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Prostaglandin E2 Regulates the Level and Stability of Cyclooxygenase-2 mRNA through Activation of p38 Mitogen-activated Protein Kinase in Interleukin-1β-treated Human Synovial Fibroblasts*

  • Wissam H. Faour
    Affiliations
    Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, H2l 4M1 Québec, Canada and
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  • Yulan He
    Affiliations
    Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, H2l 4M1 Québec, Canada and
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  • Qing Wen He
    Affiliations
    Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, H2l 4M1 Québec, Canada and
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  • Manon de Ladurantaye
    Affiliations
    Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, H2l 4M1 Québec, Canada and
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  • Maritza Quintero
    Affiliations
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  • Arturo Mancini
    Affiliations
    Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, H2l 4M1 Québec, Canada and
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  • John A. Di Battista
    Correspondence
    To whom correspondence should be addressed: Unité de Recherche en Arthrose, Center Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, 1560 Rue Sherbrooke Est, Montréal, Québec H2L 4M1, Canada. Tel.: 514-281-6000, ext. 5119; Fax: 514-896-4681
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  • Author Footnotes
    * This work was supported in part by the Medical Research Council of Canada and the Arthritis Society of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:August 24, 2001DOI:https://doi.org/10.1074/jbc.M104036200
      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.
      COX
      cyclooxygenase
      MAP
      mitogen-activated protein
      MAPK
      mitogen-activated protein kinase
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      PGE2
      prostaglandin E2
      LTB4
      leukotriene B4
      DMEM
      Dulbecco's modified Eagle's medium
      FCS
      fetal calf serum
      rhIL-1β
      recombinant human interleukin-1β
      JNK/SAPK
      c-Jun N-terminal kinase/stress-activated protein kinase
      ATF-2
      activating transcription factor-2
      PKA
      cAMP-dependent protein kinase
      MEK3 or MKK3
      p38 MAPK kinase
      3′-UTR
      3′-untranslated region
      ARE
      AU-rich element
      IL-1
      interleukin-1
      kb
      kilobase pairs
      TNF-α
      tumor necrosis factor-α
      HSF
      human synovial fibroblasts
      PCR
      polymerase chain reaction
      RT
      reverse transcriptase
      OA
      osteoarthritic
      RA
      rheumatoid arthritic
      DIG
      digoxigenin
      ELISA
      enzyme-linked immunosorbent assay
      bp
      base pair
      NS-398
      N-[2-(cyclohexyloxy)-4-nitrophenyl)-methanesulfonamide]
      Luc
      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 (
      • Wu K.K.
      ,
      • DuBois R.N.
      • Abramson S.B.
      • Crofford L.
      • Gupta R.A.
      • Simon L.S.
      • Van de Putte L.B.
      • Lipsky P.E.
      ). 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 (
      • Goetzl E.J.
      • An S.
      • Smith W.L.
      ,
      • Paul Robertson R.
      ). 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 (
      • Samuelsson B.
      • Goldyne M.
      • Granstrom E.
      • Hamberg M.
      • Hammarstrom S.
      • Malmsten C.
      ,
      • DeWitt D.L.
      ). 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 (
      • Picot D.
      • Loll P.J.
      • Garavito M.
      ). The constitutive COX-1 gene has been ascribed a homeostatic function and indeed has a GC-rich housekeeping promoter (
      • Wang L.H.
      • Hajibeigi A.
      • Xu X.M.
      • Loose-Mitchell D.
      • Wu K.K.
      ). 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 (
      • Hla T.
      • Neilson K.
      ,
      • O'Banion M.K.
      • Winn V.D.
      • Young D.A.
      ,
      • Appleby S.B.
      • Ristimäki A.
      • Neilson K.
      • Narko K.
      • Hla T.
      ). It behaves much like an immediate-early gene, and its regulation has been shown to occur at both transcriptional and post-transcriptional levels (
      • Ryseck R.P.
      • Raynoscheck C.
      • Macdonald-Bravo H.
      • Dorfman K.
      • Mattei M.G.
