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Phosphatidylinositol 3-Kinase Activity Negatively Regulates Stability of Cyclooxygenase 2 mRNA*

  • Martha M. Monick
    Correspondence
    To whom correspondence should be addressed: Division of Pulmonary, Critical Care, and Occupational Medicine, Rm. 100, EMRB, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242. Tel.: 319-335-7590; Fax: 319-335-6530
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Pamela K. Robeff
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Noah S. Butler
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Dawn M. Flaherty
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • A. Brent Carter
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Michael W. Peterson
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Gary W. Hunninghake
    Affiliations
    University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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  • Author Footnotes
    * This work was supported in part by a Veterans Administration Merit Review grant, National Institutes of Health Grants HL-60316 and ES-09607, Environmental Protection Agency Grant R826711 (to G. W. H.), National Institutes of Health Grant HL-03860 (to A. B. C.), and Grant RR00059 from the General Clinical Research Centers Program, NCRR, National Institutes of Health.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:June 18, 2002DOI:https://doi.org/10.1074/jbc.M203218200
      Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.
      LPS
      lipopolysaccharide
      COX
      cyclooxygenase
      PG
      prostaglandin
      PLA
      phospholipase
      PI3K
      phosphatidylinositol 3-kinase
      NFκB
      nuclear factor κB
      MAP
      mitogen-activated protein
      ERK
      extracellular signal-regulated kinase
      JNK
      c-Jun NH2-terminal kinase
      ELISA
      enzyme-linked immunosorbent assay
      Ct
      threshold cycles
      MKK3/6
      mitogen-activated protein kinase kinase 3/6
      MAPKAP2
      mitogen-activated protein kinase activated protein kinase-2
      HPRT
      hypoxanthine phosphorilosyltransferase
      PI
      phosphatidylinositol
      P
      phosphate
      Alveolar macrophages are major effector cells of the innate immune system. They play a central role in the response to Gram-negative bacteria in the lung. Endotoxin (LPS)1 is the principle activating component of the Gram-negative cell wall and is a major activator of these macrophages. After initial exposure to LPS, expression of prostaglandin endoperoxide H synthase 2 (COX2) and production of prostaglandin E2 (PGE2) appears at 12 to 24 h after stimulation (
      • Monick M.
      • Glazier J.
      • Hunninghake G.W.
      ). Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid and O2 to PGH2. It is the rate-limiting step in the metabolism of arachidonic acid to prostanoid products. Arachidonic acid is a 20-carbon unsaturated fatty acid that is hydrolyzed from membrane-bound phospholipids by the actions of phospholipases (PLA) (secretory PLA2 and cytosolic PLA2). Both COX1 and -2 catalyze the same step in the arachidonic acid pathway (a cyclooxygenase reaction in which arachidonic acid is converted to PGG2 and a peroxidase reaction in which PGG2 is reduced to PGH2) (
      • Smith W.L.
      • DeWitt D.L.
      • Garavito R.M.
      ,
      • Smith W.L.
      • Garavito R.M.
      • DeWitt D.L.
      ). COX1 is a constitutively present enzyme, and its products are thought to be important in gastric and renal homeostasis (
      • Smith W.L.
      • Langenbach R.
      ,
      • Brock T.G.
      • McNish R.W.
      • Peters-Golden M.
      ). It is the only cyclooxygenase present in platelets and has been linked to platelet production of thromboxane A2. In contrast, COX2 is induced by inflammatory mediators and has been linked to inflammation, fever, pain, and a number of cancers (
      • Tilley S.L.
      • Coffman T.M.
      • Koller B.H.
      ). The preferred prostanoid products of COX2 are prostacyclin and PGE2 (
      • Brock T.G.
      • McNish R.W.
      • Peters-Golden M.
      ). We have shown that in alveolar macrophages LPS induces COX2 expression and PGE2 release (
      • Monick M.
      • Glazier J.
      • Hunninghake G.W.
      ,
      • Hempel S.L.
      • Monick M.M., He, B.
      • Yano T.
      • Hunninghake G.W.
      ,
      • Hempel S.L.
      • Monick M.M.
      • Hunninghake G.W.
      ,
      • Hempel S.L.
      • Monick M.M.
      • Hunninghake G.W.
      ).
      Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric dual function lipid and protein kinase that has been linked to cell survival, transcription factor activation, and multiple signaling pathways (
      • Toker A.
      • Cantley L.C.
      ,
      • Fruman D.A.
      • Mauvais-Jarvis F.
      • Pollard D.A.
      • Yballe C.M.
      • Brazil D.
      • Bronson R.T.
      • Kahn C.R.
      • Cantley L.C.
      ,
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Wells C.
      • Waterfield M.D.
      • Ridley A.J.
      ). Class 1A PI3Ks (found in alveolar macrophages) consist of a p85-kDa subunit protein (α and β) and a p110-kDa catalytic subunit (α, β, and δ) or a p101-kDa regulatory unit and a p110-kDa catalytic unit (γ). The p85 regulatory unit is activated via interaction of the SH2 domain with YXXM motifs of multiple receptors. The p101 regulatory unit is activated by γβ subunits of G proteins downstream of G protein-coupled receptors (
      • Toker A.
      • Cantley L.C.
      ). Once activated PI3K catalyzes the transfer of ATP to thed-3 position of the inositol ring of membrane-localized phosphoinositides. This results in the production of a number of bioactive lipid species including PI3P, PI3,4P, and PI3,4,5P. Both PI3,4P and PI3,4,5P are absent in most unstimulated cells and increase dramatically following PI3K activation. The presence of PI3,4,5P results in the membrane recruitment of proteins containing pleckstrin homology domains. This includes PI3K-dependent kinase (PDK-1), which phosphorylates a number of biologically important substrates (Akt, protein kinase A, and multiple protein kinase C isoforms) (
      • Toker A.
      • Newton A.C.
      ). We have shown previously that LPS activates Akt via activation of PI3K in alveolar macrophages (
      • Monick M.M.
      • Carter A.B.
      • Flaherty D.M.
      • Peterson M.W.
      • Hunninghake G.W.
      ,
      • Monick M.M.
      • Carter A.B.
      • Robeff P.K.
      • Flaherty D.M.
      • Peterson M.W.
      • Hunninghake G.W.
      ,
      • Monick M.M.
      • Mallampalli R.K.
      • Carter A.B.
      • Flaherty D.M.
      • McCoy D.
      • Robeff P.K.
      • Peterson M.W.
      • Hunninghake G.W.
      ). Activation of Akt is linked to NFκB translocation and transactivation, endothelial nitric oxide synthase activation, and inhibition of a number of substrates positively involved in apoptosis. The apoptosis-related factors that are inhibited by Akt include glycogen synthase kinase 3, forkhead transcription factors, Bad, and caspase 9 (
      • Ivanov V.N.
      • Krasilnikov M.
      • Ronai Z.
      ). Glycogen synthase kinase 3 inhibition results in increased signaling from a number of transcription factors, β catenin, nuclear factor of activated T-cells, CCAAT/enhancer binding protein, GATA 4, and some of the activator protein 1 proteins (
      • Piwien-Pilipuk G.
      • Van Mater D.
      • Ross S.E.
      • MacDougald O.A.
      • Schwartz J.
      ). Activation of PI3K is therefore linked to multiple biological effects. One possible role of PI3K activity is as a modulator of MAP kinase signaling. Akt, in some conditions, has been shown to negatively regulate c-Raf (part of the ERK pathway in some cells) (
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ), p38 (
      • Blum S.
      • Issbruker K.
      • Willuweit A.
      • Hehlgans S.
      • Lucerna M.
      • Mechtcheriakova D.
      • Walsh K.
      • von der Ahe D.
      • Hofer E.
      • Clauss M.
      ), and stress-activated protein kinase kinase (upstream of JNK) (
      • Park H.S.
      • Kim M.S.
      • Huh S.H.
      • Park J.
      • Chung J.
      • Kang S.S.
      • Choi E.J.
      ).
      The MAP kinases are a family of evolutionarily conserved enzymes that connect cell surface receptors to regulatory targets that include both cytoplasmic and nuclear proteins. The three major MAP kinase families are the ERK (1 and 2), p38 (α, β, γ, and δ), and the JNK (1, 2, and 3) (
      • Kyriakis J.M.
      • Avruch J.
      ). LPS exposure leads to the activation of all three MAP kinase pathways (ERK, p38, and JNK) (
      • Carter A.B.
