen in Fig. 3C, overexpres-sion of either the DNA binding mutant or the leucinezipper mutant resulted in a 50% reduction of lumines-cence from pGL-3BF2 following stimulation by TNF-a.However, unlike the effect of adenoviral-mediated in-fections with TAM67 on the luminescence of pAP1Luc,the overexpression of DBMT and LZMT was less effec-tive in suppressing IL-8 promoter activity. This out-come may have resulted from lower transfection effi-ciencies in cotransfection experiments compared tothose in adenoviral-mediated infections using TAM67.These results, however, demonstrate that AP-1, withintact DNA binding and leucine zipper domains, isessential for TNF-a-mediated activation of the IL-8promoter.

Effects of nitric oxide on AP-1 DNA binding. Wehave previously demonstrated that NO suppressesIL-8 transcription in cytokine-activated ECV304 cellsby altering binding of NF-kB to its cognate site on theIL-8 promoter [19]. In the current study we wanted todetermine whether NO affects AP-1 binding in restingor cytokine-activated ECV304 cells. To achieve this,ECV304 cells were preincubated in medium alone or inmedium containing the NO donor GSNO (1 mM) for24 h. Cells were then exposed to TNF-a (2 ng/ml) for4 h prior to harvest in buffer A. EMSA was performedas described above. As seen in Fig. 4 (lane 2), there isbasal AP-1 DNA binding activity in ECV304 cells ex-posed to medium alone. No difference in DNA bindingoccur following exposure to the NO donor GSNO (1mM) (lane 4). Exposure to TNF-a resulted in an ex-pected increase in AP-1 DNA binding activity (lane 3).Pretreatment with GSNO failed to alter AP-1 DNAbinding activity (lane 5). Thus, NO does not affect theDNA binding ability of transcription factor AP-1 inbasal or cytokine-activated ECV304 cells.

Effects of nitric oxide on c-jun NH2 terminal kinaseactivity. Our next aim was to determine whether NOaffects an upstream activator of AP-1. Transcriptionalactivity of AP-1 proteins is regulated by phosphoryla-tion of transactivation domains and by their binding toprotein kinases [32]. In this study, we targeted c-JunNH2 terminal kinase, since TNF-a is a known activatorof JNK in many cells lines. We first sought to deter-mine whether TNF-a activated JNK in ECV304 cells.To achieve this, ECV304 cells in T-25 flasks were in-cubated with TNF-a (2 ng/ml) for indicated times priorto harvesting in buffer A. JNK (three known isoforms,JNK1, JNK2, and JNK3) was immunoprecipitated andused for the JNK assay with GST–c-Jun as substrate.
As seen in Fig. 5A, TNF-a strongly activated JNK

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activity in ECV304 cells (400% increase) by 20 min.Next, we tested the effects of the nitric oxide donor,GSNO, on JNK activity over a similar time frame inresting endothelial cells. ECV304 cells were incubatedwith GSNO (1 mM) for the indicated times prior toharvest and immunoprecipitation for JNK assay. Asseen in Fig. 5B, GSNO alone did not affect JNK activityin unstimulated endothelial cells. Although a slightdecrease in JNK activity occurred after 10 min, thisdecrease was not statistically significant. Finally, wedetermined the effects of NO on TNF-a-activated en-dothelium. To achieve this, ECV304 cells were prein-cubated for 24 h with differing concentrations of GSNO(250 mM, 500 mM, 1 mM). Cells were then exposed toTNF-a (2 ng/ml) for the indicated period of time. FigureC shows that JNK activity in TNF-a-activated

ECV304 was unchanged by a 5-min exposure to 250mM GSNO and then decreased slightly after 15 min. At500 mM, GSNO decreased JNK activity significantly(38%) at the 15-min time point. Finally, at 1 mMGSNO, JNK activity decreased by 60% after 5 min andby 80% after 15 min. Thus, NO represses JNK activityin a concentration-dependent fashion in activated en-dothelium.

