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Originally published In Press as doi:10.1074/jbc.M608197200 on November 29, 2007

J. Biol. Chem., Vol. 283, Issue 4, 1974-1984, January 25, 2008
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Signaling by the Cysteinyl-Leukotriene Receptor 2

INVOLVEMENT IN CHEMOKINE GENE TRANSCRIPTION*

Charles Thompson{ddagger}, Alexandre Cloutier§, Ynuk Bossé{ddagger}, Caroline Poisson{ddagger}, Pierre Larivée§, Patrick P. McDonald§, Jana Stankova{ddagger}, and Marek Rola-Pleszczynski{ddagger}1

From the {ddagger}Immunology Division, Department of Pediatrics, and §Pulmonary Division, Department of Medicine, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

Received for publication, August 25, 2006 , and in revised form, November 18, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-{kappa}B, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-{kappa}B and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative I{kappa}B{alpha} prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-{kappa}B and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKC{epsilon} prevented p65 phosphorylation, whereas dnPKC{delta} prevented AP-1 binding. Moreover, dnPKC{delta}, dnPKC{epsilon}, and dnPKC{zeta} prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-{kappa}B and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cysteinyl-leukotrienes (cysLTs)2 are lipid mediators implicated in several inflammatory processes, including allergy and asthma. Leukotriene (LT) C4, LTD4, and LTE4 act on two G-protein-coupled receptors, CysLT1 and CysLT2 (1–5). Asthmatic patients show increased production of cysLTs (6–9), mainly by eosinophils and mast cells (10). CysLT1 shares 38% homology with CysLT2 and has been shown to promote bronchoconstriction and mucus secretion in response to cysLTs (11–13). Selective antagonists for CysLT1 are in clinical use for the treatment of asthma. Conversely, the function of the CysLT2 receptor is, as yet, poorly defined.

Various reports have suggested a role for CysLT2 in a number of pathologies. The CysLT2 gene is localized on chromosome 13q14, a region linked to atopic asthma (3, 14). Interestingly, a coding polymorphism in the CysLT2 receptor, which decreases responsiveness to LTD4, has been associated with resistance to asthma (15). Furthermore, another report established an association of a CysLT2 variant with atopy in the Tristan da Cunha population (16). An implication of this receptor in vascular permeability and bleomycin-induced pulmonary fibrosis has been suggested, using cyslt2 receptor-null mice (17). Lotzer and colleagues (18) proposed an involvement of the CysLT2 receptor in inflammation and atherogenesis. In this regard, Qiu and colleagues (19) recently demonstrated the up-regulation of CysLT2 receptor expression in carotid plaques of mice susceptible to atherosclerosis (ApoE–/–).

In humans, distribution of CysLT2 mRNA shows a high expression in peripheral blood leukocytes, adrenals, heart, brain, lymph nodes, and spleen (3, 4). In situ hybridization indicates the expression of the CysLT2 receptor in the human lung, specifically in interstitial macrophages (3). Moreover, expression of CysLT2 has been detected on eosinophils, mast cells, and mononuclear cells in the upper airways of seasonal allergic rhinitis patients (20). Affinity of cysLTs for the CysLT2 receptor is LTC4 = LTD4 > LTE4, as demonstrated by binding studies (3, 4) and cell populations that express CysLT1 usually coexpress CysLT2. No selective CysLT2 antagonist is available, however, at this time, making this receptor more difficult to study.

Inflammation is characterized by cellular infiltration, orchestrated by a cytokine/chemokine network. IL-8 (or CXCL8) is an important member of the chemokine superfamily, which plays a major role in the recruitment and activation of polymorphonuclear cells, thus promoting inflammation (21). Several pathologies, including severe asthma and chronic obstructive pulmonary disease, are associated with elevated levels of IL-8 (22–24). Regulation of IL-8 expression usually occurs at the transcriptional level. Its promoter region is well characterized and contains NF-{kappa}B, AP-1, and NF-IL-6 transcription sites (25). NF-{kappa}B is involved in the expression of several genes controlling the immune and inflammatory responses (26). NF-{kappa}B complexes are dimer combinations of Rel family members composed of NF-{kappa}B1(p50), NF-{kappa}B2(p52), RelA(p65), and/or c-Rel (27, 28). Activator Protein 1 (AP-1) is also a dimeric transcription factor, composed of the JUN (c-Jun, JunB, and JunD) and FOS (c-Fos, FosB, Fra1, and Fra-2) families, which regulate the expression of many genes, including IL-8, c-Jun (29), and COX-2 (30). On the other hand, nuclear factor IL-6 (NF-IL-6) also named CCAAT enhancer-binding protein (C/EBP), is usually characterized as a cofactor, cooperating with other transcription factors such as NF-{kappa}B (31–33).

We hypothesized that LTC4 could transcriptionally up-regulate IL-8 and MCP-1 expression via the CysLT2 receptor. Furthermore, we investigated the signaling events following LTC4 stimulation, in the context of IL-8 transcription. Human umbilical vein endothelial cells (HUVEC) at first passage as well as HEK-293 cells stably transfected with CysLT2 were used. Most of the studies that were performed with transfected HEK-293 cells could not have been done with early passage endothelial cells and thus the best surrogate cell model was used. Here, we provide the first evidence that the human CysLT2, in response to LTC4, activates specific PKC isozymes, as well as the NF-{kappa}B and AP-1 transcription factors, and that this activation results in chemokine up-regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—LTC4 and LTD4 were purchased from Cayman Chemical (Ann Arbor, MI). GF109203X and rottlerin were obtained from Biomol Research Laboratory (Plymouth Meeting, PA), and dimethyl sulfoxide (Me2SO) from Fischer Scientific. Specific antibodies against p-I{kappa}B{alpha} Ser-32/36(9246S), p-p65 Ser-536(3031S), and I{kappa}B{alpha} (9242) were from Cell Signaling Technology (Beverly, MA) and p65 (sc-8008X), p65 (sc-372), p50 (sc-7178X), p52(sc-298X), c-Rel (sc-70X), c-Jun (sc-1694X), c-Fos (sc-52X), JunD (sc-74X), JunB (sc-46X), FosB (sc-48X), CREB1 (sc-240X), and CREB2 (sc-200X) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Aprotinin, 4-(2 aminoethyl)benzenesulfonyl fluoride, leupeptin, NaF, soybean trypsin inhibitor, and Na3VO4 were from Sigma; Geneticin (G-418 Sulfate) (Invitrogen Canada Inc.) and FuGENE6 were purchased from Roche.

