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15-Deoxy-Δ12,14-prostaglandin J2 and Thiazolidinediones Activate the MEK/ERK Pathway through Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells*

  • Kotaro Takeda
    Affiliations
    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
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  • Toshihiro Ichiki
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-92-642-5361; Fax: 81-92-642-5374
    Affiliations
    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
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  • Tomotake Tokunou
    Affiliations
    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
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  • Naoko Iino
    Affiliations
    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
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  • Akira Takeshita
    Affiliations
    Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
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  • Author Footnotes
    * This study was supported in part by Grants-in-aid 12877113 and 11770355 for Scientific Research from the Ministry of Education, Science and Culture, Japan.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:December 28, 2001DOI:https://doi.org/10.1074/jbc.M108722200
      Peroxisome proliferator-activated receptor (PPAR) γ belongs to the nuclear receptor superfamily of ligand-dependent transcription factors. Recent results have shown that the ligands for nuclear receptors have rapid effects so called “nongenomic” effects, which are observed within minutes after stimulation. We examined whether 15-deoxy-Δ12,14-prostaglandin J2(15-d-PGJ2) had rapid effects on cultured vascular smooth muscle cells. Phosphorylation of ERK and c-fos mRNA expression were determined by Western and Northern blot analyses, respectively. PPARγ agonists 15-d-PGJ2 and thiazolidinediones such as pioglitazone and troglitazone elicited rapid activation of ERK within 15 min and induced c-fos mRNA expression within 30 min, whereas the PPARα agonist bezafibrate failed to activate ERK. 15-d-PGJ2-induced expression of c-fos mRNA was blocked by PD98059 or U0126, two ERK kinase inhibitors, suggesting that the MEK/ERK pathway mediates 15-d-PGJ2-induced c-fos gene expression. Furthermore, pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, inhibited 15-d-PGJ2-induced ERK activation and c-fos mRNA expression, suggesting that PI3-kinase is involved in the process. An electrophoretic mobility shift assay showed that 15-d-PGJ2 enhanced AP-1 binding activity to AP-1 consensus sequence in a time-dependent manner. 15-d-PGJ2 increased thymidine incorporation in a PI3-kinase-dependent manner. Taken together, our findings show that 15-d-PGJ2 and thiazolidinediones activate the MEK/ERK pathway through PI3-kinase and lead to c-fosmRNA expression and DNA synthesis. These findings indicate a novel regulatory mechanism of gene expression by 15-d-PGJ2 and thiazolidinediones.
      PPAR
      peroxisome proliferator-activated receptor
      PPRE
      PPAR response element
      VSMC
      vascular smooth muscle cell
      15-d-PGJ2
      15-deoxy-Δ12,14-prostaglandin J2
      TZD
      thiazolidinedione
      Pio
      pioglitazone
      Tro
      troglitazone
      ERK
      extracellular signal-regulated protein kinase
      MAP
      mitogen-activated protein
      MKP
      MAP kinase phosphatase
      MEK
      MAP kinase/ERK kinase
      PMA
      phorbol 12-myristate 13-acetate
      AP-1
      activating protein 1
      PKC
      protein kinase C
      Peroxisome proliferator-activated receptor (PPAR)1 belongs to steroid/thyroid hormone nuclear receptor transcription factor superfamily, and three isoforms, designated α, γ, δ, have been identified (
      • Schoonjans K.
      • Martin G.
      • Staels B.
      • Auwerx J.
      ). PPARγ is highly expressed in adipocytes and activated macrophages and is involved in fatty acid metabolism, adipocyte differentiation (
      • Tontonoz P.
      • Hu E.
      • Spiegelman B.M.
      ), and inhibition of macrophage activation (
      • Ricote M.
      • Li A.C.
      • Willson T.M.
      • Kelly C.J.
      • Glass C.K.
      ). PPARγ is also expressed in vascular smooth muscle cells (VSMCs) (
      • Iijima K.
      • Yoshizumi M.
      • Ako J.
      • Eto M.