      • Bravo R.
      ,
      • Newton R.J.
      • Seybold J.
      • Kuitert L.M.
      • Bergmann M.
      • Barnes P.J.
      ,
      • Dean J.L.E.
      • Brook M.
      • Clark A.R.
      • Saklatvala J.
      ). 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 (
      • Appleby S.B.
      • Ristimäki A.
      • Neilson K.
      • Narko K.
      • Hla T.
      ). The latter sequences are believed to be associated with message instability, translational efficiency, and rapid turnover (
      • Beelman C.A.
      • Parker R.
      ,). 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) (
      • Appleby S.B.
      • Ristimäki A.
      • Neilson K.
      • Narko K.
      • Hla T.
      ). 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.
      • Martel-Pelletier J.
      • Di Battista J.A.
      • Lajeunesse D.
      ). 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 (
      • Crofford L.J.
      • Wilder R.L.
      • Ristimaki A.P.
      • Sano H.
      • Remmers E.F.
      • Epps H.R.
      • Hla T.
      ) and endothelial cells (
      • Jones D.A.
      • Carlton D.P.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      )), 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 (
      • Rock F.L.
      • Hardiman J.C.
      • Timans R.
      • Kasteelin R.
      • Bazan F.J.
      ). 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 (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Muzio M.
      • Ni J.
      • Feng P.
      • Dixit D.V.
      ,
      • Muzio M.
      • Natoli G.
      • Saccani S.
      • Levrero M.
      • Mantovani A.
      ,
      • Zhang F.X.
      • Kirschning C.J.
      • Mancinellli R.
      • Xu X.-P.
      • Jin Y.
      • Faure E.
      • Mantovani A.
      • Rothe M.
      • Muziom M.
      • Arditi M.
      ). With the phosphorylation and degradation of IκBα, NF-κB is released to the nucleus and increases COX-2 promoter activity (
      • Crofford L.J.
      • Tan B.
      • McCarthy C.J.
      • Hla T.
      ,
      • Schmedtje Jr., J.F.
      • Ji Y.S.
      • Liu W.L.
      • DuBois R.N.
      • Runge M.S.
      ), 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 (
      • Schmedtje Jr., J.F.
      • Ji Y.S.
      • Liu W.L.
      • DuBois R.N.
      • Runge M.S.
      ,
      • Yamamoto K.
      • Arakawa T.
      • Ueda N.
      • Yamamoto S.
      ).
      The p38 MAPKs (four isoforms) are members of the MAPK family that are typically activated by environmental stresses and pro-inflammatory cytokines (
      • Herlaar E.
      • Brown Z.
      ). 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 (
      • Ichijo H.
      ,
      • Tibbles L.A.
      • Woodgett J.R.
      ). p38 kinase can phosphorylate trans-acting factors like ATF-2 and CREB-1 that render them transcriptionally competent (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ,
      • Raingeaud J.
      • Gupta S.
      • Rogers J.S.
      • Dickens M.
      • Han J.
      • Ulevitch R.J.
      • Davis R.J.
      ). An alternative but perhaps less well appreciated mechanism for the mediation of IL-1β signaling is in fact the modulation of p38 MAPK activity (
      • Freshney N.W.
      • Rawlinson L.
      • Guesdon F.
      • Jones E.
      • Cowley S.
      • Hsuan J.
      • Saklatvala J.
      ,
      • Geng Y.
      • Valbracht J.
      • Lotz M.
      ). 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 (
      • Dean J.L.E.
      • Brook M.
      • Clark A.R.
      • Saklatvala J.
      ,
      • Guan Z.
      • Buckman S.Y.
      • Pentland A.P.
      • Templeton D.J.
      • Morrison A.R.
      ,
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ). 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 (
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ). The latter nucleotide sequence interacted with a protein identified as an AU-rich element/poly(U)-binding factor I (
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ).
      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.

      DISCUSSION

      Normally not expressed, the elevated levels of COX-2 mRNA, COX-2 protein, and PGE2 release observed in OA/RA-affected synovial membranes have been associated etiologically with the disease process (
      • Martel-Pelletier J.
