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
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m212790200
ISSN1083-351X
AutoresWissam H. Faour, Arturo Mancini, Qing He, John A. Di Battista,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoAlthough 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 ( 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. Human interleukin-17 (IL-17), 1The 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; IκB-α, inhibitor of NF-κB; 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-α, tumor necrosis factor-α; RT, reverse transcription; β-gal, β-galactosidase; EMSA, electrophoretic mobility shift assay; c/EBP, CCAAT-enhancer-binding protein; RLU, relative light unit. 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 (1Rouvier E. Luciani M.F. Mattei M.G. Denizot F. Golstein P. J. Immunol. 1993; 150: 5445-5456PubMed Google Scholar, 2Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar, 3Fossiez 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. Lebecque S. J. Exp. Med. 1996; 183: 2593-2603Crossref PubMed Scopus (1259) Google Scholar). The IL-17 gene is expressed as a 155-amino acid chain that is glycosylated and manifests biological activity as a covalent homodimer (2Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar). 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 (4Lee J. Wei-Hsien H. Marouka M. Corpuz R.T. Baldwin D.T. Foster J.S. Goddard A.D. Yansura D.G.V len R.L. Wood W.I. Gurney A.L. J. Biol. Chem. 2001; 276: 1660-1664Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 5Shi 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. Ebner R. J. Biol. Chem. 2000; 275: 19167-19176Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 6Li H. Chen J. Huang A. Stinson J. Heldens S. Foster J. Dowd P. Gurney A.L. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 773-778Crossref PubMed Scopus (281) Google Scholar). The biological activity of the IL-17 homologs is believed to overlap with that of IL-17 (4Lee J. Wei-Hsien H. Marouka M. Corpuz R.T. Baldwin D.T. Foster J.S. Goddard A.D. Yansura D.G.V len R.L. Wood W.I. Gurney A.L. J. Biol. Chem. 2001; 276: 1660-1664Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 5Shi 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. Ebner R. J. Biol. Chem. 2000; 275: 19167-19176Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 6Li H. Chen J. Huang A. Stinson J. Heldens S. Foster J. Dowd P. Gurney A.L. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 773-778Crossref PubMed Scopus (281) Google Scholar). 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. 7Aggarwal S. Gurney A.L. J. Leukocyte Biol. 2002; 71: 1-8PubMed Google Scholar and 8Ye 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. Kolls J.K. J. Exp. Med. 2001; 194: 519-527Crossref PubMed Scopus (1236) Google Scholar). 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) (7Aggarwal S. Gurney A.L. J. Leukocyte Biol. 2002; 71: 1-8PubMed Google Scholar, 8Ye 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. Kolls J.K. J. Exp. Med. 2001; 194: 519-527Crossref PubMed Scopus (1236) Google Scholar, 9Chabaud M. Garnero P. Dayer J.-M. Guerne P.-A. Fossiez F. Miossec P. Cytokine. 2000; 12: 1092-1099Crossref PubMed Scopus (236) Google Scholar, 10Cai L. Yin J.P. Starovasnik A. Hogue D.A. Hillan K.J. Mort J.S. Filvaroff E.H. Cytokine. 2001; 16: 10-21Crossref PubMed Scopus (96) Google Scholar). 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 (11Ziolkowska M. Koc A. Luszczykiewicz G. Ksiezopolska-Pietrzak K. Klimczak E. Chwalinska-Sadowska H. Maslinski W. J. Immunol. 2000; 164: 2832-2838Crossref PubMed Scopus (526) Google Scholar, 12Jovanovic D.V. Martel-Pelletier J. Di Battista J.A. Mineau F. Jolicoeur F.-C. Benderdour M. Pelletier J.-P. Arthritis Rheum. 2000; 43: 1134-1144Crossref PubMed Scopus (154) Google Scholar, 13Kotake S. Udagawa N. Takahashi N. matsuzaki K. Itoh K. Ishiyama S. Saito S. Inoue K. Kamatani N. Gillespie M.T. Martin T.J. Suda T J. Clin. Invest. 1999; 103: 1345-1352Crossref PubMed Scopus (1390) Google Scholar), 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-α and IL-1β by infiltrating macrophages, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, GRO-α, 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 (14Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.-C. He Y. Zhang M. Mineau F. Pelletier J.-P. J. Immunol. 