T-cell-derived Interleukin-17 Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of the p38 Mitogen-activated Protein Kinase Cascade

ROLE OF DISTAL SEQUENCES IN THE 3'-UNTRANSLATED REGION OF COX-2 mRNA*

Wissam H. Faour {ddagger}, Arturo Mancini §, Qing Wen He ¶ and John A. Di Battista § ¶ ||

From the {ddagger}Molecular Biology Program, University of Montreal, and the Departments of Medicine and §Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 1A1, Canada

Received for publication, December 16, 2002 , and in revised form, May 12, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although interleukin-17 (IL-17) is the pre-eminent T-cell-derived pro-inflammatory cytokine, its cellular mechanism of action remains poorly understood. We explored novel signaling pathways mediating IL-17 induction of the cyclooxygenase-2 (COX-2) gene in human chondrocytes, synovial fibroblasts, and macrophages. In preliminary work, recombinant human (rh) IL-17 stimulated a rapid (5–15 min), substantial (>8-fold), and sustained (>24 h) increase in COX-2 mRNA, protein, and prostaglandin E2 release. Screening experiments with cell-permeable kinase inhibitors (e.g. SB202190 and p38 inhibitor), Western analysis using specific anti-phospho-antibodies to a variety of mitogen-activated protein kinase cascade intermediates, co-transfection studies using chimeric cytomegalovirus-driven constructs of GAL4 DNA-binding domains fused to the transactivation domains of transcription factors together with Gal-4 binding element-luciferase reporters, ectopic overexpression of activated protein kinase expression plasmids (e.g. MKK3/6), or transfection experiments with wild-type and mutant COX-2 promoter constructs revealed that rhIL-17 induction of the COX-2 gene was mediated exclusively by the stress-activated protein kinase 2/p38 cascade. A rhIL-17-dependent transcriptional pulse (1.76 ± 0.11-fold induction) was initiated by ATF-2/CREB-1 transactivation through the ATF/CRE enhancer site in the proximal promoter. However, steady-state levels of rhIL-17-induced COX-2 mRNA declined rapidly (<2 h) to control levels under wash-out conditions. Adding rhIL-17 to transcriptionally arrested cells stabilized COX-2 mRNA for up to 6 h, a process compromised by SB202190. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that rhIL-17 increased reporter gene mRNA stability and protein synthesis via distal regions (–545 to –1414 bases) of the 3'-untranslated region. This response was mediated entirely by the stress-activated protein kinase 2/p38 cascade. As such, IL-17 can exert direct transcriptional and post-transcriptional control over target proinflammatory cytokines and oncogenes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human interleukin-17 (IL-17),1 previously referred to as cytotoxic T-cell lymphocyte-associated antigen 8 (CTLA 8), is a widely recognized prototypic CD45+RO+ T-cell (CD4+)-derived pro-inflammatory cytokine (13). The IL-17 gene is expressed as a 155-amino acid chain that is glycosylated and manifests biological activity as a covalent homodimer (2). Recent cloning and sequencing studies have led to the identification of several genes (IL-17 B–F) that bear close homology to IL-17, highlighted by four to six functionally conserved cysteines that adopt cysteine knot structural conformations (46). The biological activity of the IL-17 homologs is believed to overlap with that of IL-17 (46).

It has become increasing clear that interleukin-17 occupies an important place in the hierarchy of proinflammatory cytokines associated with inflammatory, immune, malignant, and arthritic diseases (reviewed in Refs. 7 and 8). Indeed, there is now wide agreement, based on in vitro and in vivo studies, that the T-cell-derived cytokine may play a fundamental role in the pathophysiology of rheumatoid arthritis (RA) and possibly osteoarthritis (OA) (710). The cytokine is found in high levels in the synovial fluid of RA and OA patients, and synovial tissues express and produce abundant IL-17 (1113), likely from infiltrating T-cell populations. The gamut of target genes and cells include the IL-17-dependent stimulation of the pro-inflammatory cytokines TNF-{alpha} and IL-1{beta} by infiltrating macrophages, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, GRO-{alpha}, and ICAM-1 from mononuclear phagocytes, endothelial cells and fibroblasts, matrix-destructive metalloproteases (e.g. collagenases and aggrecanases) from activated synovial fibroblasts and cartilage chondrocytes, NO from endothelial cells and chondrocytes, and inflammatory modulators such as eicosanoids (e.g. PGE2) from may sources (1422). Thus, IL-17 stimulates tissue damage and cartilage degradation (joint failure) directly or indirectly by recruiting activated inflammatory cells (e.g. neutrophils via adhesion molecules) and inducing the synthesis of proinflammatory cytokines in the inflamed tissue.

The effector cascades mediating the proinflammatory actions of IL-17 are currently under study, and although the IL-17 receptor (IL-17R) is a type I transmembrane protein (130 kDa) with no intrinsic kinase activity (23), it can transduce a signal through the activation of protein kinase A (PKA), JNK, ERK1/2, JAK/STAT, and the NF-{kappa}B cascades (20, 2427). Interleukin-17-treated bovine chondrocytes express increased levels of inducible nitric-oxide synthase mRNA, inducible nitric-oxide synthase protein, and NO release, a process associated with PKA, ERK1/2, and, to a lesser extent, JNK activation (20). Cyclooxgenase-2 mRNA expression and PGE2 release is concomitant with the stimulation of JNK1 and JNK2 by IL-17 in bovine chondrocytes (20). Interleukin-17 activated release of MMP-9 by human monocyte/macrophages followed a time course coincident with the phosphorylation of ERK1/2 and the transcription factors STAT1 and STAT3 (12). Using the same cell and culture conditions, IL-17 stimulated macrophagic release of IL-1{beta}, TNF-{alpha}, and IL-6 was related to rapid calcium flux and a more delayed increase in NF-{kappa}B DNA binding (14). Studies using TRAF-2 or TRAF-6-deficient mouse embryonic fibroblasts suggest strongly that TRAF6 is a critical mediator of IL-17 signaling, implying the involvement of NF-{kappa}B and/or JNK cascades (26).

In the present study, we examined the IL-17-dependent signaling events using as a paradigm the IL-17 induction of COX-2 gene in human synovial fibroblasts, chondrocytes, and, where indicated, macrophages. The COX-2 protein represents the rate-limiting step in the activated biosynthesis of prostanoids, the latter playing a cardinal role as pleiotropic immune and inflammatory modulators (2830). The COX-2 gene is an inducible immediate early gene regulated at both transcriptional (promoter based) and post-transcriptional levels (3133) and, once induced, can be largely controlled by a positive feedback loop involving PGE2 (34). We report that the magnitude and duration of the induction of COX-2 mRNA, COX-2 protein, and PGE2 release by rhIL-17 is primarily the result of IL-17-dependent stabilization of COX-2 mRNA, although transcriptional mechanisms are also involved in the initial phase of induction. Essentially, rhIL-17 mitigates COX-2 mRNA decay normally mediated by the 3'-UTR of COX-2 mRNA. Finally, we provide evidence that the transcriptional and stabilization processes involve a restricted MAPK profile, the MKK3/6/SAPK2/p38 cascade.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Crystalline dexamethasone (9-fluoro-11{beta},17,21-trihydroxy-16-methylpregna-1,4, diene-3, 20-dione), sodium fluoride, leupeptin, aprotinin, pepstatin, phenylmethylsulfonyl fluoride, actinomycin D, dithiothreitol, sodium orthovanadate, and bovine serum albumin were products of Sigma-Aldrich. NS-398 N-[2-(cyclohexyloxy)-4-nitrophenyl)-methanesulfonamide], pyrrolidinedithiocarbamate, PGE2, L-N6-(1-iminoethyl)lysine, 2HCl, PD98059, SB202190, KT-5720, phorbol 12-myristate 13-acetate, forskolin, and rolipram were purchased from Calbiochem (La Jolla, CA), whereas Bay 11-7082 was from Biomol (Plymouth Meeting, PA). SDS, acrylamide, bis-acrylamide, ammonium persulfate, and Bio-Rad protein reagent originated from Bio-Rad Laboratories. Tris-base, EDTA, MgCl2, CaCl2, chloroform, Me2SO, anhydrous ethanol (95%), methanol (99%), formaldehyde, and formamide were obtained from Fisher. rhIL-17, rhIL-10, rh interferon-{gamma}, and rh TNF-{alpha} were purchased from R & D Systems (Minneapolis, MN). Dulbecco's modified Eagle's medium (DMEM), phosphate-free and phenol red-free DMEM, Trizol reagent, heat-inactivated fetal bovine serum, and an antibiotic mixture (10,000 units of penicillin (base), 10,000 µg of streptomycin (base)) were products of Invitrogen.