DISCUSSION

The goal of the current study was to further eluci-

FIG. 4. Electrophoretic mobility shift assay for AP-1. ConfluentCV304 cells in 60-mm dishes were preincubated in medium aloner in medium containing the NO donor GSNO (1 mM) for 24 h andhen exposed to TNF-a (2 ng/ml) for 4 h prior to harvest in buffer A.

g-32P-labeled AP-1 oligonucleotide (10,000–20,000 cpm) was incu-bated with 5 mg nuclear extract and subjected to EMSA as describedunder Materials and Methods. The arrow indicates the position ofthe shifted band in the representative autoradiogram. Lane 1, neg-ative control; lane 2, medium; lane 3, TNF-a (2 ng/ml, 4 h); lane 4,GSNO (1 mM, 24 h); lane 5, GSNO (1 mM, 24 h) 1 TNF-a (2 ng/ml,4 h).

date the role of nitric oxide in the transcriptional reg-

ulation of IL-8. New information suggests that theAP-1 site on the IL-8 promoter contributes to bothbasal and inducible IL-8 promoter activity and hasbeen shown to be critical in some epithelial cell lines[11]. However, the role of AP-1 in endothelial cells hasbeen undefined. In this report, we show that coopera-tion between the NF-kB, NF-IL-6, and AP-1 sites onthe IL-8 promoter is essential for TNF-a-mediated ac-tivation of IL-8 in ECV304 cells. This work has shownthat nitric oxide, besides attenuating binding of NF-kBto its cognate binding site, also attenuates IL-8 tran-scription by inhibition of the activation of c-Jun byinhibiting its upstream kinase, c-Jun NH2 terminalkinase. Inactivation of c-Jun results in down-regula-tion of transcription of the IL-8 gene in cytokine-acti-vated ECV304 cells.

Cooperation between transcription factors is essen-tial for IL-8 gene transcription. Recent reports demon-strate cooperation between NF-kB and NF-IL-6 to ac-tivate IL-8 transcription [8, 33, 34]. Cooperationbetween C/EBP, the transcription factor that binds tothe NF-IL-6 site, and c-Jun was also essential for bothbasal and inducible IL-8 promoter activity in Caco-2cells [35]. Mastronarde et al. showed AP-1 to be the

referred transcription factor over NF-IL-6 for cooper-tive interactions with NF-kB in respiratory syncitial

virus-induced IL-8 production in A549 cells [14]. Coop-erativity between NF-kB and AP-1 is also proposed forIL-8 production in other transformed human cell linesstimulated with TNF-a and interferon [10, 31]. Todate, no previous reports have examined the role ofAP-1 in TNF-a-induced IL-8 production from ECV304.In the present study, we used mutational analysis of anIL-8 promoter construct to demonstrate cooperativitybetween AP-1, NF-IL-6, and NF-kB. We showed thatboth AP-1 and NF-IL-6 mutant plasmids failed to ac-tivate IL-8 transcription following TNF-a exposure de-spite the presence of an intact NF-kB site.

Mukaida et al. suggested that NF-kB was the criticaltranscription factor for IL-8 transcription and that NF-IL-6 was the preferred factor for cooperation withNF-kB [10]. Mukaida proposed that AP-1 was utilizedonly in the absence of NF-IL-6 or in the presence of amutated NF-IL-6 site. Our data from a previous report[19] support that model, in that NF-kB seems to be thecritical transcription factor for IL-8 gene expression.The current study shows that NF-kB alone was unableto drive TNF-a-stimulated IL-8 transcription in thepresence of mutated AP-1 or NF-IL-6 sites. Therefore,both AP-1 and C/EBP are essential for “cooperation”with NF-kB in cytokine-mediated IL-8 gene transcrip-tion in ECV304 cells.