      • Kim S.
      • Hashimoto M.
      • Sugimoto N.
      • Liang Y.Q.
      • Sudoh N.
      • Toba K.
      • Ouchi Y.
      ). PPARγ is activated by a natural ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2) (
      • Forman B.M.
      • Tontonoz P.
      • Chen J.
      • Brun R.P.
      • Spiegelman B.M.
      • Evans R.M.
      ), and synthetic ligands, thiazolidinediones (TZDs) (
      • Spiegelman B.M.
      ) such as pioglitazone (Pio) and troglitazone (Tro). Ligand-activated PPARγ forms a heterodimer with retinoid X receptor, binds to a specific DNA sequence, PPAR response element (PPRE), and activates target gene transcription (
      • Schoonjans K.
      • Martin G.
      • Staels B.
      • Auwerx J.
      ). These effects are referred to as “genomic” action, which requires relatively long periods.
      A growing body of evidences suggests that steroid hormones have more rapid effects so-called “nongenomic” effects, which may be mediated by membrane-bound steroid receptors (
      • Schmidt B.M.
      • Gerdes D.
      • Feuring M.
      • Falkenstein E.
      • Christ M.
      • Wehling M.
      ). Estrogen is well characterized for the rapid nongenomic action. Estrogen induces an increase in the intracellular cGMP level and the release of nitric oxide within a few minutes of administration in endothelial cells (
      • Caulin-Glaser T.
      • Garcia-Cardena G.
      • Sarrel P.
      • Sessa W.C.
      • Bender J.R.
      ). Estrogen also activates extracellular signal-regulated protein kinase (ERK) within 15 min (
      • Singh M.
      • Setalo G.
      • Guan X.
      • Warren M.
      • Toran-Allerand C.D.
      ). These time courses are too rapid to encompass the classical genomic mechanism mediated by estrogen response element-dependent gene transcription. Consistent with these findings, bovine serum albumin-conjugated estrogen, which is impermeable to cell membrane, has been also reported to activate the ERK pathway in endothelial cells (
      • Russell K.S.
      • Haynes M.P.
      • Sinha D.
      • Clerisme E.
      • Bender J.R.
      ) and in a neuroblastoma cell line (
      • Watters J.J.
      • Campbell J.S.
      • Cunningham M.J.
      • Krebs E.G.
      • Dorsa D.M.
      ). These findings suggest that nongenomic actions of estrogen via novel membrane-bound estrogen receptor play a pivotal role in mediating intracellular signals.
      Phosphatidylinositol 3 (PI3)-kinase, a heterodimer of an adapter subunit (p85) and a catalytic subunit (p110), is activated by many growth factors (
      • Wymann M.P.
      • Pirola L.
      ). PI3-kinase phosphorylates the D-3 position of the inositol ring of PI to generate phosphatidylinositol 3-phosphate (
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). One of the effector molecules downstream from PI3-kinase is Akt/PKB, a serine/threonine kinase (
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ). The signaling cascade from PI3-kinase to Akt mediates cell proliferation and the cell survival signal (
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ). It has been reported that activation of the ERK kinase (MEK) ERK pathway is regulated through PI3-kinase by several stimuli (
      • Pandey S.K.
      • Theberge J.F.
      • Bernier M.
      • Srivastava A.K.
      ,
      • Miura K.
      • MacGlashan D.W.
      ,
      • Nishida M.
      • Maruyama Y.
      • Tanaka R.
      • Kontani K.
      • Nagao T.
      • Kurose H.
      ). We have demonstrated in the present study that PPARγ agonists such as 15-d-PGJ2, Pio, and Tro rapidly activated the MEK/ERK pathway and led to c-fos mRNA induction in VSMCs. We have also demonstrated that PI3-kinase was responsible for ERK activation by 15-d-PGJ2. To our knowledge, this is the first report to reveal that PPARγ agonists activate the MEK/ERK pathway in VSMCs. PPARγ agonist-induced ERK activation provides a novel regulatory mechanism of gene expression by PPARγ agonists in VSMCs.