      • Monick M.M.
      • Hunninghake G.W.
      ,
      • Matsuguchi T.
      • Musikacharoen T.
      • Johnson T.R.
      • Kraft A.S.
      • Yoshikai Y.
      ), and activation of ERK and p38 has been linked to COX2 expression (
      • Subbaramaiah K.
      • Hart J.C.
      • Norton L.
      • Dannenberg A.J.
      ,
      • Subbaramaiah K.
      • Chung W.J.
      • Dannenberg A.J.
      ). p38 phosphorylates both cytoplasmic and nuclear substrates. Non-transcription factor effects of p38 include increasing mRNA stability and phosphorylation of basal transcription complex components (
      • Lasa M.
      • Mahtani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Carter A.B.
      • Knudtson K.L.
      • Monick M.M.
      • Hunninghake G.W.
      ). p38 activity has been linked to regulation of cytokine mRNA stability via its inhibitory actions on the mRNA destabilizing protein tristetraprolin (
      • Carballo E.
      • Cao H.
      • Lai W.S.
      • Kennington E.A.
      • Campbell D.
      • Blackshear P.J.
      ). More specifically, p38 activation has been linked to COX2 mRNA stability (
      • Lasa M.
      • Mahtani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Lasa M.
      • Brook M.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Jang B.C.
      • Sanchez T.
      • Schaefers H.J.
      • Trifan O.C.
      • Liu C.H.
      • Creminon C.
      • Huang C.K.
      • Hla T.
      ).
      In these studies, we found that inhibition of the PI3K pathway increased LPS-induced COX2 and PGE2. Lack of PI3K activity increased the stability of COX2 mRNA, and this increased stability correlated with increased PGE2 release. We found PI3K activity to be correlated inversely with LPS-induced p38 activity. Inhibition of PI3K resulted in increased p38 activity. These studies suggest that constitutive and LPS-induced PI3K activity in alveolar macrophages delays and decreases the production of COX2 and release of PGE2.

      DISCUSSION

      We have shown previously that LPS induces production of PGE2 in human alveolar macrophages by increasing amounts of COX2 protein and mRNA (
      • Monick M.
      • Glazier J.
      • Hunninghake G.W.
      ,
      • Brock T.G.
      • McNish R.W.
      • Peters-Golden M.
      ,
      • Tilley S.L.
      • Coffman T.M.
      • Koller B.H.
      ). We have also shown that LPS increases the activity of PI3K and its downstream kinase, Akt (
      • Toker A.
      • Newton A.C.
      ,
      • Monick M.M.
      • Carter A.B.
      • Flaherty D.M.
      • Peterson M.W.
      • Hunninghake G.W.
      ). In this study, we show that PI3K activity regulates expression of COX2 and release of PGE2 negatively. A significant effect of active PI3K is to decrease stability of COX2 mRNA. We further showed that the PI3K pathway suppressed p38 MAPK activity, and active p38 regulates stability of the COX2 mRNA positively. Of interest, we also found that human alveolar macrophages have high constitutive PI3K activity, as well as LPS-induced PI3K activity. The expression of COX2 did not increase after LPS stimulation until there was a decrease in PI3K activity to below baseline level. This occurred at late time points after LPS stimulation. These studies suggest that PI3K activity must decrease below a threshold level to permit expression of COX2.
      Fig. 12 shows the role we feel PI3K plays in COX2 expression. The figure shows formation of the well described Toll-like receptor (TLR 4) signaling pathway after macrophage LPS exposure (
      • Medzhitov R.
      ,
      • Janeway C.A., Jr.
      • Medzhitov R.
      ,
      • Horng T.
      • Barton G.M.
      • Medzhitov R.
      ). Downstream of this complex, signaling intermediates activate NFκB, all three MAP kinases (this diagram focuses on p38 only), and PI3K. The exact mechanism of PI3K activation after LPS is not known at this time, but it is known that there are two major pathways downstream of TLR 4, MyD88-dependent and MyD88-independent (
      • Toshchakov V.
      • Jones B.W.
      • Perera P.Y.
      • Thomas K.
      • Cody M.J.
      • Zhang S.
      • Williams B.R.
      • Major J.
      • Hamilton T.A.
      • Fenton M.J.
      • Vogel S.N.