Specimen Selection and Cell Culture—Synovial lining cells (human synovial fibroblasts (HSF)) were isolated from synovial membranes (synovia) and chondrocytes from articular cartilage. Both were obtained at necropsy from donors with no history of arthritic disease (mean age, 30 ± 27). Additional experiments were conducted with specimens obtained from OA and RA patients undergoing arthroplasty who were diagnosed based on the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA/RA (mean age, 67 ± 19) (35). Human synovial fibroblasts and chondrocytes were released by sequential enzymatic digestion with 1 mg/ml Pronase (Roche Applied Science) for 1 h, followed by 6 h with 2 mg/ml collagenase (type IA; Sigma) at 37 °C in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin (3638). Released HSF were incubated for 1 h at 37 °C in tissue culture flasks (Primaria 3824, Falcon, Lincoln Park, NJ), allowing the adherence of nonfibroblastic cells possibly present in the synovial preparation, particularly from OA and RA synovia. In addition, flow cytometric analysis (Epic II, Coulter, Miami, FL), using the anti-CD14 (fluorescein isothiocyanate) antibody, was conducted to confirm that no monocytes/macrophages were present in the synoviocyte preparation (3638). The cells were seeded in tissue culture flasks and cultured until confluence in DMEM supplemented with 10% FCS and antibiotics at 37 °C in a humidified atmosphere of 5% CO2, 95% air. The cells were incubated in fresh medium containing 0.5–1% fetal bovine serum for 24 h before the experiments, and only second or third passaged HSF were used. Human monocyte/macrophage cultures were prepared from the freshly drawn blood of healthy volunteers as previously described (14).

Preparation of Cell Extracts and Western Blotting—50–100 µg of cellular protein extracted in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mM sodium orthovanadate, and 1 mM NaF) or hot SDS-PAGE loading buffer, from control and treated cells, were subjected to SDS-PAGE through 10% gels (final concentration of acrylamide, 16 x 20 cm) under reducing conditions and transferred onto nitrocellulose membranes (Amersham Biosciences). Following blocking with 5% BLOTTO for 2 h at room temperature and washing, the membranes were incubated overnight at 4 °C with polyclonal anti-human COX-2 (Cayman Chemical Co., Ann Arbor, MI; 1:7500 dilution) in TTBS containing 0.25% BLOTTO. The second anti-rabbit antibody-horseradish peroxidase conjugate (1:10,000 dilution) was subsequently incubated with membranes for 1 h at room temperature, washed extensively for 30–40 min with TTBS, and then rinsed with Tris-buffered saline at room temperature. Following incubation with an ECL chemiluminescence reagent (Amersham Biosciences), the membranes were prepared for autoradiography, exposed to Kodak (Rochester, NY) X-Omat film, and subjected to a digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada, Mississauga, Canada) for semi-quantitative measurements. In addition to the anti-COX-2 and COX-1 (Cayman) antisera, the following polyclonal antibodies were used (New England Biolabs Ltd., Mississauga, ON, Canada): total (independent of phosphorylation state) and anti-phospho-p38 MAPK (Thr180/Tyr182), anti-phospho-MKK3/6 (Ser189/207), total and anti-phospho-I{kappa}B-{alpha} (Ser32), anti-phospho-ATF-2 (Thr69/71), anti-phospho-CREB-1 (Ser133), anti-phospho-c-Jun (Ser63), anti-phospho-JNK/SAPK (Thr183/Tyr185), anti-phospho-Mnk1 (Thr197/201), total and anti-phospho-eIF4E (Ser209), total and anti-phospho-p44/42 (Thr202/Tyr204), and total and anti-phospho-STAT3 (Ser727).

Northern Blot Analysis of mRNA—Total cellular RNA was isolated (1 x 106 cells = 10–20 µg of RNA) using the Trizol (Invitrogen) reagent. Generally, 5 µg of total RNA were resolved on 1.2% agarose-formaldehyde gel and transferred electrophoretically (30 V overnight at 4 °C) to Hybond-NTM nylon membranes (Amersham Biosciences) in 0.5x Tris/acetate/EDTA buffer, pH 7. After prehybridization for 24 h, the hybridizations were carried out at 50–55 °C for 24–36 h, followed by high stringency washing at 68 °C in 0.1x SSC, 0.1% SDS. The following probes, labeled with digoxigenin-dUTP by random priming, were used for hybridization: human COX-2 cDNA (1.8 kb; Cayman Chemical Co.) initially cloned into the EcoRV site of pcDNA 1 (Invitrogen) that was released by PstI and XhoI digestion resulting in a 1.2-kb cDNA fragment and a 780-bp PstI/XbaI fragment from GAPDH cDNA (1.2 kb; American Type Culture Collection, Rockville, MD) that was initially cloned into a PstI of pBR322 vector. This latter probe served as a control of RNA loading because GAPDH is constitutively expressed in cells used in these experiments. All of the blots were subjected to a digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada) for semi-quantitative measurements, and changes in COX-2 expression were always considered as a ratio, COX-2/GAPDH mRNA.

Transfection Experiments—Transient transfection experiments were conducted in 4-, 6-, or 12-well cluster plates as previously described (34, 38). Transfections were conducted using the FuGENE 6TM (Roche Applied Science) or LipofectAMINE 2000TM reagents (Invitrogen) method for 6 h according to the manufacturers' protocol with cells at around 30–40% confluence. The cells were re-exposed to a culture medium with 1% FCS for 2 h prior to the addition of the biological effectors. Transfection efficiencies were controlled in all experiments by co-transfection with 0.5 µg of pCMV-{beta}-gal, a {beta}-galactosidase reporter vector under the control of CMV promoter (Stratagene, La Jolla, CA). A COX-2 promoter (–2390 to + 34)-LUC plasmid was kindly provided by Dr. Stephen Prescott (University of Utah) (39), and site-directed mutagenesis was performed with the Bsu36I COX-2 promoter fragment (–415 to + 34) using the QuikChangeTM (Stratagene) kit and involved modifying the ATF-CRE site at bp –53/–54 (CA -> TC) and the NF-{kappa}B site at bp–215/–216 (CC -> GG). Chimeric luciferase reporter plasmids fused with the human COX-2 mRNA 3'-UTR (1451 bp), AU-rich elements (429 bp of which the first 116 bp contain an AU-cluster), the 3'-UTR minus the AU-rich element cluster, or a construct completely devoid of the COX-2 3'-UTR but containing the SV40 poly(A) signal (40). The plasmids are designated LUC-3'-UTR, LUC-+ARE, LUC-{Delta}ARE, LUC-{Delta}3'-UTR, respectively, and were a kind gift of Dr. D. Dixon (University of Utah).

In our signal transduction pathway reporting systems (Stratagene), a reporter plasmid containing the 17-bp (5x) GAL4 DNA-binding element (UAS) fused to a TATA box upstream from the luciferase gene (pFR-LUC) was co-transfected with a construct containing the transactivation domains of transcription factors (e.g. ATF-2 and c-Jun) fused to GAL4 DNA-binding domain and driven by a CMV promoter (e.g. pFA-ATF-2). In addition, plasmids (Stratagene) harboring NF-{kappa}B or interferon-stimulated response element enhancer elements (5x) fused to a TATA box upstream from the luciferase gene were used to assess transactivation processes activation by IL-17 in the cell phenotypes tested. Finally, wild-type and activated MKK3/6 expression plasmids (Stratagene) were overexpressed to determine the role on COX-2 expression. The luciferase values, expressed as enhanced relative light units, were measured in a Lumat LB 9507 luminometer (EG&G, Stuttgart, Germany) and normalized to the level of {beta}-galactosidase activity (optical density at 450 nm after 24 h of incubation) and cellular protein (bicinchoninic acid procedure; Pierce).

RT-PCR for Luciferase and GAPDH—The oligonucleotide primers for PCR were prepared with the aid of a DNA synthesizer (Cyclone Model, Biosearch Inc., Montreal, Canada) and used at a final concentration of 200 nmol/liter. The sequences for the Luciferase primers were as follows: 5'-ACGGATTACCAGGGATTTCAGTC-3' and 5'-AGGCTCCTCAGAAACAGCTCTTC-3' (antisense) for the luciferase fragment of 367 bp (40). The sequences for the GAPDH (which served as a standard of quantitation) primers were 5'-CAGAACATCATCCCTGCCTCT-3', which corresponds to position 604–624 bp of the published sequence, and 5'-GCTTGACAAAGTGGTCGTTGAG-3', from positions 901–922 bp, for an amplified product of 318 bp (36). Two µg of total RNA, extracted with the Trizol reagent, was reverse transcribed and then subjected to PCR as previously described (34). RT and PCR assays were carried out with the enzymes and reagents of the GeneAmP RNA PCR kit manufactured by PerkinElmer Life Sciences. Both the RT and PCR reactions were done in a Gene ATAQ Controller (Amersham Biosciences).

The amplification process was conducted over 10–30 cycles to define the linear range of product amplification. The first cycle consisted of a denaturation step at 95 °C for 1 min, followed by annealing and elongation at 60 °C for 30 s, and 72 °C for 1.5 min, respectively. All subsequent cycles were executed under the same conditions, with the exception of the last cycle, where the elongation step was extended to 7 min. We found a linear range (log luciferase/GAPDH versus log cycle number) between 10 and 17; as such we chose 11–13 cycles depending on the type of experiment.

The PCR products were analyzed and verified by electrophoresis on 1.15% agarose gels in a Tris-borate-EDTA buffer system as previously described (34). All gel photos were subjected to a digital imaging system (see above) for semi-quantitative measurements, and the results were expressed as a ratio of luciferase/GAPDH PCR fragments.