Plasmids—The plasmid encoding for I{kappa}B{alpha} mutated at serines 32 and 36 was generously provided by Dr. Christian Jobin (University of North Carolina, Chapel Hill, NC). pCMVintron-cmyc-CysLT2R encoding for Myc-labeled human CysLT2 receptor and geneticin resistance, stabilized by a 5' β-globin intron and under the control of a CMV promoter, was constructed in our laboratory. The human IL-8 promoter luciferase constructs IL-8 WT, IL-8-{Delta}AP-1, IL-8-{Delta}NF-{kappa}B, and IL-8-{Delta}NF-IL-6 were generously provided by Dr. Allan R. Brasier from the University of Texas Medical Branch (34). PKC dominant negative (dn) constructs (PKCβII, PKC{delta}, PKC{epsilon}, and PKC{zeta}) were a kind gift from Drs. Gilles Dupuis and Jean-Guy Lehoux (35) from the Université de Sherbrooke; they were shown to be capable of effectively inhibiting selective PKC activities (36).

Cells—Human embryonic kidney (HEK)-293 cells (American Type Culture Collection (ATCC), Rockville, MD) were cultured in Dulbecco's modified Eagle's medium with glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma), and gentamicin sulfate (40 µg/ml). Transient transfections were carried out with FuGENE 6, and experiments were performed 48 h post-transfection.

HEK-293 Stably Expressing CysLT2—HEK-293 cells grown at 50% confluence in 60-mm Petri dishes were stably transfected with 4 µg of pCMVintron-cmyc-CysLT2R using 10 µl of FuGENE 6. Forty-eight h following transfections, geneticin was added at a final concentration of 800 µg/ml. Cells were then cultured for 2 weeks in medium containing geneticin and isolated for clonal selection. Cell clones were analyzed for Myc expression by FACScan flow cytometer (BD Bioscience). Positive clones were maintained in 800 µg/ml geneticin and referred to as 293LT2. Untransfected or empty vector-transfected HEK293 cells expressed neither CysLT1 nor CysLT2 receptors.

HUVEC—Umbilical cords were obtained after uneventful deliveries, following informed consent from the mother. HUVEC were freshly isolated from umbilical veins by collagenase type V digestion as previously described (37). Cells were cultured in Iscove's medium supplemented with fetal bovine serum (20%), endothelial cell grow supplement (VWR, Ville Mont-Royal, QC, Canada), and antibiotics. Experiments were performed with cell cultures at the first passage.

Northern Blot Analysis—Total cellular RNA was extracted using TriPure according to the manufacturer's instructions (Roche Diagnostics), 15 µg of total RNA was separated by electrophoresis on 1% agarose and transferred onto a Hybond N+ (Amersham Biosciences) membrane. Human IL-8 cDNA probe (0.5-kb EcoRI fragment) and human MCP-1 cDNA probe were obtained from the ATCC. 28 S or 18 S were used as internal controls. The probes were labeled with a Ready-to-Go DNA Labeling Beads (dCTP) (Amersham Biosciences) using [{alpha}-32P]dCTP (specific activity 3000 Ci/mmol; Amersham Biosciences). Membranes were prehybridized for 4 h in a mixture containing 120 mM Tris (pH 7.4), 600 mM NaCl, 8 mM EDTA (pH 8), 0.1% sodium pyrophosphate, 0.2% SDS, and 100 µg/ml heparin; hybridization was performed overnight at 71 (IL-8) or 68 °C (MCP-1) in the same mixture in which the concentration of heparin was increased to 625 µg/ml and dextran sulfate at 10% was added. The membranes were then washed once at room temperature for 20 min in 2x SSC (1x SSC: 0.15 M NaCl, 0.15 M sodium citrate (pH 7)) and once with 0.1x SSC. The membranes were exposed to Hyperfilm MP (Amersham Biosciences) with intensifying screens at –80 °C.

Reverse Transcription and Quantitative PCR—For reverse transcription, 1 µg of total RNA was mixed with 0.5 µg of dT oligos (Amersham Biosciences) and 0.5 mM dNTPs (Roche) in Moloney murine leukemia virus reverse transcription buffer and incubated at 65 °C for 5 min. Ten mM dithiothreitol (Invitrogen) and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) were then added and tubes were incubated at 42 °C for 50 min, followed by a 70 °C incubation for 15 min.

For quantitative PCR analysis, 1 µl of reverse transcription product was mixed with 5 µM forward primer, 5 µM reverse primer, 0.5 units of recombinant Taq polymerase (Amersham Biosciences), 0.25 mM dNTP, 1.25 mM MgCl2, and 1:30000 SYBR Green (Molecular Probes) final dilution in supplied Taq buffer, in a final volume of 20 µl. PCR was started with a 3-min hold step at 95 °C, followed by 50 times repeat of the cycling step: 15 s at 95 °C, 20 s at 58 °C, and 20 s at 73 °C (acquiring on Fam/Syb) and ended by a melt step rising from 72 to 95 °C. Experiments were performed on RotorGene 3000 PCR machine from Corbett Research (Montreal Biotech Inc., Kirkland, QC, Canada) using Rotor-gene 6 (build 21) software. Data analysis was performed according to the 2–{Delta}{Delta}CT method as previously described (38, 39). The primer sequences were: GAPDH forward, 5'-GATGACATCAAGAAGGTGGTGAA-3'; GAPDH reverse, 5'-GTGTTACTCCTTGGAGGCCATGT-3'; IL-8 forward, 5'-TCTGCAGCTCTGTGTGAAGG-3'; IL-8 reverse, 5'-AGTTTTCCTTGGGGTCCAGA-3'.