      ). Future studies should determine the exact upstream activators of PI3K. PI3K, via Akt, is known to be a positive regulator of NFκB activity, suggesting the need for some PI3K activity in COX2 transcription (
      • Madrid L.V.
      • Wang C.Y.
      • Guttridge D.C.
      • Schottelius A.J.
      • Baldwin A.S., Jr.
      • Mayo M.W.
      ,
      • Koul D.
      • Yao Y.
      • Abbruzzese J.L.
      • Yung W.K.
      • Reddy S.A.
      ,
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). Our studies, however, focus on the negative regulation of p38 by PI3K and its consequence (decreased COX2 mRNA stability). The end effect of PI3K activity in LPS-treated alveolar macrophages is the decreased production of PGE2.
      Figure thumbnail gr12
      Figure 12PI3K activity negatively regulates COX2 mRNA stability via inhibition of p38. This is a diagram demonstrating the role of PI3K in LPS-induced COX2 and PGE2 production. Our data show that PI3K regulates p38 activity negatively leading to decreased mRNA stability, less COX2 production, and decreased PGE2 release. AA, arachidonic acid; IRAK, interleukin 1 receptor-associated kinase; LBP, lipopolysaccharide-binding protein; MyD88, myeloid differentiation factor 88; TIRAP, Toll-IL-1R-like homology domain-containing adaptor protein; TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6.
      This study doesn’t exclude an effect on COX2 transcription by PI3K inhibition. In fact, we have shown that an active p38 is necessary for expression of both NFκB and AP-1 luciferase activity after LPS (NFκB) or phorbol myristate acetate (AP-1) (
      • Carter A.B.
      • Knudtson K.L.
      • Monick M.M.
      • Hunninghake G.W.
      ,
      • Carter A.B.
      • Tephly L.A.
      • Hunninghake G.W.
      ). This effect of p38 is mediated by phosphorylation of TATA-binding protein, which permits this component of the basal transcription complex to bind to the TATA box and interact physically with NFκB and AP-1 proteins. However, we believe the major regulatory effect of PI3K on COX2 is at the message stability level. Mestre et al. (
      • Mestre J.R.
      • Mackrell P.J.
      • Rivadeneira D.E.
      • Stapleton P.P.
      • Tanabe T.
      • Daly J.M.
      ) have shown in a recent study that there is significant redundancy in the pathways that lead to COX2 transcription in monocyte/macrophages. They found that mutations in the NFκB, NFIL-6, or CRE sites alone did not change COX2 promoter activity. They also showed that dominant negative MAP kinases (ERK, p38, or JNK) did not decrease COX2 promoter activity.
      Several previous studies have suggested a role for p38 in the stabilization of some mRNAs. A study by Guan et al. (
      • Guan Z.
      • Buckman S.Y.
      • Pentland A.P.
      • Templeton D.J.
      • Morrison A.R.
      ) in 1998 links p38 activation to production of COX2. They showed that a dominant negative p38 upstream kinase would reduce expression of COX2. This study was followed in 2000 by a group of studies showing that p38 activity had a positive effect on COX2 mRNA stability (
      • Lasa M.
      • Mahtani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ,
      • Jang B.C.
      • Sanchez T.
      • Schaefers H.J.
      • Trifan O.C.
      • Liu C.H.
      • Creminon C.
      • Huang C.K.
      • Hla T.
      ). The study by Clark and colleagues (
      • Lasa M.
      • Mahtani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ) used a tetracycline-regulated reporter system to investigate the regulation of COX2 mRNA stability. They found that a chimeric transcript with the COX2 3′ region was stabilized by a constitutively active MKK6, as well as an active MAPKAP2. They localized the p38 effect to the first 123 nucleotides 3′ to the stop codon (
      • Lasa M.
      • Mahtani K.R.
      • Finch A.
      • Brewer G.
      • Saklatvala J.
      • Clark A.R.
      ). This same group later showed that dexamethasone inhibition of COX2 was because of a decrease in COX2 mRNA stability subsequent to inhibition of p38 (
      • Lasa M.
      • Brook M.
      • Saklatvala J.
      • Clark A.R.
