The p38 MAPK mediates transcriptional and post-transcriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E2 (PGE2)-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1β-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE2 release following a recombinant human (rh) IL-1β signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE2 completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1β-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1β stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1β for 3–4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE2, unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE2 monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE2. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3′-untranslated region reporter constructs revealed that IL-1β increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE2/p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE2 release by rhIL-1β is primarily the result of PGE2-dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase. The p38 MAPK mediates transcriptional and post-transcriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E2 (PGE2)-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1β-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE2 release following a recombinant human (rh) IL-1β signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE2 completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1β-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1β stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1β for 3–4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE2, unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE2 monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE2. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3′-untranslated region reporter constructs revealed that IL-1β increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE2/p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE2 release by rhIL-1β is primarily the result of PGE2-dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase. cyclooxygenase mitogen-activated protein mitogen-activated protein kinase glyceraldehyde-3-phosphate dehydrogenase prostaglandin E2 leukotriene B4 Dulbecco's modified Eagle's medium fetal calf serum recombinant human interleukin-1β c-Jun N-terminal kinase/stress-activated protein kinase activating transcription factor-2 cAMP-dependent protein kinase p38 MAPK kinase 3′-untranslated region AU-rich element interleukin-1 kilobase pairs tumor necrosis factor-α human synovial fibroblasts polymerase chain reaction reverse transcriptase osteoarthritic rheumatoid arthritic digoxigenin enzyme-linked immunosorbent assay base pair N-[2-(cyclohexyloxy)-4-nitrophenyl)-methanesulfonamide] luciferase Cellular activation by external proinflammatory stimuli results in, among other responses, increased phospholipid-derived eicosanoid synthesis that is believed to play a cardinal role in the etiopathogenesis of many immune and inflammatory diseases (1Wu K.K. J. Lab. Clin. Med. 1996; 128: 242-245Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 2DuBois 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). Additionally, acting locally in an intracrine, autocrine, or paracrine fashion, eicosanoids initiate and modulate cell and tissue responses involved in many physiological processes affecting essentially all organ systems in the human organism (3Goetzl E.J. An S. Smith W.L. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (249) Google Scholar, 4Paul Robertson R. Trends Endocrinol. Metab. 1995; 6: 293-297Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Although synthesized through the concerted activity of multiple enzyme systems, the rate-limiting step in the formation of prostanoids is the conversion of arachidonic acid to prostaglandin H2 by cyclooxygenase (COX)1 (5Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (975) Google Scholar, 6DeWitt D.L. Biochim. Biophys. Acta. 1991; 1083: 121-134Crossref PubMed Scopus (606) Google Scholar). A constitutive and inducible form of COX has been identified, and x-ray crystallographic analyses suggest strongly that they are monotopic, endoplasmic reticulum-associated homodimeric enzymes that possess heme-dependent peroxidase and cyclooxygenase activity (7Picot D. Loll P.J. Garavito M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1154) Google Scholar). The constitutive COX-1 gene has been ascribed a homeostatic function and indeed has a GC-rich housekeeping promoter (8Wang L.H. Hajibeigi A. Xu X.M. Loose-Mitchell D. Wu K.K. Biochem. Biophys. Res. Commun. 