      • Di Battista J.A.
      • Lajeunesse D.
      ,
      • Crofford L.J.
      • Wilder R.L.
      • Ristimaki A.P.
      • Sano H.
      • Remmers E.F.
      • Epps H.R.
      • Hla T.
      ). Thus understanding the mechanisms responsible for aberrant COX-2 expression is of considerable clinical concern. It now seems clear from published evidence and the present study that theCOX-2 gene is regulated through both 5′ (transcriptional) and 3′ (post-transcriptional) regulatory elements following IL-1β signal activation in both normal (this study) and transformed cell phenotypes. We believe that the levels of IL-1β-induced COX-2 mRNA are the result of a more minor element of transcriptional control supported by robust post-transcriptional regulation (i.e. message stability and/or translational control). The COX-2 protein levels always mirrored precisely COX-2 mRNA in our studies, suggesting precise coordination between message expression and translation. Our data confirm previous work (
      • Dean J.L.E.
      • Brook M.
      • Clark A.R.
      • Saklatvala J.
      ,
      • Guan Z.
      • Buckman S.Y.
      • Pentland A.P.
      • Templeton D.J.
      • Morrison A.R.
      ,
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ) implicating the p38 MAPK cascade as critical to the magnitude and duration of cellular COX-2 mRNA. We now add, however, that the COX-2 product, PGE2, is largely responsible for this activation of p38 MAPK cascade resulting in the stabilization of COX-2 mRNA and increased COX-2 protein synthesis. In this regard, the exact contribution of the cAMP/PKA pathway (see “Results”) will require further investigation but is likely to be another example of signaling redundancy (
      • Mestre J.R.
      • Mackrel P.J.
      • Rivadeneira D.E.
      • Stapleton P.P.
      • Tanabe T.
      • Daly J.M.
      ). In this connection, our COX-2 promoter experiments seem to suggest that p38 MAP kinase and the cAMP/PKA synergize to activate transcription so that expression of the COX-2 gene may be controlled transcriptionally and post-transcriptionally by the latter two signaling pathways. The data are also consistent with a positive feedback loop involving released PGE2 binding to the prostaglandin EP4 receptor in our cell cultures. Therefore, considering the impressive amounts of PGE2 released by human OA/RA synovial membranes (in explant cultures (

      He, W., Pelletier, J. P., Martel-Pelletier, J., Di Battista, J. A., (2001) Arthritis Rheum., in press.

      )), it is conceivable that the COX-2 gene expression levels are sustained in this cyclic, positive-feedback loop.
      There are contrary reports (
      • Akarasereenont P.
      • Tachatrisak K.
      • Chotewuttakorn S.
      • Thaworn A.
      ,
      • Callejas N.A.
      • Castrillo A.
      • Bosca L.
      • Martin-Sanz P.
      ) of PGE2 inhibiting IL-1β-induced COX-2 expression in human umbilical vein endothelial cells presumably through a negative feedback loop. The design of the latter studies was different to the extent that high concentrations of exogenous PGE2 were added in co-incubation with IL-1β and, in some cases, in the absence of any inhibition of endogenous prostanoid production (e.g. by NS-398). One explanation could be that added PGE2 inhibits IL-1β-induced NF-κB-driven transactivation of the COX-2 promoter by interacting with IκB kinase, although this may require metabolism of PGE2 to cyclopentenone derivatives by human umbilical vein endothelial cells (
      • Rossi A.
      • Kapahl P.
      • Natoli G.
      • Takahashi T.
      • Chen Y.
      • Karin M.
      • Gabriella Santoro M.
      ). It is curious that IL-1β-induced PGE2 release occurs in tandem with increases with COX-2 mRNA and protein, whereas exogenously added PGE2reverses the up-regulation. Compared with HSF, human umbilical vein endothelial cells release much less PGE2 and thus COX-2 expression could not depend on PGE2 accumulation (
      • Callejas N.A.