      ). These studies on regulation of COX2 mRNA are consistent with our observation that PI3K inhibition increased p38 activity and stability of COX2 mRNA. p38 activation alone is not enough to induce COX2 production. In our cells, PI3K inhibition without LPS induced an increase in p38 activity but did not increase either COX2 or PGE2. This suggests that alternative pathways are activated by LPS (ERK, JNK, NFκB) that are necessary for transcriptional activation of the COX2 gene.
      A possible negative role for PI3K in MAP kinase signaling has been described. A study by Park et al. (
      • Park H.S.
      • Kim M.S.
      • Huh S.H.
      • Park J.
      • Chung J.
      • Kang S.S.
      • Choi E.J.
      ) shows that Akt (downstream of PI3K) can inhibit SEK and JNK. Gratton et al.(
      • Gratton J.P.
      • Morales-Ruiz M.
      • Kureishi Y.
      • Fulton D.
      • Walsh K.
      • Sessa W.C.
      ) have shown, in bovine aortic endothelial cells, that Akt phosphorylates and inactivates MEKK3 leading to a down-regulation of MKK3/6 and p38 activity. In Gratton's study, vascular endothelial growth factor-induced PI3K activity inhibits p38, protecting the cells from apoptosis. These studies, combined with our data, suggest that Akt may decrease activation of the stress kinases.
      PI3K negative regulation of COX2 production is a possible explanation for the observation that IL-10 decreases COX2 (
      • Berg D.J.
      • Zhang J.
      • Lauricella D.M.
      • Moore S.A.
      ). IL-10, like IL-13, has been shown to activate PI3K (
      • Pahan K.
      • Khan M.
      • Singh I.
      ,
      • Zhou J.H.
      • Broussard S.R.
      • Strle K.
      • Freund G.G.
      • Johnson R.W.
      • Dantzer R.
      • Kelley K.W.
      ), and in a separate study, IL-10 has also been shown to decrease p38 activity (
      • Kontoyiannis D.
      • Kotlyarov A.
      • Carballo E.
      • Alexopoulou L.
      • Blackshear P.J.
      • Gaestel M.
      • Davis R.
      • Flavell R.
      • Kollias G.
      ). This, in addition to our in vitro macrophage data, would suggest that modulation of PI3K activity levels may play an in vivo role in regulation of PGE2 production.
      A few studies have evaluated regulation of COX2 by PI3K. Three of these studies described outcomes opposite of ours. Tang et al.(
      • Tang Q.
      • Gonzales M.
      • Inoue H.
      • Bowden G.T.
      ) found that a dominant negative Akt suppresses UV-induced COX2 in keratinocytes. In the Tang study, PI3K inhibition increased COX2 expression. A study by Sheng et al. (
      • Sheng H.
      • Shao J.
      • Dubois R.N.
      ) looked at intestinal epithelial cells and showed that a PI3K inhibitor blocked COX2 production by activated K-Ras. Finally, a study in colon carcinoma cells found that active Akt increased COX2 expression (
      • Shao J.
      • Sheng H.
      • Inoue H.
      • Morrow J.D.
      • DuBois R.N.
      ). The difference in findings between these studies and ours could be because of cell specificity (none of these cells are immune cells) or different stimuli. The only study that has shown PI3K inhibition increasing COX2 is a study by Weaver et al. (
      • Weaver S.A.
      • Russo M.P.
      • Wright K.L.
      • Kolios G.
      • Jobin C.
      • Robertson D.A.
      • Ward S.G.
      ). Using colonic epithelial cell lines, they showed that the inhibitor wortmannin increased COX2 protein, and the inhibitor LY294002 increased COX2 mRNA. Their data were limited to these observations. Our study utilizing human alveolar macrophages and a sepsis-relevant stimulus (LPS) is the first to show negative regulation of COX2 by PI3K activity in a primary human cell. In addition, it is the first study to show a link between PI3K and destabilization of COX2 mRNA.
      In summary, the novel findings of this study include constitutive PI3K activity in primary macrophages (alveolar), PI3K-dependent suppression of p38 activity and COX2 mRNA stability, and increased LPS-induced COX2 and PGE2 production with PI3K inhibition. These observations suggest that there is both constitutive and inducible early negative regulation of COX2 activity. The late rise in COX2 activity coincides with an increase in p38 activity and a decline in PI3K activity.

      Acknowledgments

      We thank Dave Fultz for graphics assistance.

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