1993; 190: 406-411Crossref PubMed Scopus (107) Google Scholar). In contrast, the COX-2 gene (mRNAs 4.6 and 2.8 kb) is rapidly induced by tumor promoters, growth factors, cytokines, and mitogens in many cell model systems (9Hla T. Neilson K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7384-7388Crossref PubMed Scopus (1488) Google Scholar, 10O'Banion M.K. Winn V.D. Young D.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4888-4892Crossref PubMed Scopus (806) Google Scholar, 11Appleby S.B. Ristimäki A. Neilson K. Narko K. Hla T. Biochem. J. 1994; 302: 723-727Crossref PubMed Scopus (460) Google Scholar). It behaves much like an immediate-early gene, and its regulation has been shown to occur at both transcriptional and post-transcriptional levels (12Ryseck R.P. Raynoscheck C. Macdonald-Bravo H. Dorfman K. Mattei M.G. Bravo R. Cell Growth Differ. 1992; 3: 443-450PubMed Google Scholar, 13Newton 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, 14Dean 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). In this regard, the COX-2 message has an extensive 3′-UTR having at least two distinct polyadenylation sites and 22 Shaw-Kamen 5′-AUUUn-A-3′ motifs (11Appleby S.B. Ristimäki A. Neilson K. Narko K. Hla T. Biochem. J. 1994; 302: 723-727Crossref PubMed Scopus (460) Google Scholar). The latter sequences are believed to be associated with message instability, translational efficiency, and rapid turnover (15Beelman C.A. Parker R. Cell. 1995; 81: 179-183Abstract Full Text PDF PubMed Scopus (573) Google Scholar,16Proudfoot N. Cell. 1996; 87: 779-781Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Furthermore, sequence analysis of the 5′-flanking region has shown several potential transcription regulatory sequences, including a TATA box, a c/EBP motif, two AP-2 sites, 3 SP-1 sites, two NF-κB sites, a CRE motif, and an Ets-1 site (no AP-1 site) (11Appleby S.B. Ristimäki A. Neilson K. Narko K. Hla T. Biochem. J. 1994; 302: 723-727Crossref PubMed Scopus (460) Google Scholar). Nevertheless, despite this wealth of structural information, it is still not totally clear how the COX-2 gene is regulated transcriptionally by external stimuli particularly in terms of the relevant signaling pathways and the transcription factors acting on 5′-flanking sequences. Even less is known about post-transcriptional regulation, although it is apparently critical in determining the amplitude and duration of the inductive process. Interleukin-1 (IL-1) occupies a prominent place in the hierarchy of proinflammatory cytokines associated with inflammatory, immune, and arthritic diseases. Indeed, there is now wide agreement, based on animal models and clinical studies, that the macrophage-derived cytokine plays a fundamental role in the development of osteoarthritis (reviewed in Ref. 17Martel-Pelletier J. Di Battista J.A. Lajeunesse D. Reginster J.-Y. Henrotin Y. Martel-Pelletier J. Pelletier J.-P. Osteoarthritis: Experimental and Clinical Aspects. Springer-Verlag, Heidelberg1999: 156-187Crossref Google Scholar). Among the plethora of genes under IL-1 control, COX-2 is particularly sensitive and is induced rapidly. In many cell types (e.g. synovial fibroblasts (18Crofford L.J. Wilder R.L. Ristimaki A.P. Sano H. Remmers E.F. Epps H.R. Hla T. J. Clin. Invest. 1994; 93: 1095-1101Crossref PubMed Scopus (663) Google Scholar) and endothelial cells (19Jones D.A. Carlton D.P. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar)), IL-1β induces COX-2 gene expression by binding to a specific cell-surface receptor (IL-1RI) that has been shown to be the mammalian homolog of the Drosophila Toll protein (20Rock F.L. Hardiman J.C. Timans R. Kasteelin R. Bazan F.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 95: 558-592Google Scholar). The binding event is followed by the activation of a signaling cascade involving the adapter protein MyD88 that recruits IL-1R-associated kinase and IL-1R-associated kinase 2 to the receptor complex. The latter Ser/Thr kinases interact with the adapter molecule TNF receptor-activated factor-6 that bridges them to the NF-κB-inducing kinase that in turn activates IκB kinases α and β (21DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar, 22Muzio M. Ni J. Feng P. Dixit D.V. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar, 23Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar, 24Zhang F.X. Kirschning C.J. Mancinellli R. Xu X.-P. Jin Y. Faure E. Mantovani A. Rothe M. Muziom M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). With the phosphorylation and degradation of IκBα, NF-κB is released to the nucleus and increases COX-2 promoter activity (25Crofford L.J. Tan B. McCarthy C.J. Hla T. Arthritis Rheum. 1997; 40: 226-236Crossref PubMed Scopus (249) Google Scholar, 26Schmedtje Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), although this has not been conclusively shown in human synovial fibroblasts. In addition, c/EBP enhancer sequences are also believed to play a role and c/EBPβ/δ synergize transcriptionally with NF-κB for full activation of the human COX-2 promoter (26Schmedtje Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 27Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Crossref PubMed Scopus (606) Google Scholar). The p38 MAPKs (four isoforms) are members of the MAPK family that are typically activated by environmental stresses and pro-inflammatory cytokines (28Herlaar E. Brown Z. Mol. Med. Today. 1999; 5: 439-447Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). The signal is initiated by membrane-proximal small GTPases of the Rho family, activation of an MAPKKK (e.g.MEKK1 and MLK), and phosphorylation and activation of an MAPKK (e.g. MKK3/6 or MEK3/6) that in turn phosphorylates and activates p38 kinase (29Ichijo H. Oncogene. 1999; 18: 6087-6093Crossref PubMed Scopus (474) Google Scholar, 30Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (555) Google Scholar). p38 kinase can phosphorylate trans-acting factors like ATF-2 and CREB-1 that render them transcriptionally competent (31Tan Y. Rouse J. Zhang A. Cariati S. Cohen P. Comb M.J. EMBO J. 1996; 15: 4629-4642Crossref PubMed Scopus (568) Google Scholar, 32Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). An alternative but perhaps less well appreciated mechanism for the mediation of IL-1β signaling is in fact the modulation of p38 MAPK activity (33Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 34Geng Y. Valbracht J. Lotz M. J. Clin. Invest. 1996; 98: 2425-2430Crossref PubMed Scopus (200) Google Scholar). It was shown that p38 not only mediates a transcriptional response, presumably at the level of the COX-2 promoter, but also at the level of COX-2 mRNA stability (14Dean 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, 35Guan Z. Buckman S.Y. Pentland A.P. Templeton D.J. Morrison A.R. J. Biol. Chem. 1998; 273: 12901-12908Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 36Lasa M. Maktani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar). Indeed, the strength and duration of COX-2 expression was largely attributed to the posttranscriptional regulatory phase in which a short (123-nucleotide) fragment of the COX-2 3′-UTR was necessary and sufficient for the regulation of mRNA stability by a p38/MAPKAPK-2/hsp 27 cascade (36Lasa M. Maktani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar). The latter nucleotide sequence interacted with a protein identified as an AU-rich element/poly(U)-binding factor I (36Lasa M. Maktani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar). In the present study, we report that the magnitude and duration of the induction of COX-2 mRNA, COX-2 protein, and PGE2release by rhIL-1β is primarily the result of PGE2-dependent stabilization of COX-2 mRNA in primary cultures of human synovial fibroblasts. In addition, PGE2 mitigates COX-2 mRNA decay and inhibition of COX-2 protein translation normally mediated by the 3′-UTR region of COX-2 mRNA. Finally, we provide evidence that the stabilization process involves a positive feedback loop and is mediated by PGE2-dependent up-regulation of the p38 MAPK cascade via the prostaglandin EP4 receptor. N-[2-(Cyclohexyloxy)-4-nitrophenyl)-methanesulfonamide] (NS-398), nimesulide,N-[4-nitro-2-phenoxyphenyl)-methanesulfonamide], indomethacin 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid, prostaglandin E2, 15-deoxy-Δ12,14-prostaglandin J2, prostaglandin D2, leukotriene B4, butaprost, and the anti-PGE2 monoclonal anti-body were purchased from Cayman Chemical (Ann Arbor, MI). Crystalline dexamethasone (9-fluoro-11β,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione), sodium