      • Castrillo A.
      • Bosca L.
      • Martin-Sanz P.
      ). The phenomenon may be particular to endothelial cells since we have observed only PGE2-dependent positive feedback in human macrophages, chondrocytes, and skin fibroblasts.2
      The present data are consistent with a more minor element of transcriptional regulation of the COX-2 gene by IL-1β through the p38 kinase pathway occurring within the 1st hour or so. By using nuclear run-on assays, however, others have shown (
      • Newton R.
      • Kuitert L.
      • Bergmann M.
      • Adcock I.
      • Barnes P.
      ,
      • Ristimaki A.
      • Garfinkel S.
      • Wessendorf J.
      • Maciag T.
      • Hla T.
      ) 5–15-fold increases in COX-2 transcriptional rates following IL-1β stimulation which would better reflect steady-state mRNA levels normally observed. We were able to measure only a 2-fold increase in human COX-2 promoter (−1840 bp) activity after IL-1β stimulation of transiently transfected HSF; addition of NS-398, PGE2, or 15-deoxy-Δ12,14-prostaglandin J2 had no effect, although SB202190 was capable of reversing promoter induction. Furthermore, MEK3 overexpression induced human COX-2 promoter activity to levels similar those induced by rhIL-1β, and this was also quite sensitive to SB202190. The p38 MAP kinase pathway synergized with the cAMP/PKA cascade to elicit transcriptional activation. Saklatvala and co-workers (
      • Ridley S.H.
      • Dean J.L.E.
      • Sarsfield S.J.
      • Brook M.
      • Clark A.R.
      • Saklatvala J.
      ) observed, using nuclear run-on technology and HeLa cells, that IL-1β induced a 2-fold increase in transcription of theCOX-2 gene and that the effect was refractory to inhibitors of p38 kinase. Since PGE2 incubated alone doesn't stimulate transcription of the COX-2 gene, the massive increase and persistence of COX-2 mRNA is most likely due, in large part, to post-transcriptional regulation. Nevertheless, even considering the strong evidence for COX-2 mRNA stabilization, one must include some transcriptional control since both NS-398 and SB202190 inhibit 80–90%, not 100%, of IL-1β-induced COX-2 mRNA. Although PGE2 stabilized COX-2 mRNA for up to 16 h in transcriptionally arrested cells, 24 h COX-2 mRNA levels in the presence of PGE2 were no different from controls. We attribute this to rapid PGE2 metabolism (
      • Oliw E.H.
      ,
      • Oliw E.H.
      • Fahlstadius P.
      • Hamberg M.
      ), an effect that would not be as critical with cells constantly releasing PGE2 as in the presence of IL-1β for 0–48 h (see Fig. 1).
      There is some circumstantial evidence suggesting that NF-κB mediates a transcriptional induction of the COX-2 gene by IL-1 in human RA synovial fibroblasts (
      • Crofford L.J.
      • Tan B.
      • McCarthy C.J.
      • Hla T.
      ). These latter studies, however, were not designed to address the mechanisms controlling steady-state COX-2 mRNA levels and were focused on the notion of NF-κB-dependent promoter-based regulation. However, no data were reported to show a direct activation of the COX-2 reporter by NF-κB via its cognate response element(s). Evidence of this nature has been demonstrated in osteoblasts and vascular endothelial cells, as mentioned earlier (
      • Schmedtje Jr., J.F.
      • Ji Y.S.
      • Liu W.L.
      • DuBois R.N.
      • Runge M.S.
      ,
      • Yamamoto K.
      • Arakawa T.
      • Ueda N.
      • Yamamoto S.
      ). Nevertheless, it could be argued that the NF-κB cascade acts as a “priming” mechanism in our cell cultures to initiate transcription of the gene, while the PGE2-dependent p38 kinase signaling pathway stabilizes, augments, and maintains the levels of COX-2 mRNA found in cells after an IL-1β signal. The results with TNF-α may be instructive in this regard. Consider that the TNF-α-induced response, in terms of COX-2 mRNA/protein and PGE2 release, amounts to about 2.5–5% of the cellular response normally induced by IL-1β2; it is not suppressed by co-incubations in the presence of NS-398, and further additions of PGE2potentiate the effect to the extent that the steady-state levels of COX-2 mRNA become indistinguishable from those generated by IL-1β. In HSF, TNF-α is an extremely potent activator of the NF-κB cascade (
      • Crofford L.J.