Western Blot Analysis—293LT2 cells in 6-well plates were incubated in medium without serum for 24 h, stimulated with LTC4 (20 nM) for the indicated times, and lysed in buffer: 50 mM Tris (pH 7.5), 1 mM EGTA, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 0.25% sodium deoxycholate, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, for 30 min on ice. Total lysates were separated on 10% SDS-PAGE, and transferred to Trans-Blot nitrocellulose membrane (Bio-Rad). Membranes were blocked with Tris-buffered saline with 5% dry milk for 1 h and incubated with specific antibodies in Tris-buffered saline, 0.1% Tween, and 5% dry milk overnight at 4 °C. After washing and incubation with secondary antibodies, an ECL detection system was used for protein detection (Amersham Biosciences). Membranes were stripped by incubation in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 10 mM 2-mercaptoethanol for 30 min at 50 °C. After washing, membranes were reprobed with the appropriate antibodies and developed as described above.

Electrophoretic Mobility Shift Assay (EMSA)—293LT2 cells were cultured in 6-well plates until nearly confluent; cells were starved overnight. Cells were then stimulated with LTC4, LTD4, or EtOH for the indicated times and incubations were stopped by adding an equal volume of ice-cold phosphate-buffered saline containing 10 mM NaF and 1 mM Na3VO4. Cells were collected by gentle scraping and centrifuged at 1000 x g for 3 min at 4 °C. The resulting cell pellets were resuspended in ice-cold lysis buffer (10 mM HEPES (pH 7.90), 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM EGTA, and 0.5 mM dithiothreitol) containing an antiprotease mixture (0.5 mM diisopropyl fluorophosphate, 0.5 mM 4-(2 aminoethyl)benzenesulfonyl fluoride, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, final concentrations). After a 10-min incubation on ice, an equal volume of lysis buffer containing the antiprotease mixture as well as 0.2% Nonidet P-40 was added (to yield a final concentration of 0.1% Nonidet P-40). Samples were immediately vortex mixed for 15 s before centrifugation at 1200 x g (5 min at 4 °C). The resulting nuclear pellets were washed once with lysis buffer containing the antiprotease mixture before being resuspended in ice-cold nuclear extraction buffer (20 mM HEPES (pH 7.90), 400 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, and 10% (v/v) glycerol) containing the antiprotease mixture. After a 20-min incubation on ice (with frequent mixing), samples were spun (15,000 x g for 15 min at 4 °C), and supernatants (the nuclear extracts) were snap-frozen in liquid nitrogen and stored at –80 °C. Extracts were routinely processed for protein content determination. The sequences of the sense strands of the oligonucleotides used for EMSA were as follows: 5'-AGTTGAGGGGACTTTCCCAGGC-3' (NF-{kappa}B), 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1) from Promega (Madison, WI)) and 5'-AGTTGAGGCGACTTTCCCAGGC-3' (NF-{kappa}B mutant) 5'-CGCTTGATGACTTGGCCGGAA-3' (AP-1 mutant) from Santa Cruz Biotechnology. For supershift experiments, binding reactions were conducted in the presence of specific antisera to individual c-Jun, JunB, JunD, c-Fos, FosB, CREB1, CREB2 or p65, p50, p52, c-Rel proteins (30 min at 4 °C), before the addition of {gamma}-32P-labeled probes. Samples were electrophoresed on 6% acrylamide gels at 4 °C in 0.5x TBE; dried gels were then exposed to Hyperfilm MP (Amersham Biosciences) with intensifying screens at –80 °C.

Luciferase Assays—293LT2 cells were plated in 12-well tissue culture plates 24 h before transfection with 0.5 µg of plasmid DNA per well, using 1.5 µl of FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions. The day after transfection, cells were serum-starved overnight before stimulation with LTC4 (20 nM) or EtOH for 6 h. Cell lysates were assayed for luciferase activity as previously described (40).

Enzyme-linked Immunosorbent Assay—293LT2 cells were cultured in 12-well culture plates and serum-starved overnight before stimulation with LTC4 for the indicated times. Cell-free culture supernatants were carefully collected, snap-frozen in liquid nitogen, and stored at –80 °C. IL-8 concentrations were determined using the Opt-EIA Human IL-8 enzyme-linked immunosorbent assay kit (BD Pharmingen).

Statistical Analyses—Where mentioned, statistical significance was assessed using the Student's t test for paired data (one-tailed) or analysis of variance, as required, using PRISM4 software (GraphPad Software Inc., San Diego, CA). Differences were considered significant at p ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LTC4 Regulates IL-8 and MCP-1 Expression in 293LT2 Cells—Because several primary cell types express both CysLT2 and CysLT1 receptors and because of a lack of selective CysLT2 antagonists, we generated a cell line that stably expresses only the CysLT2 receptor, referred to as 293LT2 cells, as described under "Experimental Procedures." This provided a useful tool to selectively study the signaling pathways downstream of the CysLT2 receptor. Many of the studies that were performed with the 293LT2 cells could not have been done with early passage primary cells and thus the best surrogate cell model was used. Untransfected or empty vector-transfected HEK293 cells expressed neither CysLT1 nor CysLT2 receptors. Time course experiments were conducted to elucidate the effects of LTC4 on IL-8 and MCP-1 expression in 293LT2 cells. As shown in Fig. 1A, LTC4 induced IL-8 mRNA expression within 1 h of stimulation, with a maximum after 8 h. The effect of LTC4 on IL-8 mRNA was rapid, but transient, as demonstrated by a reduced expression after 24 h of stimulation. Kinetics of MCP-1 mRNA expression in response to LTC4 are shown in the middle panel of Fig. 1A. Increased IL-8 protein expression in response to LTC4 was also time-dependent, starting at 2 h and maintained through 24 h of LTC4 stimulation (Fig. 1B). Empty vector-transfected HEK293 cells showed no response to LTC4 stimulation (data not shown).