Extraction of Nuclear Proteins and EMSA Experiments—Confluent control and treated cells in 4-well cluster plates (3–5 x 106 cells/well) were carefully scraped into 1.5 ml of ice-cold phosphate-buffered saline and pelleted by brief centrifugation. The nuclear extracts were prepared as previously described (36).

Double-stranded oligonucleotides containing wild-type and mutant sequences were from Invitrogen, annealed in 100 nM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA buffer at 65 °C for 10 min, cooled for 1–2 h at room temperature, and finally end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI). The sense sequences of the oligonucleotides tested were as follows: NF-{kappa}B (COX-2), 5'-CAG GAG AGT GGG GAC TAC CCC CTC TGC TC-3'; NF-{kappa}B mut, 5'-CAG GAG AGT GGC GAC TAG GCC CTC TGC TC-3'; ATF/CRE (COX-2), 5'-GGC GGA AAG AAA CAG TCA TTT CGT CAC ATG GGC TTG G-3'; ATF/CRE mut, 5'-GGC GGA AAG AAA CAG TCA TTT CGT TCC ATG GGC TTG-3'; NF-IL6, 5'-CTA GGG CTT GCG CAA TCT ATA TTC G-3'; and NF-IL6 mut, 5'-CTA GGG CTT GCT ACC CCT ATA TTC G-3'. Binding buffer consisted of 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol, and 2.5 µg of poly(dI-dC). The binding reactions were conducted with 15 µg of nuclear extract (± 1 µg c/EBP{alpha}/{beta}/{gamma}/{delta} antibodies in supershift analysis) and 100,000 cpm of 32P-labeled oligonucleotide probe at 22 °C for 20 min in a final volume of 10 µl. The binding complexes were resolved by nondenaturing polyacrylamide gel electrophoresis through 6% gels in a Tris-borate buffer system, after which the gels were fixed, dried, and prepared for autoradiography.

Statistical Analysis—All of the results were expressed as the means ± S.D. or the means and the coefficient of variation of three to five separate experiments as indicated. The transfection experiments were performed in triplicate. Statistical treatment of the data was performed parametric (Student's t test) or by nonparametric (Mann-Whitney) analysis if Gaussian distribution of the data could not be confirmed. Significance was acknowledged when the probability that the Null Hypothesis was satisfied at <5%.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Human IL-17 Induction of COX-2 mRNA Expression and Synthesis: Time and Dose Dependence—To better characterize the properties of IL-17 cell signaling, we performed both dose response and time course studies on the induction of the COX-2 gene. As shown in Fig. 1A, the EC50 for rhIL-17-dependent COX-2 protein synthesis in our HC culture system was ~10 ng/ml (n = 3), and as such, this concentration was chosen for all subsequent experimentation unless otherwise indicated. Time course studies revealed that increases in COX-2 mRNA expression reached 2.1-fold at 15 min and greater than 7-fold after 2 h, attained steady state at 4–8 h, and thereafter declined very gradually for the next 16 h as shown in Fig. 1B. In fact, COX-2 mRNA was detectable within 5 min after rhIL-17 treatment (data not shown). The induction of COX-2 protein was perceptible within 30 min following stimulation with rhIL-17 and thereafter followed a pattern of biosynthesis closely resembling COX-2 mRNA expression. Similar experiments conducted with HSF and human macrophages yielded identical results (data not shown). The rhIL-17-stimulated PGE2 release profile indicated that eicosanoid levels were increased after 1 h and by 24 h more than 30 ng/106 cells were detected (n = 3).



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FIG. 1.
Dose response (A) and time course (B) of rhIL-17 stimulation of COX-2 protein and COX-2 mRNA. Cultured confluent primary articular HC (1.2 x 106 cells in 6-well cluster plates) were preincubated for 24 h in DMEM supplemented with 1% FCS plus antibiotics at 37 °C to assure synchrony and quiescence. The cells were then treated with increasing concentrations of rhIL-17 (0–200 ng/ml) for 6 h (A) and for varying time periods (0–24 h) (B). Monolayers were extracted for protein or RNA; 50 µg of protein were analyzed for COX-2 protein by Western blotting using a specific polyclonal anti-COX-2 antiserum, whereas 5 µg of total RNA were analyzed for COX-2 mRNA by Northern hybridization using a specific digoxigenin-labeled cDNA probe as described under "Experimental Procedures."

 

SAPK2/p38 MAPK Activity and rhIL-17-dependent Regulation of COX-2 Gene Expression—Previous studies in our laboratory and others demonstrate that IL-17 may signal through activation of MAPK (e.g. p44/42 MAPK) and/or NF-{kappa}B cascades, although it is presently unclear how the COX-2 gene responds (12, 14, 20). To delineate post-receptor signaling pathways activated by IL-17, we chose, as a first approach, to use cell-permeable chemical inhibitors. As shown in Fig. 2A, SB202190 (SB, SAPK2{alpha}/{beta}/p38{alpha}/{beta} MAPK inhibitor) suppressed rhIL-17-induced COX-2 mRNA and protein synthesis by ~90% (87 ± 9%, mean ± S.D., n = 4); PGE2 release was suppressed by greater than 95% (data not shown). The MEK1/2 inhibitor PD98059, and the I{kappa}B kinase inhibitor, Bay 11-7802 were without significant effect, whereas the PKA inhibitor KT-5720 had a modest albeit inconsistent inhibitory activity. The clinically useful anti-inflammatory steroid, dexamethasone completely suppressed rhIL-17-induced COX-2 mRNA and protein synthesis, whereas pyrrolidinedithiocarbamate and L-N6-(1-iminoethyl)lysine, 2HCl, inhibitors of reactive oxygen radicals and nitric oxide production respectively, were seemingly without effect (Fig. 2A). We previously reported that IL-1 induction of the COX-2 gene was mediated by a PGE2-dependent feed-forward mechanism and as such could be abrogated by co-incubating IL-1 with a preferential COX-2 inhibitor like NS-398 (34). The present data show that NS-398 does not block to any significant degree the rhIL-17 induction of the COX-2 gene (Fig. 2A). Identical results were obtained with HSF in culture (data not shown).



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FIG. 2.
rhIL-17 stimulation of COX-2 protein and mRNA was blocked by SAPK/p38 MAPK inhibitor (A) in a dose-dependent fashion (B). Quiescent HC were treated with vehicle (C, lane 1) or with 10 ng/ml (570 pmol/liter) of rhIL-17 (lane 2) for 8 h in the presence of either SB202190 (SB, 1 µmol/liter), a p38 MAPK inhibitor; PD98059 (PD, 50 µmol/liter), a MEK1/2 inhibitor; KT-5720 (KT, 2 µmol/liter), a PKA inhibitor; dexamethasone (DEX, 100 nmol/liter); NS-398 (NS, 100 nmol/liter), a preferential COX-2 inhibitor; pyrrolidinedithiocarbamate (100 µmol/liter), an oxygen radical scavenger; L-N6-(1-iminoethyl)lysine, 2HCl (l-NIL, 1 µmol/liter), an inducible nitric-oxide synthase inhibitor; and Bay 11-7082 (BAY, 5 µmol/liter), an IKK inhibitor. In B, the cells were incubated with or without rhIL-17 in the absence or presence of increasing concentrations of SB202190. Monolayers were extracted for protein or RNA; 50 µg of protein were analyzed for COX-2 protein by Western blotting using a specific polyclonal anti-COX-2 antiserum as described under "Experimental Procedures," whereas 5 µg of total RNA were analyzed for COX-2 mRNA and GAPDH mRNA by Northern hybridization using specific digoxigenin-labeled cDNA probes.

 

Given the apparent role of SAPK2/p38 in the induction of COX-2 by rhIL-17, we endeavored to determine the degree of sensitivity of the inductive process to SB202190 by carefully controlled dose-response studies. As shown in Fig. 2B, the inhibitor blocked rhIL-17 stimulation of steady-state levels of COX-2 mRNA in a dose-dependent fashion, and the IC50, calculated by plotting the log optical density COX-2/GAPDH mRNA versus the concentration of SB202190, was 36 ± 4 nM (n = 3).

Cell Signaling by rhIL-17 Is Restricted to the SAPK2/p38 MAPK Cascade—To pursue further the results obtained with regard to rhIL-17 and SAPK2/p38 activation, we performed additional investigative experimentation. As shown in Fig. 3A, rhIL-17 triggered a bi-phasic pattern of MKK3/6 (Ser189/207) and SAPK2/p38 phosphorylation with the initial phase reaching a zenith at 20 min and then falling to essentially control levels within the next 40 min (i.e. 1 h post-stimulation). The second phase was initiated within the next 60 min (i.e. 2 h post-stimulation) and attained a maximum at 8 h. There were no changes in the cellular protein level of SAPK2/p38 (Fig. 3A). In addition, ATF-2, a transcription factor whose transcriptional activity is modulated by phosphorylation at Thr69 and Thr71 by SAPK2/p38 (41), was phosphorylated at the latter sites in a time course essentially identical to that of SAPK2/p38/MKK3/6 (Fig. 3A). A similar pattern was observed with CREB-1/ATF-1 (Ser133) phosphorylation following IL-17 stimulation, although the second phase was less evident. Fragmentation of both SAPK2/p38 and ATF-2 was observed in the initial phosphorylation phase. The downstream kinases MAPK-APK2 and MSK1 were not phosphorylated by rhIL-17 treatment, although the pro-inflammatory cytokine rhIL-1{beta} phosphorylated both (data not shown). The MAPK signal-integrating kinase-1 (Mnk1) and its putative substrate, the eukaryotic initiation factor 4E (eIF-4E), were avidly phosphorylated by rhIL-17 at Thr197/202 and Ser209, respectively (Fig. 3B). The latter rhIL-17-induced post-translational modifications were substantially blocked in the presence of SB202190 (Fig. 3C). Finally, overexpression of a constitutively activated MKK3 construct (p{Delta}MKK3) stimulated COX-2 protein synthesis, an effect completely abrogated by SB202190 (Fig. 3D).