AP-1-induced transcription is dependent upon phos-phorylation of serine residues in the transactivationdomain of c-Jun. In this report, we show that c-Jun
lacking the transactivation domain is sufficient to

irpda

t

cf

w(see Materials and Methods). Immunoprecipitates were then used for

TTmep


stimulate transcription of IL-8 following TNF-a activa-tion. To accomplish this, we infected ECV304 with arecombinant adenovirus encoding for a dominant neg-ative c-Jun mutant lacking its transactivation domain(TAM67). TAM67 forms heterodimers with wild-typec-Jun and c-Fos and binds to DNA as a homodimer orheterodimer [27]. TAM67 suppresses AP-1-mediatedprocesses through a “quenching” mechanism by inhib-iting endogenous Jun and/or Fos protein function. Inour studies, TAM67 effectively suppressed transcrip-tion of pAP1Luc, a plasmid solely dependent upon AP-1for transcription, but failed to suppress transcriptionby the IL-8 promoter construct pGL-3BF2. Interest-ingly, IL-8 transcription increased at the highestTAM67 (m.o.i. of 3, Fig. 3B). Stein et al. showed thatthe bZip regions of c-Fos and c-Jun interact with theNF-kB p65 subunit through the Rel homology domainand synergistically activate IL-8 transcription [29].Our work thus suggests that TAM67 binds to the AP-1site on the IL-8 promoter, interacts with C/EBP and/orwith NF-kB subunits through the leucine zipper do-main, and drives IL-8 transcription. On the contrary,overexpression of dominant negative mutants of c-Junlacking either the DNA binding domain or the leucinezipper dimerization domain suppressed cytokine-in-duced IL-8 promoter activity (Fig. 3C). Therefore, itappears that DNA binding and the ability to interactwith other transcription factors are essential for cyto-kine induction of IL-8 promoter activity by AP-1.Taken together with the mutation data discussed pre-viously, it appears that AP-1 is critical for cytokine-induced IL-8 transcription in ECV304 cells.

Nitric oxide is a key signaling molecule in differentphysiological processes. We and others have shownthat NO inhibits cytokine-induced IL-8 expression in avariety of cell types through its effects on NF-kB activ-ty [18, 19, 23, 36, 37]. To our knowledge, all previouseports on the effects of NO on AP-1 binding have beenerformed under in vitro conditions. In this study weetermined the effects of NO on AP-1 binding andctivation under in vivo (i.e., intact cell) conditions.Electrophoretic mobility shift assays to determine

he effect of NO on AP-1 DNA binding activity showed

a JNK assay with 10 mg GST-c-Jun as substrate in the presence of 10mCi [g-32P]ATP for 20 min at 37°C. Phosphorylated GST–c-Jun wasresolved on 10% SDS–PAGE, the gels were dried and autoradio-graphed, and the radioactivity was incorporated in GST–c-Jun de-termined by densitometry and expressed as relative units or percent-age control. Lane 1, TNF-a, 5 min; lane 2, 250 mM GSNO 1 TNF-a,5 min; lane 3, 500 mM GSNO 1 TNF-a, 5 min; lane 4, 1 mM GSNO 1

NF-a, 5 min; lane 5, TNF-a, 15 min; lane 6, 250 mM GSNO 1NF-a, 15 min; lane 7, 500 mM GSNO 1 TNF-a, 15 min; lane 8, 1M GSNO 1 TNF-a, 15 min. Representative autoradiograms for

ach treatment are shown. Values are means 6 SEM of three inde-

FIG. 5. Effect of TNF-a and GSNO on JNK activity. ECV304ells were grown to confluence in T-25 flasks. Cells were treated asollows: (A) TNF-a (2 ng/ml) for the indicated times, (B) GSNO (1

mM) for the indicated times, and (C) GSNO (250 mM, 500 mM, 1 mM)for 24 h followed by TNF-a (2 ng/ml) for the indicated times. Cells

ere harvested and JNK1 and JNK2 isoforms immunoprecipitated
endent observations.

soscvpti3eN


no difference in basal or inducible c-Jun DNA bindingin the presence of NO (Fig. 4). Though AP-1 DNAbinding activity can be measured in this fashion,changes in AP-1 DNA binding activity do not mirror itstranscriptional activity [38]. The level of AP-1 activa-tion can be measured by determining the ability ofupstream kinases to activate c-Jun in the presence orabsence of NO. The phospho-acceptor sites on the N-terminal transactivation domain of c-Jun are majortargets for JNK [12, 13]. Once activated, JNK migratesto the nucleus where it phosphorylates serines 63 and73 of c-Jun and stimulates its activity as a transcrip-tion factor.