Figure 1
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  		FIGURE 1.
LTC4 induces time-dependent IL-8 and MCP-1 synthesis in 293LT2 cells. A, 293LT2 cells were stimulated with LTC4 (20 nM) or EtOH for the indicated times. IL-8 and MCP-1 mRNA expression is shown by Northern blot analysis using specific human IL-8 and MCP-1 probes. Data are representative of three independent experiments. B, 293LT2 cells were treated with LTC4 (20 nM) or EtOH at the indicated times and supernatants were collected to measure IL-8 protein by enzyme-linked immunosorbent assay. Data are expressed as the mean ± S.E.; n=3.

 
LTC4 and LTD4 Induce IL-8 Promoter Activity in a Concentration-dependent Manner—To determine whether LTC4-induced IL-8 gene expression occurs at the transcriptional level, 293LT2 cells were transfected with a luciferase reporter gene driven by the IL-8 promoter and the promoter activity was measured following stimulation with LTC4 or LTD4. As shown in Fig. 2A, LTC4 strongly up-regulated IL-8 transactivation in a concentration-dependent manner, with a maximal effect at 200 nM LTC4 for 6 h. LTD4 also induced IL-8 transactivation, with 200 nM giving the highest induction (Fig. 2A). Interestingly, LTC4 was more potent than LTD4 and induced equivalent IL-8 promoter activity at 10-fold lower concentrations. IL-8 promoter activity in response to cysLT stimulation was not induced in untransfected HEK293 cells (data not shown).


Figure 2
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  		FIGURE 2.
CysLTs increase IL-8 promoter activity: implication of NF-{kappa}B, AP-1, and NF-IL-6 binding sites. A, 293LT2 cells were transiently transfected with 0.5 µg/well pOLUC (promoterless vector) or pIL-8-WT promoter constructs. Cells were incubated for 6 h with EtOH, LTC4, or LTD4 at the indicated concentrations before measurement of luciferase activity. B, 293LT2 cells were transiently transfected with 0.5 µg/well pOLUC (promoterless vector), IL-8-WT, IL-8-{Delta}NF-{kappa}B, IL-8-{Delta}AP-1, or IL-8-{Delta}NF-IL-6 promoter constructs. Cells were incubated for 6 h with EtOH or LTC4 at 20 nM before measurement of the luciferase activity. Data are expressed as -fold increase relative to EtOH control. ***, p< 0.001 relative to wild-type control, using Student's t test; n = 3.

 
LTC4-induced IL-8 Promoter Activity Is Dependent on NF-{kappa}B, AP-1, and NF-IL-6 Sites—To determine the transcription factor binding sites involved in the induction of IL-8 transactivation in response to LTC4, site-specific mutants of the 5'-flanking region of the IL-8 gene were used (34). As shown in Fig. 2B, the NF-{kappa}B site mutation reduced drastically (by 90%) the IL-8 promoter activity induced by LTC4. Mutation within the AP-1 site resulted in a 75% reduction of the LTC4-induced IL-8 transactivation, whereas the NF-IL-6 site mutation caused only a 40% reduction.

LTC4 Activates Different NF-{kappa}B Pathway Elements—Because we observed the involvement of NF-{kappa}B, we addressed the elements of this pathway that could be activated by LTC4. Fig. 3A illustrates the induction of I{kappa}B{alpha} phosphorylation following LTC4 stimulation with a maximal induction after 15 min. As also shown in Fig. 3A (top panel), LTC4 induced a time-dependent phosphorylation of the p65 NF-{kappa}B subunit, on serine 536. To assess whether I{kappa}B{alpha} activation was required for LTC4-induced IL-8 promoter transactivation, 293LT2 cells were cotransfected with a dominant-negative form of I{kappa}B{alpha} and the IL-8-luc reporter construct and then stimulated with LTC4. As illustrated in Fig. 3B, overexpression of dominant-negative I{kappa}B{alpha} inhibited IL-8 promoter activity by 90% in response to LTC4.


Figure 3
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  		FIGURE 3.
The NF-{kappa}B pathway is involved in the induction of IL-8 promoter activity by LTC4. A, 293LT2 cells were stimulated with LTC4 or EtOH for the indicated times. Total proteins were separated by electrophoresis and Western blots were performed using the indicated antibodies. At the bottom of the panel, total p65 protein expression served as internal control. B, transient transfection of 293LT2 cells with 0.5 µg/well pIL-8-WT and pcDNA or dominant negative I{kappa}B{alpha} construct. Cells were incubated for 6 h with EtOH or LTC4 at 20 nM before measurement of luciferase activity. Data are expressed as -fold increase relative to EtOH control, ***, p< 0.001 relative to pcDNA-transfected cells stimulated with LTC4 using Student's t test; n = 3.

 
LTC4 Increases NF-{kappa}B DNA Binding Activity—Based on the above results, we investigated whether LTC4 could up-regulate NF-{kappa}B DNA binding. Fig. 4A illustrates the kinetics of LTC4-induced NF-{kappa}B DNA binding, indicating an induction that could be seen within 15 min of stimulation. As seen above (Fig. 2A), LTC4 was also a better agonist than LTD4 in inducing NF-{kappa}B binding (Fig. 4A). Analysis of the DNA binding complex following LTC4 stimulation was performed using specific antibodies. Supershift experiments indicated that the NF-{kappa}B binding complex contained p50 and p65 subunits (Fig. 4B).

LTC4 Stimulates the DNA Binding of AP-1—We also investigated whether LTC4 stimulation of the 293LT2 cells could modulate AP-1 DNA binding activity. As shown in Fig. 5A, 293LT2 cells exposed to 20 nM LTC4 for the indicated periods of time showed a time-dependent induction of AP-1 DNA binding activity, which was evident at 60 min. Fig. 5A also indicates a stronger induction of AP-1 DNA binding activity by LTC4 than by LTD4. Supershift experiments showed that the AP-1 binding complex was composed of c-JUN and c-FOS proteins (Fig. 5B).