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FIG. 3.
Time course of SAPK2/p38 MAPK cascade activation by rhIL-17. A and B, quiescent HC treated for varying time periods (0–16 h; 16C, 16 h of control) with 10 ng/ml (570 pmol/liter) of rhIL-17 (top panels). Monolayers were extracted for protein, and 50 µg were analyzed for total and phospho-SAPK2/p38 MAPK intermediates by Western blotting using specific rabbit polyclonal antisera: anti-phospho-p38 MAPK(Thr180/Tyr182), anti-phospho-MKK3/6 (Thr180/Tyr182), anti-phospho-Mnk1 (Thr197/202), anti-phospho-eIF-4E (Ser209), anti-phospho-CREB-1 (Ser133), and anti-phospho-ATF-2 (Thr69/71). In C, the cells were treated with 10 ng/ml (570 pmol/liter) of rhIL-17 in the absence or presence of 0.1 µM of SB202190 for 0, 0.33, 1, and 2 h, after which time the cell extracts were prepared for Western blotting (top panel) using anti-phospho-Mnk1 (Thr197/202), anti-phospho-eIF-4E (Ser209), and total anti-eIF-4E. Bottom panel, densitometric scanning was performed on Western blots (average of two determinations with a coefficient of variation of 11.3%). In D, the cells were plated at 40% confluence in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin and transfected with 50 ng each of pCMV, pFCMKK3, p{Delta}MKK3, or p{Delta}MKK3 in the presence of 0.1 µM of SB202190. The cells were lysed after 24 h, and COX-2 and COX-1 were analyzed by Western blotting using specific antisera as described under "Experimental Procedures."

 

To confirm whether rhIL-17 could increase the transactivational capacity of ATF-2, we conducted experiments in which a luciferase reporter construct harboring 5'-flanking GAL4 DNA-binding elements (5x) was co-transfected with a chimeric plasmid containing the N-terminal (1–96) ATF-2 transactivation domain fused to GAL4 DNA-binding domain and driven by a CMV promoter. When the cells were then incubated with rhIL-17 for 6 h, a 5.3 ± 0.7 increase (n = 3, mean ± S.D.) in luciferase activity was observed (Fig. 4A), an effect completely blocked by co-incubations with SB202190 (control, 2808 ± 312 RLU; SB202190, 2689 ± 675 RLU). This response was quite specific because when co-transfections were performed with chimeric constructs containing the transactivation domain of c-Jun (1–223), c-Fos (206–313), or Elk-1 (307–428), no increases in luciferase activity were observed following rhIL-17 stimulation. In contrast, phorbol 12-myristate 13-acetate potently stimulated transactivation by c-Fos and c-Jun, whereas rhIL-1{beta} activated the transactivational capacity of Elk1 (Fig. 4B). Interestingly, rhIL-17 stimulated reporter transactivation by CREB by a modest but statistically significant 2.1 ± 0.2, whereas the combination of forskolin (adenylate cyclase activator) and rolipram (cAMP-dependent phosphodiesterase type IV inhibitor) provoked a 6.2 ± 0.45 (n = 3, mean ± S.D.) increase (Fig. 4A). The absence of induction by rhIL-17 of c-Jun, c-Fos, or Elk-1 transactivational activity was supported by the observations that the cytokine had little or no effect (in contrast to rhIL-1{beta}) on the phosphorylation (activity) of the signaling intermediates ERK1/2 and JNK, which are known to phosphorylate and increase the transactivational capacity of the latter transcription factors (42, 43) (Fig. 4C).



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FIG. 4.
rhIL-17 stimulation of ATF-2/CREB-1 transactivation. HSF were plated in 12-well cluster plates at 30% confluence in complete medium and then co-transfected with 1 µg of pFC-LUC reporter, 0.5 µg of pCMV-{beta}-gal, and 50 ng each of pFA-CMV, pFA-ATF-2, pFA-CREB-1 (A), pFA-c-Jun, pFA-c-Fos, or pFA-Elk-1 (B) as described under "Experimental Procedures." After 24 h, the cells were stimulated for 6 h with or without rhIL-17 (10 ng/ml) or forskolin (60 µmol/liter) and rolipram (100 µmol/liter), phorbol 12-myristate 13-acetate (200 nmol/liter), or rhIL-1{beta} (100 pg/ml) as indicated in Fig. 3. The cells were lysed, and the luciferase activity, {beta}-gal activity, and protein content were determined as described under "Experimental Procedures." The values are expressed as the means ± S.D. of three determinations in triplicate of luciferase activity (relative light units) divided by {beta}-gal activity (optical density). Probabilities (p) were performed by Student's t test. In C, quiescent HC were treated for 20 min with rhIL-17 (0–200 ng/ml) or rhIL-1{beta} (10 or 100 pg/ml). Monolayers were extracted for protein, and 50 µg were analyzed for total and phospho-ERK1/2 (p42/44) or phospho-JNK (p46/p54) by Western blotting using specific rabbit polyclonal antisera.

 

Many studies have suggested that the IL-17 signal is transduced by the JAK/STAT and NF-{kappa}B cascades in a variety of cell types (12, 20, 25). To address this issue in the context of our cell culture models and the control of COX-2 gene expression, we measured the phosphorylation (activation) state of critical intermediates and performed transactivational analysis with reporter constructs. As shown in Fig. 5A, rhIL-17 weakly stimulated the phosphorylation of I{kappa}B-{alpha}, which reached a maximum after 10 min with no measurable change in total cellular I{kappa}B-{alpha}. In contrast, TNF-{alpha}, a prototypical pro-inflammatory cytokine and potent activator of the NF-{kappa}B cascade potently induced I{kappa}B-{alpha} phosphorylation in less than 2 min with a concomitant reduction in total I{kappa}B-{alpha} of more than 50%. Interleukin-17 stimulated a time-dependent phosphorylation of the transcription factor STAT3 (but not STAT1) with detectable Ser727 phosphorylation observed between 1–2 h, a zenith at 4 h, and gradual decay thereafter. The prototypic STAT inducer IL-10 caused a similar magnitude of phosphorylation but much sooner (after 20 min) (Fig. 5B). Furthermore, although TNF-{alpha} stimulated a 7–10-fold increase in luciferase activity in cells transfected with a reporter construct harboring five tandem NF-{kappa}B consensus sequences, rhIL-17 had no statistically significant effect (Fig. 5C). However, when identical protocols were repeated in transiently transfected HSF, rhIL-17 increased reporter activity by 1.78 ± 0.31-fold (n = 4 determinations). 6 h post-stimulation, rhIL-10, interferon-{gamma} and rhIL-17 all increased luciferase activity to a significant degree in cells transfected with a reporter construct harboring five interferon-stimulated response element tandem sequences (Fig. 5C).



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FIG. 5.
Role of NF-{kappa}B and JAK/STAT cascades as mediators of rhIL-17 induction of the COX-2 gene. Quiescent confluent HC were treated with 10 ng/ml of rhIL-17 from 0 to 240 min or for 2 min with 10 ng/ml of TNF-{alpha} (A). Alternatively the cells were exposed to 10 ng/ml of rhIL-17 for 0–16 h or with rhIL-10 (1 ng/ml) for 20 min (B). Monolayers were extracted for protein and prepared for Western blotting and probed with specific anti-phospho-I{kappa}B-{alpha} and anti-I{kappa}B-{alpha} (A) or anti-phospho-STAT3 and anti-STAT3 (B) antibodies as indicated. The blots are representative of three separate experiments that gave essentially identical results. In C, the cells were transfected with 1 µg each of either 5x NF-{kappa}B-tatata-LUC or 5x interferon-stimulated response element-tatata-LUC and 0.5 µg of pCMV-{beta}-gal as described under "Experimental Procedures." The cells were then exposed to either rhIL-17 (10 ng/ml), rhTNF-{alpha} (10 ng/ml), rhIL-10 (1 ng/ml), or interferon-{gamma} (1 ng/ml) for 6 h. The cells were lysed and prepared for measurement of luciferase activity, {beta}-gal activity, and protein content as described under "Experimental Procedures." The values are expressed as the means ± S.D. of four determinations in triplicate of luciferase activity (relative light units) divided by {beta}-gal activity (optical density). Probabilities (p) were performed by Student's t test.