fluoride, okadaic acid, leupeptin, aprotinin, pepstatin, pyrrolidinedithiocarbamate, phenylmethylsulfonyl fluoride, actinomycin D, dithiothreitol, and bovine serum albumin were products of Sigma. Verapamil, nifedipine, PD98059, SB202190, KT-5720, and rolipram were purchased from Calbiochem, and Bay 11-7082 was from Biomol (Plymouth Meeting, PA). SDS, acrylamide, bisacrylamide, ammonium persulfate, and Bio-Rad protein reagent originated from Bio-Rad. Tris base, EDTA, MgCl2, CaCl2, chloroform, dimethyl sulfoxide (Me2SO), anhydrous ethanol (95%), methanol (99%), formaldehyde, and formamide were obtained from Fisher. Human recombinant IL-1β (rhIL-1β) and recombinant human tumor necrosis factor-α (TNF-α) were purchased from Genzyme Corp. (Cambridge, MA). 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 Life Technologies, Inc. Synovial lining cells (human synovial fibroblasts, HSF) were isolated from synovial membranes (synovia) obtained at necropsy from donors with no history of arthritic disease (mean age 30 ± 27). Additional experiments were conducted with synovia from osteoarthritic (OA) and rheumatoid arthritic (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). Human synovial fibroblasts were released by sequential enzymatic digestion with 1 mg/ml Pronase (Roche Molecular Biochemicals) 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 (37Di 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, 38Alaaeddine 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). 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 (38Alaaeddine 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, 39Fahmi H. He Y. Zhang M. Martel-Pelletier J. Pelletier J.-P. Di Battista J.A. Osteoarthritis Cartilage. 2001; 9: 332-340Abstract Full Text PDF PubMed Scopus (22) 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. 50–100 µg of cellular extract (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 mmsodium orthovanadate, and 1 mm NaF) from control and treated HSF were subjected to SDS-polyacrylamide gel electrophoresis through 10% gels (final concentration of acrylamide), under reducing conditions, and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech). 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 (1:4000 dilution) was subsequently incubated with membranes for 1 h at room temperature, washed extensively for 30–40 min with TTBS, and a final rinse with TBS at room temperature. Following incubation with an ECL chemiluminescence reagent (Amersham Pharmacia Biotech), membranes were prepared for autoradiography, exposed to Kodak X-Omat film, and subjected to digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada, Mississauga, Ontario, Canada) for semi-quantitative measurements. In addition to the anti-COX-2 antiserum, the following polyclonal antibodies were used: total and anti-phospho-p38 MAP kinase (Thr-180/Tyr-182), anti-phospho-ATF-2 (Thr-69/Thr-71), anti-phospho-c-Jun (Ser-63), and anti-phospho-JNK/SAPK (Thr-183/Tyr-85). Total cellular RNA was isolated (1 × 106 cells = 10–20 µg of RNA) using the Trizol (Life Technologies, Inc.) 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 Pharmacia Biotech) in 0.5× Tris/acetate/EDTA (TAE) buffer, pH 7. After prehybridization for 24 h, hybridizations were carried out at 50 °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 (DIG)-dUTP by random priming, were used for hybridization. Human COX-2 cDNA (1.8 kb, Cayman Chemical Co.) was cloned into the EcoRV site of pcDNA 1 (Invitrogen, Carlsbad, CA) and released by PstI and XhoI digestion; a 780-bpPstI/XbaI fragment was obtained from GAPDH cDNA (1.2 kb; American Type Culture Collection, Manassas, VA). This latter probe served as a control of RNA loading as GAPDH is constitutively expressed. All blots were subjected to a digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada, Mississauga, Ontario, Canada) for semi-quantitative measurements, and changes in COX-2 expression were always considered as a ratio, COX-2/GAPDH mRNA. Transient transfection experiments were conducted in 4-, 6-, or 12-well cluster plates with HSF as described previously (37Di 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). Transfections were conducted using the FuGene 6™ (Roche Molecular Biochemicals) or LipofectAMINE 2000™ reagents (Life Technologies, Inc.) method for 6 h according to the manufacturer's protocol with cells at around 