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Prostaglandin E2 Induced Functional Expression of Early Growth Response Factor-1 by EP4, but Not EP2, Prostanoid Receptors via the Phosphatidylinositol 3-Kinase and Extracellular Signal-regulated Kinases*

  • Hiromichi Fujino
    Affiliations
    Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207
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  • Wei Xu
    Affiliations
    Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207
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  • John W. Regan
    Correspondence
    To whom correspondence should be addressed. Tel.: 520-626-2181; Fax: 520-626-2466
    Affiliations
    Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207
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  • Author Footnotes
    * This work was supported in part by Grant EY11291 from the National Institutes of Health, Allergan Inc., and the Arizona Disease Control Research Commission.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked 舠advertisement舡 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:February 03, 2003DOI:https://doi.org/10.1074/jbc.M212665200
      Prostaglandin E2(PGE2) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP1, EP2, EP3, and EP4. The EP2 and EP4 subtypes are known to couple to Gαs and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP1 and EP3 receptors are known to couple to Gαq and Gαi, respectively. Recently we found that EP2 and EP4 receptors can activate T-cell factor signaling; however, EP2 receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP4 receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614–2619). We now report that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP4 receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE2 stimulation of EP2 receptors. These findings point to important differences in the signaling potential of the EP2 and EP4 receptors, which could be significant with respect to the potential involvement of EP4 receptors in inflammation and cancer.
      PGE2
      prostaglandin E2
      Tcf
      T-cell factor
      PKA
      cAMP-dependent protein kinase
      PI3K
      phosphatidylinositol 3-kinase
      EGR-1
      early growth response factor-1
      TNF-α
      tumor necrosis factor-α
      MAPKs
      mitogen-activated protein kinases
      ERKs
      extracellular signal-regulated kinases
      JNK
      Jun N-terminal kinase
      PKC
      protein kinase C
      HEK
      human embryonic kidney cells
      The EP2 and EP4 prostanoid receptors are two of the four primary receptor subtypes for prostaglandin E2(PGE2).1 Both of these receptors couple to Gαs and can activate adenylyl cyclase resulting in the increased formation of intracellular cyclic 3,5-adenosine monophosphate (cAMP). Prior to the molecular cloning of these receptors it was thought that the stimulation of adenylyl cyclase by PGE2 was mediated by a single receptor subtype that was defined pharmacologically as the EP2 subtype (
      • Bastien L.
      • Sawyer N.
      • Grygorczyk R.
      • Metters K.M.
      • Adams M.
      ). Thus, the first adenylyl cyclase stimulatory EP receptor to be cloned was thought to be the EP2 subtype, but it was subsequently redefined as the EP4 subtype (
      • Nishigaki N.
      • Negishi M.
      • Honda A.
      • Sugimoto Y.
      • Namba T.
      • Narumiya S.
      • Ichikawa A.
      ) when a second adenylyl cyclase stimulatory EP receptor was cloned, which had the expected pharmacology of the EP2 subtype (
      • Regan J.W.
      • Bailey T.J.
      • Pepperl D.J.
      • Pierce K.L.
      • Bogardus A.M.
      • Donello J.E.
      • Fairbairn C.E.
      • Kedzie K.M.
      • Woodward D.F.
      • Gil D.W.
      ). Surprisingly the EP2and EP4 receptors encoded by these cDNAs only shared ∼307 amino acid homology and were really no more related to each other than to other prostanoid receptor subtypes (
      • Pierce K.L.
      • Regan J.W.
      ). In fact, the EP2 receptor shows a closer phylogenetic relationship to the DP and IP receptors than it does to the EP4 receptor. The human EP4 receptor is larger than the human EP2 (488 amino acids versus 358), which is almost entirely due to a significantly longer carboxyl-terminal domain (155 versus 34 amino acids).
      Recently we have shown that PGE2 stimulation of the EP2 receptors activates a T-cell factor (Tcf) signaling pathway by a mechanism that mainly involves the activation of cAMP-dependent protein kinase (PKA) (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). PGE2stimulation of the EP4 receptors also activates Tcf signaling, but the mechanism is more complex and involves the activation of phosphatidylinositol 3-kinase (PI3K) (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). The involvement of PI3K with EP4 receptor signaling has also been suggested in studies of human colorectal carcinoma cells (
      • Sheng H.