      • Tan B.
      • McCarthy C.J.
      • Hla T.
      ,
      • Alaaeddine N.
      • Di Battista J.A.
      • Pelletier J.-P.
      • Kiansa K.
      • Cloutier J.-M.
      • Martel-Pelletier J.
      ) while having little or no discernible effect on p38 kinase. Redress to chemical blockade of IKK yielded equivocal results and suppressed only a fraction of IL-1β-induced COX-2 mRNA expression; the latter portion was not PGE2-reversible.2 We chose to interpret these experiments with due caution as Bay-11-7082 (IKK inhibitor) at high concentrations (2-fold EC50) is cytotoxic as are other NF-κB inhibitors especially those that block proteosomal activity. This observation is perhaps not surprising given that inhibition of IKK in primary and transformed cell lines causes apoptosis (
      • Wang C.Y.
      • Mayo M.W.
      • Baldwin A.S.
      ,
      • Karin M.
      • Delhase M.
      ). Using stable transfectants of HSF harboring either a wild type IκBα expression construct or a non-phosphorylatable IκBα mutant expression construct has not helped to resolve the transcriptional issue since there was no blockade of induced COX-2 expression by IL-1β or TNF-α,2 suggesting purely post-transcriptional p38-dependent mechanisms. In a recent publication (
      • Alaaeddine N.
      • Di Battista J.A.
      • Pelletier J.-P.
      • Kiansa K.
      • Cloutier J.-M.
      • Martel-Pelletier J.
      ), we showed that the T-helper cell (Th2)-derived cytokines IL-4 and IL-10 inhibit, virtually to 100%, TNF-α/IL-1β-stimulated cPLA2 protein, COX-2 mRNA, COX-2 protein; and PGE2 release; tandem experiments reveal that the cytokines have no effect on TNF-α/IL-1β-induced NF-κB as judged by gel-shift analysis. However, in the same study, TNF-α/IL-1β-induced c/EBP activation was potently reversed by IL-4; the critical role of c/EBP in COX-2 promoter activation has been described previously (
      • Schmedtje Jr., J.F.
      • Ji Y.S.
      • Liu W.L.
      • DuBois R.N.
      • Runge M.S.
      ,
      • Yamamoto K.
      • Arakawa T.
      • Ueda N.
      • Yamamoto S.
      ).
      Recently it was shown (
      • Freshney N.W.
      • Rawlinson L.
      • Guesdon F.
      • Jones E.
      • Cowley S.
      • Hsuan J.
      • Saklatvala J.
      ) that the strength and duration of COX-2 mRNA expression following IL-1β activation of HeLa cells could be traced to the posttranscriptional regulatory phase in which a short (123-nucleotide) fragment of the COX-2 3′-UTR was shown to be essential and sufficient for the regulation of mRNA stability by a p38/MAPKAPK-2/hsp 27 cascade. The latter nucleotide sequence interacted with a protein identified as an AU-rich-element/poly(U) binding factor I (
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ). The authors used posttranscriptional reporter constructs in which 3′-UTR fragments of COX-2 were cloned into a rabbit β-globin reporter plasmid under the control of a tetracycline operator sequence. Dominant-negative mutants of MAPKAPK-2 and SB203580, a p38 inhibitor with properties similar to SB202190, blocked the MKK3/6 expression vector stabilization of the β-globin mRNA. Others (
      • Dixon D.A.
      • Kaplan C.D.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      ,
      • Newton R.
      • Seybold J.
      • Liu S.F.
      • Barnes P.
      ,
      • Gou Q.
      • Liu C.H.
      • Ben-Av P.