 

COX-2 Promoter Studies—To examine for elements of transcriptional control of the COX-2 gene via rhIL-17-stimulated SAPK2/p38 signaling, we conducted transient transfection analyses with a 416-bp (Bsu36I site) human COX-2 promoter construct harboring enhancer elements (44, 45) for critical transcription factors including a ATF/CRE site (–58 to –53). We observed that rhIL-17 (10 ng/ml) stimulated a human COX-2 promoter-luciferase reporter construct by 1.76 ± 0.11 (mean ± S.D., n = 3–5)-fold (Fig. 6A). SB202190 abolished the induction completely (control, 11,739 ± 1,452 versus rhIL-17, 20,660 ± 1,291; rhIL-17 + SB202190; 7,635 ± 987 RLU). Companion experiments with a ATF/CRE mutant plasmid (see "Transfection Experiments" under "Experimental Procedures") revealed that the reporter activity was considerably lower than wild-type promoter plasmid (mutant, 6,722 ± 911 versus wild type (control), 11,739 ± 1,452 RLU, mean ± S.D., n = 3) and was refractory to rhIL-17 (compare 6,722 ± 911 versus rhIL-17, 6,945 ± 753 RLU, mean ± S.D., n = 3). To assess the role of ATF-2 in transcriptional activation of the COX-2 promoter, we used a previously described decoy strategy (46) in which the chimeric plasmid containing the N-terminal (1–96) transactivation domain of ATF-2 fused to the GAL4 DNA-binding domain was overexpressed prior to rhIL-17 stimulation. As can be seen in Fig. 6A, rhIL-17 induction of luciferase activity was completely suppressed, and indeed luciferase activity was observed to be below control values. Forced expression of other transcription factor-GAL4 chimers were without effect with the exception of pFA-CREB, where 30.3 ± 3.15%of rhIL-17-stimulated luciferase activity was blocked (Fig. 6A).



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FIG. 6.
rhIL-17 induction of the COX-2 promoter is dependent on SAPK2/p38 MAPK activation and ATF-2 transactivation (A). EMSA profiles of rhIL-17 stimulated DNA binding proteins (B). HSF were plated in 12-well cluster plates at 30–40% confluence in complete medium and then co-transfected with 1 µg of a human COX-2 promoter construct containing a portion of the proximal promoter fused to a luciferase reporter construct (Bsu36I fragment, –415, phCOX-2-LUC reporter), 0.5 µg of pCMV-{beta}-gal, and 50 ng each of pFA-CMV, pFA-ATF-2, pFA-CREB-1, pFA-c-Jun, pFA-c-Fos, and pFA-Elk-1 as described under "Experimental Procedures." After 24 h, the cells were stimulated for 6 h with rhIL-17 (10 ng/ml) and lysed, and the luciferase activity, {beta}-gal activity, and protein content were determined as described under "Experimental Procedures." The values are expressed as the means ± S.D. of three to five determinations in triplicate of luciferase activity (relative light units) divided by {beta}-gal activity (optical density). Probabilities (p) were performed by Student's t test. In B, nuclear extracts were prepared from human chondrocytes treated as indicated (Con, control), and 15 µg of protein were incubated with 32P-labeled ATF/CRE (top panel), NF-{kappa}B (middle panel), or NF-IL6 (bottom panel) oligonucleotides for 20 min at 22 °C. The binding reaction mixtures were subject to EMSA analysis as described under "Experimental Procedures."

 

Mutating the more proximal NF-{kappa}B site (–223/–214) in the human COX-2 promoter construct was without effect in terms of basal and IL-17-stimulated luciferase activity (control, 10,639 ± 1,975 versus rhIL-17, 18,332 ± 2,070 RLU; 1.72 ± 0.13-fold increase, n = 3; compare with wild type; see above) when transfections were performed with human chondrocytes. However, when HSF were used with the same protocol, basal reporter activity was reduced but not the level of rhIL-17 induction (control, 8,117 ± 1,424 versus rhIL-17, 14,998 ± 2,114 RLU; 1.85 ± 0.14-fold increase).

EMSA experiments using 32P-labeled ATF/CRE (COX-2) oligonucleotides indicated a high level of endogenous nuclear protein binding that was increased with rhIL-17 treatment (Fig. 6B, upper panel). Basal and induced binding was displaced by adding 10-fold excess cold oligonucleotide but not by the mutant oligonucleotide (see above ATF/CRE mutant COX-2 promoter studies). Furthermore, the addition of SB202190 abrogated induced and, to a large extent, basal oligonucleotide binding. Confirming promoter studies, rhIL-17 stimulation of human chondrocytes produced no increases in 32P-labeled NF-{kappa}B (COX-2) oligonucleotide binding in contrast to TNF-{alpha} (Fig. 6B, middle panel). The human COX-2 promoter harbors a proximal NF-IL-6 site that binds c/EBP transcription factors as either homodimers or heterodimers with other transcription factors (e.g. NF-{kappa}B) and can increase transcriptional activation (44, 45). Time course studies revealed that rhIL-17 increased NF-IL-6 binding after 30 min, reached a zenith after 60 min, and thereafter declined so that at 90 min binding to the oligonucleotide was similar to controls (Fig. 6B, lower panel). Supershifts with antibodies to the different isoforms of c/EBP (see "Experimental Procedures") confirmed the presence of c/EBP{beta} only (data not shown).

Interleukin-17 Stabilizes COX-2 mRNA—Judging by the accumulated data (see above), it is unlikely that IL-17 modifies steady-state COX-2 mRNA expression in our cell culture models exclusively at the transcriptional level. As such we examined post-transcriptional mechanisms involving strictly message stabilization and protein synthesis. As a first approach, we employed classical techniques involving measuring COX-2 mRNA in transcriptionally arrested cells (actinomycin D) in the absence or presence of rhIL-17. We had previously reported (34) that when HSF are activated with rhIL-1{beta} for 3–4 h (steady state) followed by wash-out and a fresh change of medium, the elevated levels of COX-2 mRNA declined rapidly such that within 2 h the levels were similar to control non-stimulated cells (t1/2 = 0.85 h). However, if PGE2 was added to fresh medium (in the presence of actinomycin D), COX-2 mRNA levels remained elevated (t1/2 = 13 h) (34). Similarly, as represented in Fig. 7, rhIL-17 stabilized COX-2 mRNA and increased its half-life to ~5.8 h, based on multiple linear regressions of optical density of COX-2 mRNA versus time (n = 3, y = 1.1–0.095x). The stabilizing effect was abrogated by co-incubations with SB202190. We included PGE2 in these experiments as a positive control and for purposes of comparison.



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FIG. 7.
rhIL-17 stabilizes COX-2 mRNA: role of SAPK2/p38 MAPK. Quiescent HSF were treated with vehicle (lane C) or with 10 ng/ml of rhIL-17 for 6 h (steady state), after which time cells were washed out and treated with actinomycin D (1 µg/ml) for 30 min, and then fresh medium was added containing either vehicle (Wo), rhIL-17 (10 ng/ml), rhIL-17 + SB202190 (0.1 µM), or PGE2 (100 nmol/liter). After an additional 2 (short) or 8 h (long) of incubation, monolayers were extracted for RNA at each time point, and 5 µg of total RNA were analyzed for COX-2 mRNA and GAPDH mRNA by Northern hybridization using specific digoxigenin-labeled cDNA probes.

 

As mentioned earlier, the COX-2 mRNA has multiple copies of the Shaw-Kamen AU-rich sequences that are believed to influence message stability. Recent studies (40, 47) have provided evidence that the AU-rich elements (6) in the first 116 bp of the 3'-UTR may mediate COX-2 mRNA instability, whereas we recently reported that proximal but also distal sequences mediate PGE2-dependent stabilization (34). To determine whether rhIL-17-dependent COX-2 mRNA stabilization was manifested through the 3'-UTR sequences, we transfected HSF with CMV-driven chimeric expression constructs containing luciferase cDNA (reporter) fused 3' to the human COX-2–3'-UTR (Luc3'-UTR), AU-rich region (Luc+ARE), AU-deleted region (Luc{Delta}ARE-3'-UTR), or complete removal of the 3'-UTR region (Luc {Delta}3'-UTR). As shown in Fig. 8A, cells transfected with Luc {Delta}3'-UTR were refractive to any kind of modulation. However, rhIL-17 increased luciferase activity 3–4-fold (n = 5) in cells transfected with Luc3'-UTR and Luc{Delta}ARE-3'-UTR constructs but not with Luc+ARE. If the cells were washed out, and fresh medium was added, luciferase activity decreased dramatically after 4 h. The latter decrease could be mitigated with the addition of rhIL-17, a response that was apparently SAPK2/p38-mediated as the induction was abrogated by co-incubations with SB202190. We obtained similar results with PGE2, included as a positive control, with the sole difference being that the ARE-rich region of the 3'-UTR is also sensitive to eicosanoid stimulation (Fig. 8A).