Figure 4
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  		FIGURE 4.
LTC4 stimulation induces NF-{kappa}B binding. A, 293LT2 cells were cultured for the indicated times in the presence of LTC4 (20 nM), LTD4 (20 nM), or EtOH before nuclear extraction and EMSA analysis. B, 293LT2 cells were stimulated with LTC4 for 60 min prior to nuclear extract preparation. Nuclear extracts were analyzed by EMSA using a consensus NF-{kappa}B oligonucleotide radiolabeled probe. The specificity of complex formation was tested by the inclusion of unlabeled competitors (cold probe, eighth lane; or cold mutated probe, ninth lane), by including specific anti-p50 (third lane), anti-p52 (fourth lane), anti-p65 (fifth lane), or anti-c-Rel (sixth lane) antibodies or an isotype-matched control antibody (seventh lane). Experiments were performed 3 times and representative data are shown.

 


Figure 5
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  		FIGURE 5.
LTC4 up-regulates AP-1 DNA binding activities. A, 293LT2 cell were cultured in the presence of LTC4 (20 nM), LTD4 (20 nM), or EtOH for the indicated times prior to nuclear extract preparation. Nuclear extracts were analyzed in EMSA using a consensus AP-1 oligonucleotide radiolabeled probe. B, cells were stimulated with 20 nM LTC4 or EtOH for 60 min before EMSA analysis. The specificity of complex formation was tested by the inclusion of unlabeled competitors (cold probe, eleventh lane1; or cold mutated probe, twelfth lane), by including specific anti-c-Jun (third lane 3), anti-JunD (fourth lane), anti-JunB (fifth lane), anti-c-Fos (sixth lane), anti-FosB (seventh lane), anti-CREB1 (eighth lane), or anti-CREB2 (ninth lane) antibodies or an isotype-matched control antibody (tenth lane). Experiments were performed 3 times, and representative data are shown.

 


Figure 6
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  		FIGURE 6.
JNK is not involved in LTC4-induced c-JUN phosphorylation. A, 293LT2 cells were pretreated or not with SP600125 at 1, 10, or 20 µM for 30 min followed by stimulation with LTC4 (20 nM) or EtOH for 60 min. Total proteins were separated by electrophoresis and Western blots were performed using anti-phospho-c-JUN and anti-p65 antibodies. Total p65 protein expression served as internal control. Results are representative of three separate experiments. B, 293LT2 cells were pretreated or not with SP600125 at 20 µM for 30 min followed by stimulation with LTC4 (20 nM) or EtOH for 60 min. Total proteins were separated by electrophoresis and Western blots were performed using anti-phospho-JNK and anti-JNK antibodies. Total JNK protein expression served as internal control. Results are representative of three separate experiments. C, cells were pretreated or not with SP600125 (10 or 20µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 6 h before measurement of luciferase activity. Data are expressed as -fold increase relative to EtOH control; n = 3.

 
LTC4 Induces the Phosphorylation of c-JUN Independently of JNK—Phosphorylation of c-Jun is necessary for full activation of the AP-1 complex. Because we observed the involvement of the c-JUN·c-FOS complex in stimulated 293LT2 cells, we next assessed whether c-JUN could be phosphorylated following stimulation with LTC4. As shown in Fig. 6A, LTC4 induced a marked phosphorylation of c-JUN. c-Jun N-terminal kinase (JNK) is a well characterized enzyme usually responsible for c-JUN phosphorylation. We thus assessed whether SP600125, a specific JNK inhibitor, could inhibit LTC4-induced c-JUN phosphorylation. As illustrated in Fig. 6A, graded concentrations of the JNK inhibitor did not prevent c-JUN phosphorylation induced by LTC4, even at 20 µM SP600125. The efficacy of the JNK inhibitor was confirmed, however, by its capacity to inhibit JNK phosphorylation induced by LTC4 (Fig. 6B). SP600125 was also unable to prevent LTC4-induced IL-8 transactivation, as shown in Fig. 6C. Interestingly, LTC4 thus appears to be an inducer of JNK activation, but this enzyme is not involved in LTC4-induced IL-8 expression.


Figure 7
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  		FIGURE 7.
PKC regulates LTC4-induced IL-8 transactivation. A, 293LT2 cells were pretreated or not with rottlerin (10 µM), GF109203X (2 µM) or Me2SO (DMSO) (vehicle) for 30 min and were then stimulated with LTC4 (20 nM) or EtOH for 6 h before measurement of luciferase activity. B, 293LT2 cells were stimulated with LTC4 (20 nM) or EtOH for 3 or 15 min. Total proteins were separated by electrophoresis and a Western blot was performed using anti-phospho-PKC{delta} or anti-p65 antibodies (n = 2). C, transient transfection of 293LT2 cells with 0.5 µg/well pIL-8-WT and pcDNA or dominant negative PKCβ, PKC{delta}, PKC{epsilon}, and PKC{zeta} constructs. Cells were incubated for 6 h with EtOH or LTC4 (20 nM) before measurement of luciferase activity. Data are expressed as -fold increase relative to EtOH control, **, p < 0.01, or ***, p < 0.001 relative to Me2SO in A and mock-transfected cells in C using Student's t test; n = 3.

 


Figure 8
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  		FIGURE 8.
PKC{delta} is involved in LTC4-induced activation of the AP-1 pathway. A, 293LT2 cells were pretreated or not with rottlerin (5 and 10 µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 60 min for phosphorylation of c-Jun. Total proteins were separated by electrophoresis and Western blots were performed using the indicated antibodies. B, 293LT2 cells were pretreated with rottlerin (10 µM), GF109203X (2 µM), or SP600125 (20 µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 60 min prior to nuclear extract preparation. The nuclear extracts were analyzed by EMSA using a consensus AP-1 oligonucleotide radiolabeled probe. C, 293LT2 cells were pretreated or not with rottlerin (10 µM) or GF109203X (2 µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 30 min to analyze phosphorylation of JNK. Total proteins were separated by electrophoresis and Western blots were performed using the indicated antibody. D, 293LT2 cells were transfected with empty vectors or with dominant-negative forms of PKC{epsilon}, PKC{delta}, or PKC{zeta} and then stimulated with LTC4 (20 nM) or EtOH for 60 min prior to nuclear extract preparation. EMSA was performed as in B, n = 3.