40–50% confluence. HSF were re-exposed to a culture medium + 1% FCS for 2 h prior to the addition of the biological effectors. Transfection efficiencies were controlled by co-transfection with 0.5 µg of pCMV-β-gal, a β-galactosidase reporter vector under the control of cytomegalovirus promoter. The COX-2 promoter (−2390 to + 34)-LUC plasmid was kindly provided by Dr. Stephen Prescott, University of Utah (40Kutchera 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), as were the chimeric luciferase reporter plasmids fused with the entire human COX-2 mRNA 3′-UTR (1451 bp), AU-rich elements (429 bp of which the first 116 bp contain an AU cluster) or a construct completely devoid of the COX-2 3′-UTR but containing the SV40 poly(A) signal (41Dixon 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-Δ3′-UTR. The p38 MAPK kinase (MEK3/MKK3) and pFCPKAcat expression vectors was purchased from Stratagene (La Jolla, CA). Luciferase values, expressed as relative light units, were normalized to 1 OD unit of β-galactosidase activity and protein content per sample for all experiments. The oligonucleotide primers for the polymerase chain reactions (PCR) were prepared with the aid of a DNA synthesizer (Cyclone model, Biosearch Inc., Montreal, Quebec, Canada) and used at a final concentration of 200 nmol/liter. The sequences for the luciferase primers are as follows: 5′-ACGGATTACCAGGGATTTCAGTC-3′ and 5′-AGGCTCCTCAGAAACAGCTCTTC-3′ (antisense) for the luciferase fragment of 367 bp (41Dixon 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 are 5′-CAGAACATCATCCCTGCCTCT-3′, which corresponds to position 604–624 bp of the published sequence, and 5′-GCTTGACAAAGTGGTCGTTGAG-3′, from position 901–922 bp, for an amplified product of 318 bp (37Di 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 described previously (37Di 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). 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 PCRs were done in a Gene ATAQ Controller (Amersham Pharmacia Biotech). The amplification process was conducted over 10–30 cycles in order 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 10–17; 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 described previously (37Di 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). All gel photos were subjected to a digital imaging system (see above) for semi-quantitative measurements, and results were expressed as a ratio of luciferase/GAPDH PCR fragments. Confluent HSF in 4-well cluster plates (3–5 × 106 cells/well) from control and treated cells were carefully scraped into 1.5 ml of ice-cold phosphate-buffered saline and pelleted by brief centrifugation. Nuclear extracts were prepared as described previously (37Di 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). Double-stranded oligonucleotides containing consensus and mutant sequences were from Geneka Biotechnology Inc. (Montreal, Quebec, Canada) and were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI). The sense sequences of the oligos tested are as follows: NF-κB p50, 5′-GCC ATG GGGGGATCCCCG AAG TCC-3′; NF-κB p50 mut, 5′-GCC ATG GGC C GATCCCCG AAG TCC-3′; ATF-2, 5′-GAT TCA ATGACATCA CGG CTG TG-3′; ATF-2 mut, 5′-GAT TAC AGA ACATAG CGG CTG TG-3′. Binding buffer consisted of 10 mm Tris-HCl, pH 7.5, 50 mmNaCl, 0.5 mm dithiothreitol, 0.5 mm EDTA, 1 mm MgCl2, 4% glycerol, and 2.5 µg of poly(dI-dC). Binding reactions were conducted with 15 µg of nuclear extract and 100,000 cpm of 32P-labeled oligonucleotide probe at 22 °C for 20 min in a final volume of 10 µl. 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. Measurements of the principal eicosanoids produced by HSF were performed by ELISA kits according to the manufacturer's instructions (R & D Systems, Minneapolis, MN). Detection limits for PGE2, LTB4, and PGD2 were 39, 3.9, and 7.8 pg/ml, respectively. Studies in our laboratory (39Fahmi H. He Y. Zhang M. Martel-Pelletier J. Pelletier J.-P. Di Battista J.A. Osteoarthritis Cartilage. 2001; 9: 332-340Abstract Full Text PDF PubMed Scopus (22) Google Scholar) indicate that the EC50 for rhIL-1β-dependent COX-2gene induction in our cell culture system is 5.7 pmol/liter or 100 pg/ml. As such, this concentration was chosen for all subsequent experimentation. Time course studies revealed that increases in COX-2 mRNA expression were detectable within 5 min (1.4-fold), reached greater than 10-fold after 1 h, attained stead