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FIG. 8.
rhIL-17-dependent stabilization of COX-2 mRNA and increased translation is mediated by the distal portion of COX-2 3'-UTR. HSF were plated in 12-well cluster plates at 30–40% confluence in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. One µg of LUC-{Delta}3'-UTR, LUC-+ARE, LUC-{Delta}ARE, or LUC-3'-UTR and 0.5 µg of pCMV-{beta}-gal were transfected as described under "Experimental Procedures." Following a change in medium containing 1% FCS for 2 h, the transfected cells were treated with vehicle (control) or with 10 ng/ml of rhIL-17 for 6 h, rinsed, and treated with actinomycin D (1 µg/ml) for 30 min, and then fresh medium was added containing either vehicle (Wo), rhIL-17 (10 ng/ml), rhIL-17 (10 ng/ml) + SB202190 (SB, 0.1 µmol/liter), or PGE2 (100 nmol/liter) alone or in the presence of SB202190 (SB, 10 µmol/liter) for an additional 4 h (A). The cells were lysed, and the luciferase activity, {beta}-gal activity, and protein content were determined as described under "Experimental Procedures." The values are expressed as the means ± S.D. of three determinations in triplicate of luciferase activity (relative light units) divided by {beta}-gal activity (optical density). Probabilities (p) were calculated by Student's t test. *, p < 0.0043; **, p < 0.0012; ***, p < 0.0021 versus control; +, p < 0.00016; ++, p < 0.0001 versus rhIL-1{beta}; +++, p < 0.0006; &, p < 0.013 versus LUC-+ARE + rhIL-17. In B, HSF were plated at 30–40% confluence in complete medium and then transfected with 1 µg of LUC-3'-UTR. Following a change in medium containing 1% FCS for 2 h, the transfected cells were treated with vehicle or with 10 ng/ml of rhIL-17 for 6 h, rinsed, and treated with actinomycin D (1 µg/ml) for 30 min, and then fresh medium was added containing either vehicle (Wo) or rhIL-17 (10 ng/ml) for an additional 2, 4, or 8 h. In C, rinsed and actinomycin D-treated cells were incubated with vehicle (Wo) or rhIL-17 (10 ng/ml) in the absence or presence of SB202190 (0.1 µM) for an additional 4 h. Monolayers were extracted for RNA, and 1 µg of total RNA was analyzed for luciferase mRNA and GAPDH mRNA by RT-PCR as described under "Experimental Procedures." Representative RT-PCR gels are shown (n = 3).

 

Although COX-2 mRNA stabilization and protein synthesis are closely coupled in our cell culture models, we verified whether our reporter system (i.e. luciferase protein (activity) and mRNA) exhibited similar coordination. As shown in Fig. 8B, in cells transfected with Luc3'-UTR and stimulated with rhIL-17, luciferase mRNA decayed rapidly under wash-out conditions but was stabilized with the addition of rhIL-17. Densitometric scanning analysis (n = 4) revealed a t1/2 for luciferase mRNA of under wash-out conditions of about 0.78 h (y = 2.15–1.38x) and t1/2 in the presence of rhIL-17 of ~6.2 h (y = 2.4–0.195x). As shown in Fig. 8B, the addition of SB202190 blocked the stabilizing effect of rhIL-17.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The preponderance of data suggests that IL-17 is integrated into the immune and inflammatory response at a much higher level of hierarchy than previously thought. In inflammatory diseases like RA, T-cells infiltrate the synovial membrane, and tissue pathogenesis occurs through complex cell-cell and cell-humoral interactions (4850). In this model, T-cells initiate the inflammatory cascade by producing, among other factors, IL-17, which provokes resident target cells to produce proinflammatory cytokines, chemokines, adhesion molecules, matrix destructive metalloproteases, acute phase proteins, lipid mediators, and free radicals.

Normally not expressed in quiescent connective tissue cells or monocytes, the elevated levels of COX-2 mRNA, COX-2 protein, and PGE2 release observed in arthritis-affected synovial membranes have also been associated etiologically with the disease process (29, 30, 51). It has been suggested that the synovium, whether in a disease or normal state, is a PGE2-dependent tissue and that COX-2 behaves as a "master control gene" (34, 52). Thus, understanding the mechanisms responsible for dysregulated COX-2 expression is of considerable clinical concern, and the role of IL-17 in this regard takes on added significance from a therapeutic perspective.

To our knowledge, this is the first report associating IL-17 action with post-transcriptional/translational control of any target gene, although the SAPK/p38 pathway is connected with both levels of control as was shown previously (33, 34, 47, 53). The bi-phasic pattern of MKK3/6/SAPK/p38/ATF-2 phosphorylation induced by rhIL-17 was observed previously with rhIL-1{beta}, and the second phase was attributed to ambient accumulation of PGE2 (34). A bifurcation of the IL-17-induced signaling pathway to Mnk1 occurs in a slower time frame (maximum 1 h) than MKK3/6/SAPK/p38/ATF-2 but is coincident with the Ser209 phosphorylation of the CAP (N7-methylguanosine)-binding protein, eIF-4E. The latter post-translational modifications of both Mnk1 and eIF-4E were apparently p38 MAPK-dependent and suggested a link between IL-17 action and translational control. However, because the role of eIF-4E phosphorylation in translational control mechanisms is unclear (5456), and more targeted experimentation is required, such a suggestion would be premature. Perhaps paradoxically, phosphorylation of eIF4E is sometimes associated with a global inhibition of protein synthesis observed during cell stress or serum deprivation (5759). This would provide a reasonable explanation for the conspicuous phosphorylation of eIF4E after 16 h in our quiescent cultures (1% serum) both in control and rhIL-17-treated wells (Fig. 3B).

Previous studies, using transformed cell lines for the most part, emphasized the role of proximal, AU-rich containing sequences in mediating COX-2 mRNA instability (6063). Pro-inflammatory cytokines, through SAPK/p38 activation, increase steady-state levels of COX-2 mRNA by stimulating the production of cognate RNA-binding proteins (e.g. HuR) that mitigate, through sequence-specific binding, mRNA degradation (Ref. 60; reviewed in Ref. 64). The precise mechanisms, however, remain ill-defined. We find that distal sequences were exclusively reactive to the stabilizing effects of IL-17 both in terms of mRNA (stabilization) and protein synthesis in our primary cell culture models. Indeed, the absence of the AU-rich element (123 nucleotides) had no effect on IL-17-dependent stabilization. Thus, the IL-17-modulated RNA-binding protein repertoire is likely to be different from that of other cytokines. In transient transfections of HSF with expression constructs of known RNA-binding proteins (AUF1, HuR, and tristetraprolin), only tristetraprolin destabilized distal COX-2–3'-UTR reporter constructs (65).2 Interestingly, tristetraprolin is targeted by SAPK/p38 for phosphorylation (60), which may alter its RNA binding properties and function.

There are a number of cis-elements found in the promoter region of the COX-2 gene that may exert transcriptional control of which ATF/CRE, c/EBP (–132/–124), and both NF-{kappa}B sites (–223/–214 and –445/–427) are the best studied (44, 45, 66). In our cell culture models, mutating the ATF/CRE site alone, is apparently sufficient to abrogate IL-17 induction of COX-2 promoter activity and also to reduce basal promoter activity compared with wild type. In many cell types, the ATF/CRE site is activated by homodimers and heterodimers of c-Jun, c-Fos, and ATF/CREB family members subsequent to serum, 12-O-tetradecanoylphorbol-13-acetate, or growth factor stimulation (66, 67). However, rhIL-17 does not stimulate the transactivating capacities of Fos/Jun proteins but clearly favors those of ATF-2 and CREB-1. Decoy ATF-2 overexpression reduced all of the induced (partial reduction with CREB-1) and some of the basal COX-2 promoter activity, although this does not necessarily mean that ATF-2 homodimers exclusively transactivate the COX-2 promoter at the ATF/CRE; such results could be obtained if ATF-2-containing heterodimers were binding. Judging by our EMSA studies, there is considerable basal binding activity at the COX-2 ATF/CRE site, which is not surprising given that ATF-2 is expressed constitutively at significant levels both in its native and, to a lesser extent, phosphorylated forms in our cell cultures (see also Ref. 41). Induced binding by IL-17 was modest (although significant), and this may be a reflection of the fact that IL-17 activation of the COX-2 promoter was less than 2-fold. More focused experiments would be required to identify IL-17 induced ATF/CRE-binding transactivating proteins.

As indicated above, several recent studies imply that the IL-17 signal is transduced by the NF-{kappa}B signaling cascade in a number of different cell types (reviewed in Ref. 7). In the present context, NF-{kappa}B may mediate transcriptional induction of the COX-2 gene by IL-17 in bovine chondrocytes (20). In contrast, we showed here that in human chondrocyes/synovial fibroblasts, IL-17-dependent activation of NF-{kappa}B is delayed and modest (compared with TNF-{alpha}) and is not related temporally to COX-2 transcription, mRNA stabilization, or protein synthesis. Basal COX-2 promoter activity was not affected when the proximal NF-{kappa}B site was mutated in transfections using human chondrocytes, nor did the mutation abrogate IL-17 induced COX-2 promoter activity. The situation differed somewhat in HSF, because IL-17 can mildly stimulate NF-{kappa}B transactivation activity, although the mutation in the COX-2 proximal site did not compromise IL-17 induction of the COX-2 promoter. Furthermore, using stably transfected HSF overexpressing dominant-negative mutants of TRAF-2, TRAF6, or I{kappa}B-{alpha}, IL-17 activation of a NF-{kappa}B reporter was abrogated to varying degrees but not the expression of COX-2 mRNA.3 Taken together, our data support the notion that the NF-{kappa}B signaling pathway does not play an important role in mediating a response in chondrocytes to the IL-17 signal. In support, IL-17 induction of the COX-2 gene in human macrophages is indirect (delayed) and is dependent on IL-17-induced TNF-{alpha} release, which in a feedback reaction (autocrine) activates NF-{kappa}B (14). Similar results were reported in osteoblasts where IL-17 induction of inducible nitric-oxide synthase was mediated by NF-{kappa}B but only in combination with TNF-{alpha} and not with IL-17 alone (68).