 
Protein Kinase C Is Important in CysLT2-mediated IL-8 Transactivation—Because JNK was not involved in LTC4-induced IL-8 expression, we investigated whether PKC could play a role in AP-1-dependent IL-8 transactivation. HEK293 cells are known to express endogenous PKCβI, PKCβII, PKC{delta}, and to a lesser extent PKC{epsilon} and PKC{zeta} isozymes (41). Pretreatment of 293LT2 cells with the broad-spectrum PKC inhibitor, GF109203X, or the PKC{delta}-specific inhibitor, rottlerin, significantly abrogated LTC4-induced IL-8 promoter activation (Fig. 7A). LTC4 was also found to induce PKC{delta} phosphorylation in a time-dependent manner (Fig. 7B). Furthermore, because of the potential risk for erroneous interpretations when using biochemical inhibitors, we resorted to dominant negative forms of PKC isoforms. As illustrated in Fig. 7C, specific dominant-negative constructs for PKC{delta}, PKC{epsilon}, and to a lesser extent, PKC{zeta} also reduced LTC4-induced IL-8 transactivation, thus suggesting the implication of these PKC isozymes in LTC4-induced IL-8 gene transcription. In contrast, the dominant-negative PKCβII construct only had a minor impact on LTC4-induced IL-8 transcription (Fig. 7C).

PKC{delta} Is Required for LTC4-induced AP-1 Pathway Activation—Interestingly, LTC4-induced phosphorylation of c-JUN was inhibited by rottlerin (Fig. 8A), suggesting that PKC{delta} was required for c-JUN activation. This was also reflected in LTC4-induced AP-1 DNA binding (Fig. 8B), with inhibition with rottlerin and GF109203X, but not with the JNK inhibitor SP600125. Furthermore, GF109203X, but not rottlerin prevented LTC4-induced phosphorylation of JNK (Fig. 8C). Finally, when 293LT2 cells were transfected with dnPKC isozymes, only PKC{delta} was capable of inhibiting LTC4-induced AP-1 DNA binding (Fig. 8D). We were unable, however, to prevent c-Jun phosphorylation using any of the dnPKC isozymes (data not illustrated).

LTC4-mediated NF-{kappa}B Activation Involves PKC{epsilon}—LTC4 was found to phosphorylate p65 in a CysLT2-dependent and a PKC-dependent manner, but without inhibition by rottlerin, suggesting that a PKC isozyme other than PKC{delta} would be involved (Fig. 9A). This was also observed in LTC4-induced NF-{kappa}B DNA binding (Fig. 9B). However, 293LT2 cells transfected with dominant-negative forms of PKC{epsilon}, PKC{delta}, or PKC{zeta} revealed that LTC4-induced phosphorylation of p65 was only dependent on PKC{epsilon} (Fig. 9C). In contrast, neither of the dominant-negative isoforms of PKC inhibited LTC4-induced NF-{kappa}B DNA binding (Fig. 9D).


Figure 9
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  		FIGURE 9.
PKC{epsilon} is involved in LTC4-induced activation of the NF-{kappa}B pathway. A, 293LT2 cells were pretreated or not with GF109203X (2 µM) or rottlerin (10 µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 15 min to analyze phosphorylation of p65. Total proteins were separated by electrophoresis and Western blotted using the indicated antibodies. B, 293LT2 cells were pretreated or not with GF109203X (2 µM), rottlerin (10 µM), or SP600125 (20 µM) for 30 min and then stimulated with LTC4 (20 nM) or EtOH for 60 min prior to nuclear extract preparation. The nuclear extracts were analyzed in EMSA using a consensus NF-{kappa}B oligonucleotide radiolabeled probe. C, 293LT2 cells were transfected with empty vectors or with dominant-negative forms of PKC{epsilon}, PKC{delta}, or PKC{zeta} and then stimulated with LTC4 (20 nM) or EtOH for 15 min to analyze phosphorylation of p65, as in A. D, 293LT2 cells were transfected with empty vectors or with dominant-negative forms of PKC{epsilon}, PKC{delta}, or PKC{zeta} and then stimulated with LTC4 (20 nM) or EtOH for 60 min prior to nuclear extract preparation. EMSA was performed as in B, n = 3.

 
LTC4 Induces IL-8 Gene Expression in Human Endothelial Cells—HUVEC readily express the CysLT2 receptor, with very low expression of CysLT1. The effects of cysLTs on HUVEC have been shown to be the consequence of CysLT2 activation, because specific CysLT1 antagonists do not prevent cysLT stimulation (18). As shown in Fig. 10A, LTC4 induced a time-dependent expression of IL-8 mRNA in HUVEC, with maximal effects observed following 2 h of LTC4 stimulation. This was not prevented by pretreatment with the CysLT1 receptor antagonist montelukast (data not illustrated). Furthermore, as shown in Fig. 10B, pretreatment of HUVEC with the broad-spectrum PKC inhibitor, GF109203X, completely abrogated LTC4-induced IL-8 transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cysteinyl-leukotrienes are lipid mediators with a proinflammatory profile and have been implicated in the pathogenesis of several types of inflammation. The role of the CysLT1 receptor is being extensively investigated and characterized. Conversely, the mechanisms responsible for inflammation driven by the CysLT2 receptor have not been reported as yet. This may be due, in part, to the fact that many cell types express both CysLT2 and CysLT1 receptors and to the lack of selective antagonists for CysLT2.

In the present study, we demonstrate that LTC4, acting through the CysLT2 receptor, can directly activate the production of chemokines IL-8 and MCP-1. These are potent mediators of inflammation and play key roles in the recruitment and activation of several immune cell populations. High levels of IL-8 have been reported in several pathologies, including severe asthma and chronic obstructive lung disease (22–24). Our results indicate that LTC4 induces a time-dependent expression of IL-8 at both the mRNA and protein levels and also induces a concentration-dependent transactivation of the IL-8 promoter. Interestingly, although LTC4 and LTD4 have equal binding affinity for CysLT2 (3, 4), we found LTC4 to be ~10-fold more potent than LTD4 for CysLT2 receptor activation at low nanomolar concentrations. This may be the result of different conformational changes induced in the CysLT2 receptor by the two structurally different ligands, despite their equivalent affinities.