      • Shao J.
      • Washington M.K.
      • DuBois R.N.
      ). Thus, PGE2 stimulation of endogenous EP4 receptors in LS-174 cells increased cellular proliferation and motility through a PI3K-dependent pathway. Given the known role of PI3K in carcinogenesis, it is interesting that a recent study of knockout mice suggested the potential involvement of EP4 receptors in colon cancer (
      • Mutoh M.
      • Watanabe K.
      • Kitamura T.
      • Shoji Y.
      • Takahashi M.
      • Kawamori T.
      • Tani K.
      • Kobayashi M.
      • Maruyama T.
      • Kobayashi K.
      • Ohuchida S.
      • Sugimoto Y.
      • Narumiya S.
      • Sugimura T.
      • Wakabayashi K.
      ).
      Early growth response factor-1 (EGR-1) is a member of the zinc finger family of transcription factors and plays a key role in cell growth and differentiation. It is recognized as an immediate early gene product that regulates the expression of a number of downstream genes, such as tumor necrosis factor-α (TNF-α), interleukin-2, and p53 (
      • Silverman E.S.
      • Collins T.
      ). The induction of EGR-1 expression is known to involve members of the family of mitogen-activated protein kinases (MAPKs). For example, the induction of EGR-1 expression by growth hormone was shown to depend on the phosphorylation and activation of extracellular signal-regulated kinases (ERKs), but not on the activation of either Jun N-terminal kinase (JNK) or p38 MAPK (
      • Hodge C.
      • Liao J.
      • Stofega M.
      • Guan K.
      • Carter-Su C.
      • Schwartz J.
      ). Similarly the calmodulin antagonist, trifluoroperazine, induced EGR-1 expression through the activation of ERKs and the downstream transcription factor, Elk-1 (
      • Shin S.Y.
      • Kim S.-Y.
      • Kim J.-H.
      • Min D.S.
      • Ko J.
      • Kang U.-G.
      • Kim Y.S.
      • Kwon T.K.
      • Han M.Y.
      • Kim Y.H.
      • Lee Y.H.
      ).
      Previous studies have also shown regulation of EGR-1 expression by protein kinase C (PKC), possibly involving members of the family of prostanoid receptors. For example, in Swiss 3T3 fibroblasts increases in PGE2 were associated with a PKC-dependent increase in EGR-1 mRNA expression (
      • Danesch U.
      • Weber P.C.
      • Sellmayer A.
      ). Likewise, in mouse MC3T3 osteoblastic cell lines PGE2 was found to increase EGR-1 mRNA expression. This increase was also PKC-dependent, but did not appear to involve PKA since the induction of EGR-1 mRNA expression was not observed following stimulation with forskolin, an agent which increases the formation of intracellular cAMP (
      • Fang M.A.
      • Noguchi G.M.
      • McDougall S.
      ). These findings would appear to exclude the participation of the adenylyl cyclase stimulatory EP2 and EP4 receptors in the induction of EGR-1 mRNA expression. However, because neither of these studies included pharmacological characterizations, the conclusions that can be drawn are limited with respect to the specific prostanoid receptor subtypes mediating these effects. We now show that the EP4 prostanoid receptor, but not the EP2, can induce the functional expression of EGR-1 through a signaling pathway involving the sequential activation of PI3K and the ERKs. These results further strengthen the involvement of PI3K with EP4receptor signaling and clearly differentiate the signaling potential of the EP2 and EP4 receptors.

      DISCUSSION

      We have previously shown that stimulation of EP2prostanoid receptors by PGE2 can activate a Tcf signaling pathway by a mechanism that mainly involves the activation of PKA (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). PGE2 stimulation of EP4 prostanoid receptors can also activate a Tcf signaling pathway, but the mechanism is more complex and involves the activation of PI3K (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). We now show that PGE2 stimulation of the EP4 receptor activates an additional signaling pathway involving PI3K. Thus, PGE2treatment of cells expressing EP4 receptor, leads to a PI3K-dependent phosphorylation of ERKs 1 and 2 followed by a de novo increase in the functional expression of EGR-1. The activation of this PI3K signaling cascade was unique for the EP4 receptor and was not observed in cells expressing EP2 receptors.