Despite studies demonstrating that IL-17 can activate ERK and JNK pathways in chondrocytes (20), we could not, under carefully controlled conditions, reproduce these data in human chondrocytes or synovial fibroblasts. Our experiments with transactivation reporter systems show that downstream transcription factors such as Elk1, c-Jun, or c-Fos were not activated in our connective tissue cell culture models. Admittedly most of the previously reported studies were performed with nonhuman cell types, and thus species differences may be implicated, although it is possible that cell phenotype may also be important. For example, we showed that the cytokine could activate ERK1/2 in human monocyte/macrophage cultures, although this occurred only after 1–2 h of stimulation and did not coincide with COX-2 induction (12). This of course does not exclude the regulation of other target genes, and in this regard IL-17 is known to promote monocyte differentiation (7, 17). Furthermore, IL-17 is a potent stimulator of T-cell proliferation and differentiation, suggesting that in cells of lymphoid and myeloid origin, the mitogenic activity of the cytokine may be manifested through the MAPK pathway (7, 17, 69).

In summary, IL-17 is a widely acknowledged regulator of the immune and inflammatory response regulating directly key target genes like COX-2. The mechanisms controlling the COX-2 gene delineated in the present study involve highly coordinated regulation of both DNA-binding (transcriptional) and RNA-binding (post-transcriptional) proteins and may provide a molecular paradigm for the control of other IL-17 target genes. Interleukin-17 could prove to be an excellent therapeutic target in the clinical management of inflammatory diseases like RA using, for example, soluble IL-17 receptor preparations.


    FOOTNOTES
 
* This work was supported in part by funds from the Canadian Institutes for Health Research and the Arthritis Society of Canada (to J. D. B.). 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

|| To whom correspondence should be addressed: Div. of Rheumatology and Clinical Immunology, Royal Victoria Hospital, McGill University Health Centre, 687 Pine Ave., W., Rm. M.11.22, Montréal, PQ H3A 1A1, Canada.

1 The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagles medium; FCS, fetal calf serum; rh, recombinant human; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; ATF-2, activating transcription factor-2; PKA, cAMP-dependent protein kinase; CREB-1, cAMP-response element binding protein; MEK3 or MKK3/6, p38 MAPK kinase; UTR, untranslated region; ARE, AU-rich element; MnK1, MAPK interacting kinase; eIF-4E, eukaryotic initiation factor 4E; I{kappa}B-{alpha}, inhibitor of NF-{kappa}B; JAK, Janus family tyrosine kinase; STAT, signal transducer and activator of transcription; ERK1/2 (p42/44), extracellular signal-regulated kinase; HC, human chondrocyte(s); HSF, human synovial fibroblast(s); CMV, cytomegalovirus; RA, rheumatoid arthritis; OA, osteoarthritis; TNF-{alpha}, tumor necrosis factor-{alpha}; RT, reverse transcription; {beta}-gal, {beta}-galactosidase; EMSA, electrophoretic mobility shift assay; c/EBP, CCAAT-enhancer-binding protein; RLU, relative light unit. Back