      DISCUSSION

      We have demonstrated in the present study that 15-d-PGJ2 induces rapid and transient activation of the MEK/ERK pathway in VSMCs. Pio and Tro, two types of TZDs, also induced rapid ERK activation, suggesting that PPARγ may be involved in this process. We also showed that 15-d-PGJ2 and TZDs increased c-fos mRNA expression via activation of the MEK/ERK pathway and that PI3-kinase is responsible for 15-d-PGJ2-induced ERK activation. 15-d-PGJ2-induced DNA synthesis was partially dependent on PI3-kinase.
      15-d-PGJ2 and TZDs induced rapid activation of ERK. The time course is too rapid to encompass PPRE-dependent gene transcription. Consistent with this finding, we demonstrated that 15-d-PGJ2 could activate ERK in the presence of actinomycin D, an inhibitor of gene transcription (Fig. 3D). The analysis of the c-fos promoter sequence up to about −1300 bp showed an absence of putative PPRE sequences. These results may suggest that 15-d-PGJ2-induced ERK activation is independent of PPRE-dependent gene transcription. An increasing body of evidence suggests that steroid hormone has rapid nongenomic effects, which may be mediated by membrane-bound receptors (
      • Schmidt B.M.
      • Gerdes D.
      • Feuring M.
      • Falkenstein E.
      • Christ M.
      • Wehling M.
      ). It is not clear from our results whether membrane-bound receptor for PPARγ agonists is present in VSMCs. Further investigation using bovine serum albumin-conjugated PPARγ agonists, which are impermeable to plasma membrane, will address the presence of putative membrane-bound receptors for PPARγ agonists. However, bovine serum albumin-conjugated 15-d-PGJ2 or TZDs is not available.
      We explored the upstream signaling involved in 15-d-PGJ2-induced ERK activation. PI3-kinase has been reported to activate the MEK/ERK pathway by several stimuli (
      • Pandey S.K.
      • Theberge J.F.
      • Bernier M.
      • Srivastava A.K.
      ,
      • Miura K.
      • MacGlashan D.W.
      ,
      • Nishida M.
      • Maruyama Y.
      • Tanaka R.
      • Kontani K.
      • Nagao T.
      • Kurose H.
      ). We demonstrated that the activation of ERK by 15-d-PGJ2 was completely suppressed by wortmannin, suggesting that this process is dependent on PI3-kinase activation. The finding that 15-d-PGJ2 induced phosphorylation of Akt, a target molecule of PI3-kinase, supports this idea. However, Goetze et al. (
      • Goetze S.
      • Kim S.
      • Xi X.P.
      • Graf K.
      • Yang D.C.
      • Fleck E.
      • Meehan W.P.
      • Hsueh W.A.
      • Law R.E.
      ) obtained a contradictory result in which Tro had no effect on Akt phosphorylation in VSMCs. The reason for this discrepancy is not clear at this time. It has been reported that PI3-kinase regulates the activity of Ras (
      • Miura K.
      • MacGlashan D.W.
      ,
      • Nishida M.
      • Maruyama Y.
      • Tanaka R.
      • Kontani K.
      • Nagao T.
      • Kurose H.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), upstream molecules of the MEK/ERK pathway. Our findings suggest that activation of PI3-kinase by 15-d-PGJ2 may activate Ras and lead to activation of the MEK/ERK pathway. PKC is reported to activate the ERK pathway (
      • Haneda M.
      • Araki S.
      • Togawa M.
      • Sugimoto T.
      • Isono M.
      • Kikkawa R.
      ). However, pretreatment with PMA for 24 h or with GF109203X did not affect 15-d-PGJ2-induced ERK activation or c-fosmRNA induction (Fig. 6). These results suggest that PKC pathway does not play a dominant role in 15-d-PGJ2-induced ERK activation and c-fos mRNA induction in VSMCs.
      PPARγ agonists regulate various gene expressions; two different mechanisms have been reported previously (
      • Schoonjans K.