A major aim of this work was to investigate the signaling pathways that could be activated via the CysLT2 receptor. Many of the assays were performed with transfected HEK-293 cells stably expressing CysLT2, because they could not have been done with first passage HUVEC and thus the best surrogate cell model was used.

NF-{kappa}B is a powerful transcription factor involved in the activation of many inflammatory genes, including the IL-8 gene, following stimulation by LPS or proinflammatory cytokines such as TNF-{alpha} and IL-1β (42). NF-{kappa}B has also been reported to be activated by G-protein-coupled receptor signaling (43, 44). Our data show that a mutation in the NF-{kappa}B site within the IL-8 reporter construct reduced the IL-8 promoter activity induced by LTC4 in 293LT2 cells by 90%, indicating a major role for the NF-{kappa}B transcription factor in CysLT2 signaling.

In unstimulated cells, the NF-{kappa}B complex is maintained in an inactive form by the binding of p65 to its inhibitory factor I{kappa}B{alpha} (45). Following activation, IKK phophorylates I{kappa}B{alpha} on serines 32 and 36, leading to rapid polyubiquitination and degradation of I{kappa}B{alpha} in the proteasome (46, 47). Free p65 subunits can then translocate to the nucleus and activate gene transcription. Our results demonstrate that LTC4 can induce the phosphorylation of I{kappa}B{alpha}, in a time-dependent manner, with a maximal induction at 15 min. Furthermore, LTC4 is also able to stimulate the phosphorylation of p65 at serine 536. Our data show as well that expression of a dominant-negative form of I{kappa}B{alpha} in 293LT2 cells abrogates by 90% LTC4-induced IL-8 promoter transactivation, indicating a major role for I{kappa}B{alpha} in CysLT2 receptor signaling. NF-{kappa}B is a homo- or heterodimer composed of different combinations of Rel family members. We determined by supershift assays that LTC4 induced the formation of a functional p50·p65 NF-{kappa}B complex.


Figure 10
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  		FIGURE 10.
LTC4 induces a time-dependent expression of IL-8 mRNA in HUVEC. A, HUVEC were treated with LTC4 (200 nM) or ethanol at the indicated times. Quantification of IL-8 mRNA levels was performed by real-time PCR. Data are expressed as -fold increase relative to the housekeeping gene GAPDH, mean ± S.E.; n = 3. Asterisks indicate p < 0.05. B, HUVEC were pretreated or not with GF109203X (2 µM) for 30 min and then stimulated with LTC4 (200 nM) or EtOH for 2 h. Quantifications were performed as in A.

 
Interestingly, AP-1 was also implicated in CysLT2 receptor signaling. AP-1 responsive elements are part of many gene promoters, including IL-8 (34), COX-2 (30), and c-Jun (29). We initially demonstrated that an AP-1 site mutation decreased by 75% the IL-8 promoter activity stimulated by LTC4. Next we showed that LTC4 strongly induced DNA binding of AP-1. Furthermore, our results provided the first evidence that LTC4 induces the formation of a functional c-JUN·c-FOS complex. The AP-1 complex can be regulated by a post-translational phosphorylation, which can influence the stability of its members, its transactivating potential, and its DNA-binding capacity (48). Here we showed a strong phosphorylation of c-JUN, following 60 min of LTC4 stimulation. Interestingly, although LTC4 stimulation also resulted in phosphorylation of JNK, which is usually responsible for c-JUN phosphorylation, inhibition of JNK activity neither affected c-Jun phosphorylation nor LTC4-induced IL-8 promoter transactivation. With the use of dominant-negative PKC isozymes, it appears that AP-1 activation and DNA binding are mediated through PKC{delta}, independently of JNK activation. At this time, we have no explanation for the apparent lack of involvement of JNK in LTC4-induced c-Jun phosphorylation and AP-1 DNA binding. Results from our laboratory, however, indicate that phosphorylation of other members of the mitogen-activated protein kinase family, namely p38 and Erk, also occurs in response to LTC4 despite their lack of implication in IL-8 transactivation.3 Interestingly, SP600125 was capable of inhibiting LTC4-induced JNK phosphorylation, possibly by inhibiting its autophosphorylation.

We studied whether PKC could be an upstream element of the CysLT2 signaling leading to IL-8 transactivation. PKCs are serine/threonine kinases that play a role in several signaling pathways implicated in cell proliferation, apoptosis, and gene activation (49, 50). The PKC family is divided into three classes of isoenzymes, the conventional PKC isoforms {alpha}, βI, βII, and {gamma} regulated by Ca2+ and diacylglycerol, the novel PKC isoforms {delta}, {epsilon}, {eta}, and {theta} dependent on diacylglycerol but not on Ca2+ and the atypical PKC isoforms {iota}, {lambda}, and {zeta}, which are both Ca2+- and diacylglycerol-independent. Several reports indicate that PKC is an upstream effector of NF-{kappa}B activating pathways leading to chemokine secretion (51, 52). Studies by Thodeti and colleagues (53) indicate that LTD4 can activate PKC{alpha}, {delta}, and {epsilon}, but not βII, in intestinal epithelial cells, a mechanism triggered by the CysLT1 receptor. Our results demonstrate an implication of PKCs in IL-8 transactivation, based on the use of dominant-negative isoforms of PKCs and biochemical inhibitors: GF109203X, a broad PKC inhibitor, and rottlerin, a reportedly selective PKC{delta} inhibitor. We also found PKC{delta} to be an upstream element of the AP-1 pathway, because both rottlerin and dnPKC{delta} inhibited AP-1 DNA binding. Although rottlerin pretreatment could also abrogate c-Jun phosphorylation following LTC4 stimulation, neither dnPKC{delta}, -{epsilon}, nor -{zeta} could. At this time, the kinase responsible for LTC4-induced c-Jun phosphorylation remains to be identified. Such a rottlerin-sensitive, but PKC{delta}-independent signaling pathway has recently been reported in B lymphocyte receptor signaling (54).