      One of the ways in which the EP2 and EP4receptors are known to differ is in the characteristics of their agonist induced desensitization and internalization. Thus, EP4 receptors undergo rapid, PGE2-mediated desensitization (
      • Nishigaki N.
      • Negishi M.
      • Ichikawa A.
      ) and internalization (
      • Desai S.
      • April H.
      • Nwaneshiudu C.
      • Ashby B.
      ); whereas EP2receptors do not. Since the internalization of some G-protein-coupled receptors is associated with a transactivation of the MAPK pathway (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ,
      • Naga Prasad S.V.
      • Barak L.S.
      • Rapacciuolo A.
      • Caron M.G.
      • Rockman H.A.
      ), it may be supposed that such a mechanism could explain the present findings of selective activation of ERK signaling by the EP4 receptors. However, in a previous study using human EP4 receptors expressed in HEK-293 cells, it was found that the phosphorylation of ERKs 1 and 2 was independent of PGE2-mediated receptor internalization (
      • Desai S.
      • Ashby B.
      ).
      Another way in which the EP2 and EP4 prostanoid receptors appear to differ is in their ability to stimulate intracellular cAMP formation. Thus, despite nearly identical levels of receptor expression, the maximal levels of PGE2-stimulated cAMP formation in EP4-expressing cells was only 207 of the level obtained in EP2-expressing cells (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). However, under the same conditions the ability of PGE2 to stimulate Tcf signaling was ∼507 greater in EP4-expressing cells as compared with EP2-expressing cells (
      • Fujino H.
      • West K.A.
      • Regan J.W.
      ). This indicates that the lower amounts of PGE2-stimulated cAMP formation in EP4-expressing cells is because of less efficient coupling to this pathway and not because of an overall impairment in the signaling potential of these receptors.
      The more efficient coupling of the EP2 receptor to intracellular cAMP formation; however, may be significant with respect to the present findings. Thus, it has been reported that the phosphorylation of Raf kinase by PKA inhibits the activity of Raf kinase and subsequently decreases Raf mediated MAPK signaling (
      • Dhillon A.S.
      • Pollock C.
      • Steen H.
      • Shaw P.E.
      • Mischak H.
      • Kolch W.
      ,
      • Sidovar M.F.
      • Kozlowski P.
      • Lee J.W.
      • Collins M.A.
      • He Y.
      • Graves L.M.
      ). In EP2-expressing cells, therefore, a robust activation of PKA may inhibit Raf kinase and block the phosphorylation and activation of ERKs.
      Our findings of a PGE2-mediated induction of EGR-1 expression by the EP4 receptor is interesting light of recent studies with knockout mice that show a potential involvement of the EP4 receptor with colon cancer and rheumatoid arthritis. For example EP4 knockout mice, but not EP2 knockout or control mice, show a reduced formation of preneoplastic lesions following treatment with azoxymethane, a known colon carcinogen (
      • Mutoh M.
      • Watanabe K.
      • Kitamura T.
      • Shoji Y.
      • Takahashi M.
      • Kawamori T.
      • Tani K.
      • Kobayashi M.
      • Maruyama T.
      • Kobayashi K.
      • Ohuchida S.
      • Sugimoto Y.
      • Narumiya S.
      • Sugimura T.
      • Wakabayashi K.
      ). EP4 knockout mice, but not EP2 knockout or control mice, also show a significantly decreased incidence and severity of collagen antibody induced arthritis, an animal model of rheumatoid arthritis (
      • McCoy J.M.
      • Wicks J.R.
      • Audoly L.P.
      ). In addition, in both colon cancer and rheumatoid arthritis prostaglandin levels are elevated and both conditions benefit to some extent by treatment with inhibitors of the cyclooxygenases. In colon cancer, the expression of cyclin D1, a key regulator of cell cycle progression, is known to be regulated by Tcf signaling (
      • Tetsu O.
      • McCormick F.
      ). However, it has also been reported that the expression of cyclin D1 is regulated by EGR-1 through a PI3K- and ERK-dependent pathway (
      • Guillemot L.
      • Levy A.
      • Raymondjean M.
      • Rothhut B.