      • Hla T.
      ) have used transient or stable transfections in immortalized cell lines using luciferase reporters with COX-2 3′-UTR fragments cloned downstream. Both stable and transient approaches came to the same conclusions about the importance of the AU-rich sequences in the 3′-UTR as targets for cytoplasmic/microsomal binding proteins that act to destabilize mRNA. However, the notion that mRNA stability was coupled to translational efficacy was presented, and it was argued that in fact AU sequence arrays regulate translation through a complex, yet-to-be-described interaction of AU-binding proteins and translational initiation factors. This notion is on solid ground based on data with AU-rich sequences from cytokine mRNAs (
      • Han J.
      • Brown T.
      • Beutler B.
      ,
      • Kruys V.I.
      • Wathelet M.G.
      • Huez G.A.
      ).
      Our work suggests that the COX-2 3′-UTR inhibits protein (reporter) translation and promotes rapid mRNA decay in HSF. The proximal AU-rich region and distal AUUUA sequences contribute more or less equally to the translation inhibitory effect, although the entire 3′-UTR was seemingly far more important to mRNA decay than the AU-rich region alone. Regardless, the inhibition of translation and mRNA decay were relieved when the cells are treated with IL-1β; this was a purely PGE2/p38 MAP kinase-dependent effect that reflects quite closely what we observed in our COX-2 mRNA experiments (Fig. 6 A). In contrast to the previous studies emphasizing AU-rich tract-dependent regulation (
      • Freshney N.W.
      • Rawlinson L.
      • Guesdon F.
      • Jones E.
      • Cowley S.
      • Hsuan J.
      • Saklatvala J.
      ,
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Dixon D.A.
      • Kaplan C.D.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      ), we find that distal AUUUA sequences were quite reactive to the stabilizing effects of PGE2 both in terms of mRNA and protein in HSF. Further studies will reveal whether the eicosanoid stimulates proteins that favor COX-mRNA stability (
      • Lasa M.
      • Maktani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Dean J.L.
      • Wait R.
      • Mahtani K.R.
      • Sully G.
      • Clark A.R.
      • Saklatvala J.
      ) and translation or inhibit those that serve to destabilize the message/translation. Whatever the case, it would not be surprising if we find that these proteins differ in quantity and/or composition from those isolated and identified from primate cells in heterologous systems (
      • Dixon D.A.
      • Kaplan C.D.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      ) or transformed human cell lines (e.g. HeLa, 36).
      We thus used cell-permeable pharmacological inhibitors like SB202190 and NS-398 to study COX-2 mRNA stability which always raise concerns about specificity and thus the validity of the data presented here. For example, SB202190 at higher concentrations can inhibit certain isoforms of JNK (
      • Wesselborg S.
      • Bauer M.K.A.
      • Vogt M.
      • Schmitz M.L.
      • Schulze-Osthoff K.
      ,
      • Clerk A.
      • Sugden P.H.
      ), which could lead to erroneous conclusions about the predominant role of p38 MAP kinase. This proved not to be of concern for us since JNK is not phosphorylated in HSF following rhIL-1β stimulation. The issue of SB202190 inhibiting NF-κB transactivation (
      • Wesselborg S.
      • Bauer M.K.A.
      • Vogt M.
      • Schmitz M.L.
      • Schulze-Osthoff K.
      ) following IL-1β (or TNF-α) cellular activation is probably related to cell context since, in our cell cultures, SB202190 actually had a mild stimulatory effect in this regard.
      In summary, this study describes a molecular paradigm in which membrane phospholipid-derived metabolites, released immediately following cytokine cellular activation, modulate cytokine-target gene expression in a fundamental way. It remains to be determined the degree to which target genes are modulated, the type of modulation, and if the phenomenon applies to other lipoidal metabolic pathways and/or cellular phenotypes, allowing the development of a unifying hypothesis in this regard.

      Acknowledgments

      We thank Dr. K. Kiansa for excellent cell culture work and Dr. S. Prescott for the human COX-2 promoter construct.

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