1998; 160: 3513-3521PubMed Google Scholar, 15Laan M. Lotvall J. Chung K.F. Linden A. Br. J. Pharmacol. 2001; 133: 200-206Crossref PubMed Scopus (172) Google Scholar, 16Witowski J. Pawlaczyk K. Breborowicz A. Scheuren A. Kuzlan-Pawlaczyk M. Wisniewska J. Polubinska A. Freiss H. Gahl G.M. Frei U. Jorres A. J. Immunol. 2000; 165: 5814-5821Crossref PubMed Scopus (273) Google Scholar, 17Cai X.-Y. Gommoll Jr., C.P. Justice L. Narula S.K. Fine J.S. Immunol. Lett. 1998; 62: 51-58Crossref PubMed Scopus (90) Google Scholar, 18Chabaud M. Fossiez F. Taupin J.L. Moissec P. J. Immunol. 1998; 161: 409-414PubMed Google Scholar, 19Albanesi C. Cavani A. Girolomoni G. J. Immunol. 1999; 162: 494-502PubMed Google Scholar, 20Shalom-Barak T. Quach J. Lotz M. J. Biol. Chem. 1998; 273: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 21Yamamura Y. Gupta R. Morita Y. He X. Pai R. Endres J. Freiberg A. Chung K. Fox D.A. J. Immunol. 2001; 166: 2270-2275Crossref PubMed Scopus (88) Google Scholar, 22Attur M.G. Patel R.N. Abramson S.B. Amin A.R. Arthritis Rheum. 1997; 40: 1050-1053Crossref PubMed Scopus (171) Google Scholar). 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 (23Yao Z. Spriggs M.K. Derry J.M.J. Strockbine L. Park L.S. VandenBos T. Zappone J. Painter S.L. Armitage R.J. Cytokine. 1997; 9: 794-800Crossref PubMed Scopus (243) Google Scholar), it can transduce a signal through the activation of protein kinase A (PKA), JNK, ERK1/2, JAK/STAT, and the NF-κB cascades (20Shalom-Barak T. Quach J. Lotz M. J. Biol. Chem. 1998; 273: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 24Shin H.C.K. Benbernou N. Esnault S. Guenounou M. Cytokine. 1998; 11: 257-266Crossref Scopus (130) Google Scholar, 25Subramaniam S.V. Cooper R.S. Adunyah S.E. Biochem. Biophys. Res. Commun. 1999; 262: 14-19Crossref PubMed Scopus (99) Google Scholar, 26Schwander R. Yamaguchi K. Cao Z. J. Exp. Med. 2000; 191: 1233-1239Crossref PubMed Scopus (290) Google Scholar, 27Awane M. Andres P.G. Li D.J. Reinecker H.C. J. Immunol. 1999; 162: 5337-5344PubMed Google Scholar). 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 (20Shalom-Barak T. Quach J. Lotz M. J. Biol. Chem. 1998; 273: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Cyclooxgenase-2 mRNA expression and PGE2 release is concomitant with the stimulation of JNK1 and JNK2 by IL-17 in bovine chondrocytes (20Shalom-Barak T. Quach J. Lotz M. J. Biol. Chem. 1998; 273: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). 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 (12Jovanovic D.V. Martel-Pelletier J. Di Battista J.A. Mineau F. Jolicoeur F.-C. Benderdour M. Pelletier J.-P. Arthritis Rheum. 2000; 43: 1134-1144Crossref PubMed Scopus (154) Google Scholar). Using the same cell and culture conditions, IL-17 stimulated macrophagic release of IL-1β, TNF-α, and IL-6 was related to rapid calcium flux and a more delayed increase in NF-κB DNA binding (14Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.-C. He Y. Zhang M. Mineau F. Pelletier J.-P. J. Immunol. 1998; 160: 3513-3521PubMed Google Scholar). 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-κB and/or JNK cascades (26Schwander R. Yamaguchi K. Cao Z. J. Exp. Med. 2000; 191: 1233-1239Crossref PubMed Scopus (290) Google Scholar). 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 (28DeWitt D.L. Biochim. Biophys. Acta. 1991; 1083: 121-134Crossref PubMed Scopus (606) Google Scholar, 29Wu K.K. J. Lab. Clin. Med. 1996; 128: 242-245Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 30DuBois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van de Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2231) Google Scholar). The COX-2 gene is an inducible immediate early gene regulated at both transcriptional (promoter based) and post-transcriptional levels (31Ryseck R.P. Raynoscheck C. Macdonald-Bravo H. Dorfman K. Mattei M.G. Bravo R. Cell Growth Differ. 1992; 3: 443-450PubMed Google Scholar, 32Newton R.J. Seybold J. Kuitert L.M. Bergmann M. Barnes P.J. J. Biol. Chem. 1998; 273: 32312-32321Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 33Dean J.L.E. Brook M. Clark A.R. Saklatvala J. J. Biol. Chem. 1999; 274: 264-269Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar) and, once induced, can be largely controlled by a positive feedback loop involving PGE2 (34Faour W.H. He Y. He Q.W. de Ladurantaye M. Quintero M. Mancini A. Di Battista J.A. J. Biol. Chem. 2001; 276: 31720-31731Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). 