      ). Furthermore it has been shown that PGE2 synthase is up-regulated by the binding of EGR-1 to the promoter region of the mouse gene encoding PGE2 synthase (
      • Naraba H.
      • Yokoyama C.
      • Tago N.
      • Murakami M.
      • Kudo I.
      • Fueki M.
      • Oh-ishi S.
      • Tanabe T.
      ). Signaling through an EP4receptor would have the potential, therefore, to increase the expression of cyclin D1 and PGE2 synthase through a PGE2-mediated induction of EGR-1 expression. Since the product of PGE2 synthase is PGE2 itself, the potential for a positive feedback loop is obvious.
      A similar situation could also explain the increased levels of PGE2 observed in rheumatoid arthritis. However, in this disease in addition to an up-regulation of the expression of PGE2 synthase, signaling through EP4 receptors could also potentially up regulate levels of TNF-α (
      • McCoy J.M.
      • Wicks J.R.
      • Audoly L.P.
      ) whose expression is under the control of EGR-1 (
      • Yao J.
      • Mackman N.
      • Edgington T.S.
      • Fan S.-T.
      ). Inhibitors of TNF-α, such as etancercept and infliximab, are used therapeutically in the treatment of rheumatoid arthritis because they slow the progression of this disease (
      • Aeberli D.
      • Oertle S.
      • Mauron H.
      • Reichenbach S.
      • Fordi B.
      • Villiger P.M.
      ). It is possible that the reduced incidence and severity of collagen antibody induced arthritis in EP4knockout mice (
      • McCoy J.M.
      • Wicks J.R.
      • Audoly L.P.
      ) is related to the loss of EP4receptor-mediated signaling through a PI3K/ERKs/EGR-1 pathway.
      Recently we have shown that the FPB prostanoid receptor can signal through a ॆ-catenin/Tcf signaling pathway (
      • Fujino H.
      • Regan J.W.
      ) and interact with PI3K (
      • Fujino H.
      • Srinivasan D.
      • Regan J.W.
      ); and thus, has potential to be involved in the pathophysiology of colon cancer. It has also been reported that EP1 receptors (
      • Watanabe K.
      • Kawamori T.
      • Nakatsugi S.
      • Ohta T.
      • Ohuchida S.
      • Yamamoto H.
      • Maruyama T.
      • Kondo K.
      • Ushikubi F.
      • Narumiya S.
      • Sugimura T.
      • Wakabayashi K.
      ) and EP2 receptors (
      • Sonoshita M.
      • Takaku K.
      • Sasaki N.
      • Sugimoto Y.
      • Ushikubi F.
      • Narumiya S.
      • Oshima M.
      • Taketo M.M.
      ) may have roles in colon cancer. In addition, gene knockout studies with the cyclooxygenases indicate that the synthesis of prostaglandins by these enzymes contributes to the pathophysiology of colon cancer (
      • Chulada P.C.
      • Thompson M.B.
      • Mahler J.F.
      • Doyle C.M.
      • Gaul B.W.
      • Lee C.
      • Tiano H.F.
      • Morham S.G.
      • Smithies O.
      • Langenbach R.
      ). Interestingly, the expression of cyclooxygenase-2 in intestinal polyps appears to be under a positive feedback through the EP2receptor (
      • Sonoshita M.
      • Takaku K.
      • Sasaki N.
      • Sugimoto Y.
      • Ushikubi F.
      • Narumiya S.
      • Oshima M.
      • Taketo M.M.
      ). Furthermore, it is clear from the findings of Sonoshitaet al. (
      • Sonoshita M.
      • Takaku K.
      • Sasaki N.
      • Sugimoto Y.
      • Ushikubi F.
      • Narumiya S.
      • Oshima M.
      • Taketo M.M.
      ) that the effects of the cyclooxygenase-2 knockout on the number and size of intestinal polyps was greater than the effects of the EP2 knockout and appear to require the involvement of another prostanoid receptor. Potential involvement of the EP4 receptor could work in concert with the EP2 receptor to increase PGE2 levels by increased expression of both cyclooxygenase-2 and PGE2synthase. Our present findings with the EP4 receptors further strengthen an association of the prostaglandins and their receptors with cancer and inflammation; further knowledge of which could have practical therapeutic benefits.

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