      • Martin G.
      • Staels B.
      • Auwerx J.
      ,
      • Ricote M.
      • Li A.C.
      • Willson T.M.
      • Kelly C.J.
      • Glass C.K.
      ). First, ligand-activated PPARγ nuclear receptor induces gene expression via binding to PPRE in the target gene promoter (
      • Schoonjans K.
      • Martin G.
      • Staels B.
      • Auwerx J.
      ), the so-called classical genomic effect. Second, ligand-activated PPARγ nuclear receptor suppresses other gene expression by antagonizing transcriptional factors such as AP-1 and NF-κB (
      • Ricote M.
      • Li A.C.
      • Willson T.M.
      • Kelly C.J.
      • Glass C.K.
      ). In the present study, we have demonstrated that PPARγ agonists induced c-fos mRNA expression in an ERK-dependent manner. This may be a novel regulatory mechanism of gene expression by PPARγ agonists. We also demonstrated that 15-d-PGJ2 enhanced AP-1 binding activity (Fig. 2C). AP-1 regulates a large number of genes associated with developmental, proliferative, and inflammatory processes. It was reported previously that estrogen suppressed gene expression of type I collagen through AP-1 in mesangial cells (
      • Neugarten J.
      • Medve I.
      • Lei J.
      • Silbiger S.R.
      ,
      • Silbiger S.
      • Lei J.
      • Neugarten J.
      ); these authors observed that estrogen stimulated AP-1 activity via ERK activation and that PD98059 inhibited ERK activity as well as suppression of collagen gene expression. It has also been reported that estrogen reduces the synthesis of type I collagen in VSMCs (
      • Silbiger S.R.
      • Neugarten J.
      ). Therefore, PPARγ agonists may reduce collagen synthesis through AP-1 activation, which may contribute to the anti-atherogenic effects of PPARγ agonists in vascular walls.
      Estrogen attenuated superoxide production and phagocytic activity in the MEK/ERK-dependent pathway in microglial cells (
      • Bruce-Keller A.J.
      • Keeling J.L.
      • Keller J.N.
      • Huang F.F.
      • Camondola S.
      • Mattson M.P.
      ). The exact mechanism by which estrogen-induced ERK activation suppresses microglial cells has not been clearly determined. One possibility is that estrogen may induce the negative feedback mechanism of ERK activation via up-regulation of MAP kinase phosphatase (MKP). It was previously reported that activated ERK induced the expression of MKP-1 (
      • Bokemeyer D.
      • Lindemann M.
      • Kramer H.J.
      ), which dephosphorylates and inactivates ERK. Therefore, it may be possible that transient MEK/ERK activation by 15-d-PGJ2 or TZDs and subsequent MKP expression may be responsible for the repression of the MEK/ERK signal induced by stimulation with other cytokines or growth factors.
      Although 15-d-PGJ2 or TZDs were reported to suppress insulin (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      )-, basic fibroblast growth factor (
      • Law R.E.
      • Goetze S.
      • Xi X.P.
      • Jackson S.
      • Kawano Y.
      • Demer L.
      • Fishbein M.C.
      • Meehan W.P.
      • Hsueh W.A.
      )-, and angiotensin II (
      • Graf K.
      • Xi X.P.
      • Hsueh W.A.
      • Law R.E.
      )-induced proliferation (or DNA synthesis) of VSMCs, the direct effects of these ligands on VSMCs have not been examined. To our surprise, 15-d-PGJ2 and TZDs were weak mitogen for VSMCs. However, it is not clear at this point whether this in vitro finding has a biological significance in vivo, and thus it requires further investigation.
      In conclusion, the present study has demonstrated that PPARγ agonists rapidly induce ERK activation and c-fos mRNA expression in VSMCs. The rapid kinetics suggest the presence of a nongenomic action of PPARγ agonists or a PPARγ-independent action of these ligands. The rapid signaling pathway of PPARγ agonists may implicate a potential mechanism responsible for the cardiovascular protective effects of PPARγ agonists.

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