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. Chemicals—Crystalline dexamethasone (9-fluoro-11β,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-N 6-(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-γ, and rh TNF-α 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) (35Altman R.D. Asch E. Bloch D.A. Bole G. Borenstein D. Brandt K. Arthritis Rheum. 1986; 29: 1039-1049Crossref PubMed Scopus (5363) Google Scholar). 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 (36Di Battista J.A. Zhang M. Martel-Pelletier J. Fernandes J.C. Alaaeddine N. Pelletier J.P. Arthritis Rheum. 1999; 42: 157-166Crossref PubMed Scopus (24) Google Scholar, 37Alaaeddine N. Di Battista J.A. Pelletier J.P. Cloutier J.M. Kiansa K. Dupuis M. Martel-Pelletier J. J. Rheumatol. 1997; 24: 1985-1994PubMed Google Scholar, 38Kalajdzic T. Faour W.H. He Q.W. Fahmi H. Martel-Pelletier J. Pelletier J.-P. Di Battista J.A. Arthritis Rheum. 2002; 46: 494-506Crossref PubMed Scopus (30) Google Scholar). 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 (36Di Battista J.A. Zhang M. Martel-Pelletier J. Fernandes J.C. Alaaeddine N. Pelletier J.P. Arthritis Rheum. 1999; 42: 157-166Crossref PubMed Scopus (24) Google Scholar, 37Alaaeddine N. Di Battista J.A. Pelletier J.P. Cloutier J.M. Kiansa K. Dupuis M. Martel-Pelletier J. J. Rheumatol. 1997; 24: 1985-1994PubMed Google Scholar, 38Kalajdzic T. Faour W.H. He Q.W. Fahmi H. Martel-Pelletier J. Pelletier J.-P. Di Battista J.A. Arthritis Rheum. 2002; 46: 494-506Crossref PubMed Scopus (30) Google Scholar). 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 (14Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.-C. He Y. Zhang M. Mineau F. Pelletier J.-P. J. Immunol. 1998; 160: 3513-3521PubMed Google Scholar). 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 × 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-IκB-α (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 × 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-N™ nylon membranes (Amersham Biosciences) in 0.5× 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 0.1× 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 (34Faour W.H. He Y. He Q.W. de Ladurantaye M. Quintero M. Mancini A. Di Battista J.A. J. Biol. Chem. 2001; 276: 31720-31731Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 38Kalajdzic T. Faour W.H. He Q.W. Fahmi H. Martel-Pelletier J. Pelletier J.-P. Di Battista J.A. Arthritis Rheum. 2002; 46: 494-506Crossref PubMed Scopus (30) Google Scholar). Transfections were conducted using the FuGENE 6™ (Roche Applied Science) or LipofectAMINE 2000™ 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-β-gal, a β-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) (39Kutchera W. Jones D.A. Matsunami N. Groden J. McIntyre T.M. Zimmerman G.A. White R.L. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4816-4820Crossref PubMed Scopus (445) Google Scholar), and site-directed mutagenesis was performed with the Bsu36I COX-2 promoter fragment (–415 to + 34) using the QuikChange™ (Stratagene) kit and involved modifying the ATF-CRE site at bp –53/–54 (CA → TC) and the NF-κB 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 (40Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). The plasmids are designated LUC-3′-UTR, LUC-+ARE, LUC-ΔARE, LUC-Δ3′-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 (5×) 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-κB or interferon-stimulated response element enhancer elements (5×) 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 β-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 (40Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). 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 (36Di Battista J.A. Zhang M. Martel-Pelletier J. Fernandes J.C. Alaaeddine N. Pelletier J.P. Arthritis Rheum. 1999; 42: 157-166Crossref PubMed Scopus (24) Google Scholar). Two μg of total RNA, extracted with the Trizol reagent, was reverse transcribed and then subjected to PCR as previously described (34Faour W.H. He Y. He Q.W. de Ladurantaye M. Quintero M. Mancini A. Di Battista J.A. J. Biol. Chem. 2001; 276: 31720-31731Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). RT and PCR assays were carried out with the enzymes and reagents of the GeneAmP RNA PCR kit manufactured by Perkin
Referência(s)