Interestingly, neither rottlerin nor dnPKC{delta} affected the NF-{kappa}B pathway, in terms of p65 phosphorylation and NF-{kappa}B DNA binding. Conversely, in other G-protein-coupled receptors signaling, PKC{delta} was reported to mediate lysophosphatidic acid- and substance P-induced NF-{kappa}B activation (51, 52).

Upstream elements affecting the NF-{kappa}B pathway following CysLT2 receptor stimulation appear to involve PKC{epsilon}. Indeed GF109203X, but not rottlerin, was able to inhibit NF-{kappa}B pathway activation by LTC4 and dnPKC{epsilon} inhibited both p65 phosphorylation and IL-8 transactivation following LTC4 stimulation. In this regard, a recent study indicated that PKC{epsilon} was required for formylmethionylleucylphenylalanine-induced activation of NF-{kappa}B (55). Interestingly, neither PKC{epsilon}, PKC{delta}, nor PKC{zeta} significantly affected LTC4-induced NF-{kappa}B DNA binding. It is known, however, that p65 phosphorylation is not essential for its translocation to the nucleus and its binding to DNA, but important for its enhanced transactivating effect (56, 57).


Figure 11
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  		FIGURE 11.
Schematic representation of CysLT2 signaling mechanisms involved in LTC4-induced chemokine production. LTC4 binding to the CysLT2 receptor results in activation of the AP-1 and NF-{kappa}B pathways, involving PKC family kinases. LTC4 induces an AP-1 complex composed of c-Jun and c-Fos, which binds to the TRE (AP-1) site to induce chemokine transcription. AP-1 pathway activation can be abrogated by rottlerin, a PKC{delta} inhibitor. LTC4 also induces the phosphorylation of I{kappa}B{alpha} and p65, and activates an NF-{kappa}B complex formed by p50/p65 subunits, which also participates in chemokine gene transcription. Dominant negative forms of PKC{epsilon} prevent LTC4-induced p65 phosphorylation and NF-{kappa}B binding to DNA. Dashed arrows represent hypothetical pathways and GF (GF109203X).

 
We have also found that dnPKC{zeta} partially prevented LTC4-induced IL-8 transactivation, but PKC{zeta} appeared not to play a role in either AP-1 or NF-{kappa}B activation following LTC4 stimulation. Several reports, however, suggest that PKC{zeta} may be involved in the activation of the NF-{kappa}B pathway. For instance, overexpression of PKC{zeta} in NIH-3T3 fibroblasts provoked a permanent translocation of active NF-{kappa}B into the nucleus (58) and PKC{zeta} has been shown to phosphorylate the p65 subunit and IKKβ (59–61).

Finally, we demonstrated the induction of IL-8 transcripts by LTC4 in primary HUVEC, which was not prevented by the CysLT1 antagonist montelukast, but was abrogated by PKC inhibition. Lotzer and colleagues (18) reported that HUVEC predominantly expressed a functional CysLT2 receptor, rather than CysLT1, as evidenced by a calcium flux in response to LTD4 that was not blocked by CysLT1 antagonists. Moreover, HUVEC express high levels of CysLT2 mRNA, but very few transcripts of CysLT1 receptor (62). During the preparation of this manuscript, Uzonyi and colleagues (63) reported that CysLT2 and protease-activated receptor 1 can activate early genes, including IL-8, in endothelial cells. Our results corroborate and extend these data. Indeed, the major findings of the present study are the definition of some of the signaling pathways induced by the CysLT2 receptor, which had not been reported as yet.

CysLT1 and CysLT2 receptors share similar types of G-protein signaling and could therefore use similar downstream signaling pathways. Indeed, we have recently shown that LTD4-induced CysLT1 activation can also lead to IL-8 production, but with a predominant involvement of AP-1 and no involvement of NF-IL-6 (40).

In summary, we provide evidence for a major involvement of the PKC isozymes {delta} and {epsilon} and transcription factors NF-{kappa}B and AP-1 in CysLT2 receptor signaling that leads to chemokine gene activation. Fig. 11 summarizes the CysLT2 signaling cascade that we found in this study. Taken together, our data suggest the potential for the CysLT2 receptor to induce additional proinflammatory genes, via its capacity to activate NF-{kappa}B and AP-1 transcription factors.


   FOOTNOTES
 
* This work was supported in part by grants (to P. P. McD., J. S., and M. R.-P.) and studentships (to C. T., A. C., and Y. B.) from the Canadian Institutes of Health Research and a grant from the Foundation for Research into Children's Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a Canada Research Chair in Inflammation. To whom correspondence should be addressed: 3001 North 12th Ave., Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-346-1110 (ext: 14851); Fax: 819-564-5215; E-mail: marek.rola-pleszczynski{at}usherbrooke.ca.

2 The abbreviations used are: CysLT, cysteinyl-leukotriene; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4; IL-8, interleukin-8; MCP-1, monocyte chemotactic protein 1; COX-2, cyclooxygenase 2; HEK293, human embryonic kidney 293; AP-1, activator protein-1; NF, nuclear factor; HUVEC, human umbilical vein endothelial cells; dn, dominant negative; CMV, cytomegalovirus; PKC, protein kinase C; EMSA, electrophoretic mobility shift assay; JNK, c-Jun N-terminal kinase; C/EBP, CCAAT enhancer-binding protein. Back

3 C. Thompson, A. Cloutier, Y. Bossé, C. Poisson, P. Larivée, P. P. McDonald, J. Stankova, and M. Rola-Pleszczynski, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Allan R. Brasier (U. of Texas) for the IL-8 promoter constructs and Dr. Christian Jobin for the dominant negative form of I{kappa}B{alpha}. PKC dominant negative constructs were a kind gift from Drs. Gilles Dupuis and Jean-Guy Lehoux (Universitéde Sherbrooke).



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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