Previous reports examining the effects of NO on JNKactivity are conflicting. Kim and colleagues showedthat HEK293 cells exposed to sodium nitroprussideexhibited activation of SEK1 (JNK kinase I), which inturn activated JNK [24]. In contrast, So et al. demon-strated that GSNO suppressed phospho-transferaseactivity of JNK2 under in vitro conditions [26]. In thistudy, we examined the effects of NO on the activationf c-Jun by JNK in ECV304. As seen in Fig. 5C, NOeverely repressed the ability of JNK to phosphorylate-Jun in a concentration-dependent manner in acti-ated endothelium. Thus, by inactivating JNK, NOrevented AP-1 activation and, thus, its activity as aranscription factor. However, AP-1 may still bind tots cognate site on the IL-8 promoter. As shown in Figs.

and 4, AP-1 binding is sufficient to drive IL-8 genexpression, likely through a complex interaction withF-kB or C/EBP [29]. Our results, therefore, suggest

that inhibition of JNK activity by NO may not have adirect consequence on IL-8 transcription. However, wehave previously reported that NO represses IL-8 tran-scription [19]. The current work indicates that NOmediates IL-8 repression via other, indirect means.Gupta et al. showed that JNK phosphorylates addi-tional transcription factors such as ATF2 [39]. VanDam and co-workers found that activated c-Jun andATF2 heterodimerize to stimulate c-jun gene tran-scription by binding to the nonconsensus TPA (12-O-tetradecanoylphorbol 13-acetate) response element onthe c-jun gene [40]. In our experiments (unpublishedobservations), we have found that NO suppressesc-Jun in activated endothelium. Thus, by blocking JNKactivity, NO may suppress subsequent c-jun gene tran-scription, which in turn could lead to suppressed IL-8transcription.

Endothelial cells lining vascular beds secrete proin-flammatory proteins to mediate physiological as wellas injury processes [41]. IL-8 secreted by the endothe-lium mediates adhesion, activation, and migration ofblood neutrophils to a site of injury. Massive IL-8 se-cretion from endothelium carries the potential for dev-astating organ injury. Thus, strategies to attenuate or
modulate IL-8 production are justified. NO, adminis-
tered, as a gas into the lungs of patients with acuterespiratory distress syndrome, produced dramatic im-provement in lung function and oxygenation [42]. Workrecently published from the authors’ laboratoryshowed that inhaled NO gas dramatically attenuatedacute pulmonary vascular inflammation and subse-quent injury resulting from bacterial sepsis in anesthe-tized swine [22].

Thus, early clinical research indicates that NO mayattenuate vascular injury by reduction of IL-8 secre-tion. Recent reports from this laboratory as well asfrom others have shown that NO exerts its anti-inflam-matory effects at the level of NF-kB DNA binding. Wehave advanced this knowledge in the current study bydemonstrating for the first time that NO exerts broadeffects on a variety of different processes that likelysuppress inflammation. Moreover, NO, being a gas, isnot spatially restricted and hence is ideally suited forbringing about these wide-ranging effects. These re-sults are of considerable importance, given the factthat clinical trials examining NO efficacy in humaninflammatory diseases are underway.

This research was supported in part by grants from the NationalInstitutes of Health (HL-61359-01), the American Lung Associationof Virginia/Virginia Thoracic Society, and the Thomas F. Jeffress andKate Miller Jeffress Memorial Trust. The authors are grateful to Dr.Paul Dent, Department of Radiation Oncology at Virginia Common-wealth University, for the generous gift of dominant negative mu-tants of c-Jun.

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eceived January 31, 2001evised version received March 12, 2001ublished online April 24, 2001

nitric oxide suppresses il-8 transcription by inhibiting c-jun n-terminal kinase-induced ap-1...

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