2 W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista, unpublished observations. Back

3 W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista, manuscript in preparation. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rouvier, E., Luciani, M. F., Mattei, M. G., Denizot, F., and Golstein, P. (1993) J. Immunol. 150, 5445–5456[Abstract/Free Full Text]
  2. Yao, Z., Painter, S. L., Fanslow, W. C., Ulrich, D., Macduff, B. M., Spriggs, M. K., and Armitage, R. J. (1995) J. Immunol. 155, 5483–5486[Abstract]
  3. Fossiez, F., Djossou, O., Chomarat, P., Flores-Romo, S., Ait-Yahia, S., Maat, C., Pin, J.-J., Garrone P., Garcia E., Saeland, S., Blanchard, D., Gaillard, C., Das Mahapatra, B., Rouvier, E., Golstein, P., Banchereau, J., and Lebecque, S. (1996) J. Exp. Med. 183, 2593–2603[Abstract]
  4. Lee, J., Wei-Hsien, H., Marouka, M., Corpuz, R. T., Baldwin D. T., Foster, J. S., Goddard, A. D., Yansura, D. G. Vandlen, R. L., Wood, W. I., and Gurney A. L. (2001) J. Biol. Chem. 276, 1660–1664[Abstract/Free Full Text]
  5. Shi, Y., Ullrich, S. J., Zhang, J., Connolly, K., Grzegorzewski, K. J., Barber, M. C., Wang, W., Wathen, K., Hodge, V., Fisher, C. L., Olsen, H., Ruben, S. M., Knyazev, I., Cho, Y. H., Kao, V., Wilkinson, K. A., Carrell, J. A., and Ebner, R. (2000) J. Biol. Chem. 275, 19167–19176[Abstract/Free Full Text]
  6. Li, H., Chen, J., Huang, A., Stinson, J., Heldens, S., Foster, J., Dowd, P., Gurney, A. L., and Wood, W. I., (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 773–778[Abstract/Free Full Text]
  7. Aggarwal, S., and Gurney, A. L. (2002) J. Leukocyte Biol. 71, 1–8[Abstract/Free Full Text]
  8. Ye, P., Rodriguez, F. H., Kanaly, S., Stockling, K. L., Schurr, J., Schwarzen-berger, P., Oliver, P., Huang, W., Zhang, P., Zhang, J., Shellito, J. E., Bagby, G. J., Nelson, S., Charrier, K., Peschon, J. J., and Kolls, J. K. (2001) J. Exp. Med. 194, 519–527[Abstract/Free Full Text]
  9. Chabaud, M., Garnero, P., Dayer, J.-M., Guerne, P.-A., Fossiez, F., and Miossec, P. (2000) Cytokine 12, 1092–1099[CrossRef][Medline] [Order article via Infotrieve]
  10. Cai, L., Yin, J. P., Starovasnik, A., Hogue, D. A., Hillan, K. J., Mort, J. S., and Filvaroff, E. H. (2001) Cytokine 16, 10–21[CrossRef][Medline] [Order article via Infotrieve]
  11. Ziolkowska, M., Koc, A., Luszczykiewicz, G., Ksiezopolska-Pietrzak, K., Klimczak, E., Chwalinska-Sadowska, H., and Maslinski, W. (2000) J. Immunol. 164, 2832–2838[Abstract/Free Full Text]
  12. Jovanovic, D. V., Martel-Pelletier, J., Di Battista, J. A., Mineau, F., Jolicoeur, F.-C., Benderdour, M., and Pelletier, J.-P. (2000) Arthritis Rheum. 43, 1134–1144[CrossRef][Medline] [Order article via Infotrieve]
  13. Kotake, S., Udagawa, N., Takahashi, N., matsuzaki, K., Itoh, K., Ishiyama, S., Saito, S., Inoue, K., Kamatani, N., Gillespie, M. T., Martin, T. J., and Suda, T (1999) J. Clin. Invest. 103, 1345–1352[Abstract/Free Full Text]
  14. Jovanovic, D. V., Di Battista, J. A., Martel-Pelletier, J., Jolicoeur, F.-C., He, Y., Zhang, M., Mineau, F., and Pelletier, J.-P. (1998) J. Immunol. 160, 3513–3521[Abstract/Free Full Text]
  15. Laan, M., Lotvall, J., Chung, K. F., and Linden, A. (2001) Br. J. Pharmacol. 133, 200–206[Abstract/Free Full Text]
  16. Witowski, J., Pawlaczyk, K., Breborowicz, A., Scheuren, A., Kuzlan-Pawlaczyk, M., Wisniewska, J., Polubinska, A., Freiss, H., Gahl, G. M., Frei, U., and Jorres, A. (2000) J. Immunol. 165, 5814–5821[Abstract/Free Full Text]
  17. Cai, X.-Y., Gommoll, C. P., Jr., Justice, L., Narula, S. K., and Fine, J. S. (1998) Immunol. Lett. 62, 51–58[CrossRef][Medline] [Order article via Infotrieve]
  18. Chabaud, M., Fossiez, F., Taupin, J. L., and Moissec, P. (1998) J. Immunol. 161, 409–414[Abstract/Free Full Text]
  19. Albanesi, C., Cavani, A., and Girolomoni, G. (1999) J. Immunol. 162, 494–502[Abstract/Free Full Text]
  20. Shalom-Barak, T., Quach, J., and Lotz, M. (1998) J. Biol. Chem. 273, 27467–27473[Abstract/Free Full Text]
  21. Yamamura, Y., Gupta, R., Morita, Y., He, X., Pai, R., Endres, J., Freiberg, A., Chung, K., and Fox, D. A. (2001) J. Immunol. 166, 2270–2275[Abstract/Free Full Text]
  22. Attur, M. G., Patel, R. N., Abramson, S. B., and Amin, A. R. (1997) Arthritis Rheum. 40, 1050–1053[Medline] [Order article via Infotrieve]
  23. Yao, Z., Spriggs, M. K., Derry, J. M. J., Strockbine, L., Park, L. S., VandenBos, T., Zappone, J., Painter, S. L., and Armitage, R. J. (1997) Cytokine 9, 794–800[CrossRef][Medline] [Order article via Infotrieve]
  24. Shin, H. C. K., Benbernou, N., Esnault, S., and Guenounou, M. (1998) Cytokine 11, 257–266[CrossRef]
  25. Subramaniam, S. V., Cooper, R. S., and Adunyah, S. E. (1999) Biochem. Biophys. Res. Commun. 262, 14–19[CrossRef][Medline] [Order article via Infotrieve]
  26. Schwander, R., Yamaguchi, K., and Cao, Z. (2000) J. Exp. Med. 191, 1233–1239[Abstract/Free Full Text]
  27. Awane, M., Andres, P. G., Li, D. J., and Reinecker, H. C. (1999) J. Immunol. 162, 5337–5344[Abstract/Free Full Text]
  28. DeWitt, D. L. (1991) Biochim. Biophys. Acta 1083, 121–134[Medline] [Order article via Infotrieve]
  29. Wu, K. K. (1996) J. Lab. Clin. Med. 128, 242–245[Medline] [Order article via Infotrieve]
  30. DuBois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., Van de Putte, L. B., and Lipsky, P. E. (1998) FASEB J. 12, 1063–1073[Abstract/Free Full Text]
  31. Ryseck, R. P., Raynoscheck, C., Macdonald-Bravo, H., Dorfman, K., Mattei, M. G., and Bravo, R. (1992) Cell Growth Differ. 3, 443–450[Abstract]
  32. Newton, R. J., Seybold, J., Kuitert, L. M., Bergmann, M., and Barnes, P. J. (1998) J. Biol. Chem. 273, 32312–32321[Abstract/Free Full Text]
  33. Dean, J. L. E., Brook, M., Clark, A. R., and Saklatvala, J. (1999) J. Biol. Chem. 274, 264–269[Abstract/Free Full Text]
  34. Faour, W. H., He, Y., He, Q. W., de Ladurantaye, M., Quintero, M., Mancini, A., and Di Battista, J. A. (2001) J. Biol. Chem. 276, 31720–31731[Abstract/Free Full Text]
  35. Altman, R. D., Asch, E., Bloch, D. A., Bole, G., Borenstein, D., Brandt, K. (1986) Arthritis Rheum. 29, 1039–1049[Medline] [Order article via Infotrieve]
  36. Di Battista, J. A., Zhang, M., Martel-Pelletier, J., Fernandes, J. C., Alaaeddine, N., and Pelletier, J. P. (1999) Arthritis Rheum. 42, 157–166[CrossRef][Medline] [Order article via Infotrieve]
  37. Alaaeddine, N., Di Battista, J. A., Pelletier, J. P., Cloutier, J. M., Kiansa, K., Dupuis, M., and Martel-Pelletier, J. (1997) J. Rheumatol. 24, 1985–1994[Medline] [Order article via Infotrieve]
  38. Kalajdzic, T., Faour, W. H., He, Q. W., Fahmi, H., Martel-Pelletier, J., Pelletier, J.-P., and Di Battista, J. A. (2002) Arthritis Rheum. 46, 494–506[CrossRef][Medline] [Order article via Infotrieve]
  39. Kutchera, W., Jones, D. A., Matsunami, N., Groden, J., McIntyre, T. M., Zimmerman, G. A., White, R. L., and Prescott, S. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4816–4820[Abstract/Free Full Text]
  40. Dixon, D. A., Kaplan, C. D., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2000) J. Biol. Chem. 275, 11750–11757[Abstract/Free Full Text]
  41. Fuchs, S., Tappin, I., and Ronai, Z. (2000) J. Biol. Chem. 275, 12560–12564[Abstract/Free Full Text]
  42. Leppa, S., and Bohmann, D. (1999) Oncogene 18, 6158–6162[CrossRef][Medline] [Order article via Infotrieve]
  43. Chang, L., and Karin, M. (2001) Nature 410, 37–40[CrossRef][Medline] [Order article via Infotrieve]
  44. Appleby, S. B., Ristimäki, A., Neilson, K., Narko, K., and Hla, T. (1994) Biochem. J. 302, 723–727[Medline] [Order article via Infotrieve]
  45. Smith, W., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145–182[CrossRef][Medline] [Order article via Infotrieve]
  46. He, Q. W., He, Y., Faour, W., and Di Battista, J. A. (2001) Osteoarthritis Cart. 9B, S9
  47. Lasa, M., Maktani, K. R., Finch, A., Brewer, G., Saklatvala, J., and Clark, A. R. (2000) Mol. Cell. Biol. 20, 4265–4274[Abstract/Free Full Text]
  48. Dayer, J.-M., and Burger, D. (1999) Arthritis Res. 1, 17–20[CrossRef][Medline] [Order article via Infotrieve]
  49. Firestein, G. S. (1996) Arthritis Rheum. 39, 1781–1790[Medline] [Order article via Infotrieve]
  50. Weyand, C. M., and Goronzy, J. J. (1997) J. Mol. Med. 75, 772–785[CrossRef][Medline] [Order article via Infotrieve]
  51. Crofford, L. J., Wilder, R. L., Ristimaki, A. P., Sano, H., Remmers, E. F., Epps, H. R., and Hla, T. (1994) J. Clin. Invest. 93, 1095–1101[Medline] [Order article via Infotrieve]
  52. He, Q. W., Pelletier, J.-P., Martel-Pelletier, J., Laufer S., and Di Battista, J. A. (2002) J. Rheumatol. 29, 546–553[Medline] [Order article via Infotrieve]
  53. Kyriakis, J. M., and Avruch, J. (2001) Physiological Rev. 81, 807–869[Abstract/Free Full Text]
  54. Scheper, G. C., and Proud, C. G. (2002) Eur. J. Biochem. 269, 5350–5359[Abstract/Free Full Text]
  55. Proud, C. G. (2002) Eur. J. Biochem. 269, 5338–5349[Abstract/Free Full Text]
  56. Raught, B., and Gingras, A. C. (1999) Int. J. Biochem. Cell Biol. 31, 43–57[CrossRef][Medline] [Order article via Infotrieve]
  57. Fraser, C. S., Pain, V. M., and Morley, S. J. (1999) Biochem. J. 342, 519–526[CrossRef][Medline] [Order article via Infotrieve]
  58. Wang, X., Flynn, A., Waskiewicz, A. J., Webb, B. L. J., Vries, R. G., Baines, I. A., Cooper, J. A., and Proud, C. G. (1998) J. Biol. Chem. 273, 9373–9377[Abstract/Free Full Text]
  59. Morley, S. J., and Naegele, S. (2002) J. Biol. Chem. 277, 32855–32859[Abstract/Free Full Text]
  60. Mahtani, K. R., Brook, M., Dean, J. L., Sully, G., Saklatvala, J., and Clark, A. R. (2001) Mol. Cell. Biol. 21, 6461–6469[Abstract/Free Full Text]
  61. Chen, C.-Y., Gherzi, R., Ong, S.-E., Chan, E. L., Raijmakers, R., Prujin, G. J. M., Stoecklin, G., Moroni, C., Mann, M., and Karin, M. (2001) Cell 107, 451–464[Medline] [Order article via Infotrieve]
  62. Newton, R., Seybold, J., Liu S. F., and Barnes, P. (1997) Biochem. Biophys. Res. Commun. 234, 85–89[CrossRef][Medline] [Order article via Infotrieve]
  63. Gou, Q., Liu, C. H., Ben-Av, P., and Hla, T. (1998) Biochem. Biophys. Res. Commun. 242, 508–512[CrossRef][Medline] [Order article via Infotrieve]
  64. Bevilacqua, A., Ceriani, M. C., Capaccioli, S., and Nicolin, A. (2003) J. Cell. Physiol. 195, 356–372[CrossRef][Medline] [Order article via Infotrieve]
  65. Sawaoka, H., Dixon, D. A., Oates, J. A., and Boutaud, O. (2003) J. Biol. Chem. 278, 13928–13935[Abstract/Free Full Text]
  66. Xie, W., and Herschman, H. R. (1996) J. Biol. Chem. 271, 31742–31748[Abstract/Free Full Text]
  67. Xie, W., and Herschman, H. R. (1995) J. Biol. Chem. 270, 27622–27628[Abstract/Free Full Text]
  68. Van Bezooijen, R. L., Papapoulos, S. E., and Lowik, C. W. G. M. (2001) Bone 28, 378–386[CrossRef][Medline] [Order article via Infotrieve]
  69. Laan, M., Cui, Z. H., Hoshino, H., Lotvall, J., Sjostand, M., Gruenert, D. C., Skoogh, B. E., and Linden, A. (1999) J. Immunol. 162, 2347–2352[Abstract/Free Full Text]