Advertisement

Down-regulation of the Phosphatidylinositol 3-Kinase/Akt Pathway Is Involved in Retinoic Acid-induced Phosphorylation, Degradation, and Transcriptional Activity of Retinoic Acid Receptor γ2*

  • Maurizio Giannı̀
    Footnotes
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Eliezer Kopf
    Footnotes
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Julie Bastien
    Footnotes
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Mustapha Oulad-Abdelghani
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Enrico Garattini
    Affiliations
    Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italia
    Search for articles by this author
  • Pierre Chambon
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Cécile Rochette-Egly
    Correspondence
    To whom correspondence should be addressed: Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 163, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France. Tel.: 33-3-88-65-34-59; Fax: 33-3-88-65-32-01;
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported by funds from the CNRS, the INSERM, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, and Bristol-Myers Squibb.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.
    ‡ Supported by short term fellowships from the Human Frontier Science Program, by the Association pour la Recherche sur le Cancer, and by FIRC (Fondazione Italiana per la Ricerca sul Cancro). Present address: Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italia.
    § Supported by Fondation Chateaubriand and by an INSERM fellowship. Present address: Sigma Israël, Plaut 3, Park-Rabin, Rehovot, Israël 76100.
    ¶ Supported by the Ministère de la Recherche et de l'Enseignement Supérieur.
Open AccessPublished:May 24, 2002DOI:https://doi.org/10.1074/jbc.C200230200
      Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARγ2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARγ2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARγ2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.
      RA
      retinoic acid
      RAR
      RA receptor
      RXR
      retinoid X receptor
      PI3K
      phosphatidylinositol 3-kinase
      MAPK
      mitogen-activated protein kinase
      MAPKK
      MAPK kinase
      MAPKKK
      MAPK kinase kinase
      m
      mouse
      CAT
      chloramphenicol acetyltransferase
      WCE
      whole cell extract
      WB
      Western blot
      da
      dominant active (constitutively active)
      dn
      dominant negative
      The effects of retinoic acid (RA)1 are mediated by two families of nuclear receptors, the retinoic acid receptors (RARα, -β, and -γ) and the retinoid X receptors (RXRα, -β, and -γ), which are ligand-dependent transcriptional regulators functioning as RAR/RXR heterodimers both in vivoand in vitro (
      • Chambon P.
      ,
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schutz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      • Evans R.M.
      ,
      • Kastner P.
      • Mark M.
      • Chambon P.
      ). A ligand-independent activation domain called AF-1, which is present in the N-terminal A/B region of RARs, contains serine residues (see Fig. 2 A) that are constitutively (i.e. in the absence of ligand) phosphorylated by the Cdk7 subunit of the general transcription factor TFIIH (
      • Rochette-Egly C.
      • Adam S.
      • Rossignol M.
      • Egly J.M.
      • Chambon P.
      ,
      • Bastien J.
      • Adam-Stitah S.
      • Riedl T.
      • Egly J.M.
      • Chambon P.
      • Rochette-Egly C.
      ). We recently demonstrated that phosphorylation of RARγ2 at these residues is markedly increased in response to RA through activation of p38MAPK.
      Giannı̀, M., Bauer, A., Garattini, E., Chambon, P., and Rochette-Egly, C. (2002) EMBO J., in press.
      2Giannı̀, M., Bauer, A., Garattini, E., Chambon, P., and Rochette-Egly, C. (2002) EMBO J., in press.
      This RA-induced phosphorylation is important for both RARγ2-mediated transcription of RA target genes and degradation of the receptor by the ubiquitin-proteasome pathway. The aim of the present study was to investigate how p38MAPK is activated in response to RA.
      Figure thumbnail gr2
      Figure 2RA increases RAR γ2 phosphorylation through activation of p38MAPK. A, schematic representation of the RARγ2 protein with theA–F regions (not to scale). The target sequence for phosphorylation by proline-directed kinases in the B region is shown, and the corresponding serine residues, which have been mutated to alanine (Ser-66 and Ser-68), are indicated. B, COS-1 cells transfected with the RARγ2 expression vector, either wild type (wt) or S66A/S68A (S66/68A), were treated for 24 h with vehicle or with RA as indicated. WCEs containing equal amounts of RARγ2, as checked by immunoblotting with RPγ(F), were immunoblotted with antibodies recognizing RARγ2 phosphorylated at serine 66 (P-RARγS1) or at serine 68 (P-RARγ-S2). C, RARγ2-transfected COS-1 cells were treated with RA alone or in combination with SB203580 (10 μm) or PD98059 (5 μm) as indicated. WCEs were immunoblotted with P-RARγS1 and RPγ(F). D, COS-1 cells cotransfected with the RARγ2 and p38MAPK expression vectors were treated with RA for 10 h. WCEs were immunoblotted with P-RARγS1, RPγ(F), and p38MAPK antibodies.
      Activation of p38MAPK has been traditionally associated with stress responses through a cascade of phosphorylation reactions involving upstream kinases (MAPKKK, MAPKK, and MAPK) (Refs.
      • Pearson G.
      • Robinson F.
      • Beers Gibson T., Xu, B.E.
      • Karandikar M.
      • Berman K.
      • Cobb M.H.
      ,
      • Chang L.
      • Karin M.
      ,
      • Martin-Blanco E.
      ,
      • Nebreda A.R.
      • Porras A.
      and references therein). However, it has been recently reported that p38MAPK activity could be regulated through cross-talks with the PI3K/Akt pathway (
      • Kim A.H.
      • Khursigara G.
      • Sun X.
      • Franke T.F.
      • Chao M.V.
      ,
      • Lawlor M.A.
      • Alessi D.R.
      ,
      • Park H.S.
      • Kim M.S.
      • Huh S.H.
      • Park J.
      • Chung J.
      • Kang S.S.
      • Choi E.J.
      ,
      • Gratton J.P.
      • Morales-Ruiz M.
      • Kureishi Y.
      • Fulton D.
      • Walsh K.
      • Sessa W.C.
      ). We show here that the RA-induced activation of p38MAPK and therefore the subsequent increase in RARγ2 phosphorylation resulted from the inhibition of the PI3K/Akt pathway. This down-regulation of the PI3K/Akt pathway was crucial for RA-induced degradation and transactivation activity of RARγ2, indicating that it is a key step in retinoid signaling.

      DISCUSSION

      We previously found that the RA-induced increase in RARγ2 phosphorylation is mediated through activation of p38MAPK.2Here we report that this activation implicates the down-regulation of the PI3K/Akt pathway. Indeed, blocking PI3K with wortmannin or LY294002 amplified the observed RA-induced increase in p38MAPK activity and RARγ2 phosphorylation. Reciprocally, stimulation of the PI3K/Akt pathway upon STI571 treatment or overexpression of da Akt down-regulated these processes.
      Our present results are in agreement with recent reports demonstrating that Akt negatively regulates p38MAPK (
      • Kim A.H.
      • Khursigara G.
      • Sun X.
      • Franke T.F.
      • Chao M.V.
      ,
      • Lawlor M.A.
      • Alessi D.R.
      ,
      • Park H.S.
      • Kim M.S.
      • Huh S.H.
      • Park J.
      • Chung J.
      • Kang S.S.
      • Choi E.J.
      ) and that disruption of the PI3K/Akt pathway prevents these effects, resulting in the activation of the p38MAPK (
      • Gratton J.P.
      • Morales-Ruiz M.
      • Kureishi Y.
      • Fulton D.
      • Walsh K.
      • Sessa W.C.
      ). How RA inhibits the PI3K/Akt pathway was recently elucidated in mouse embryocarcinoma cells (F9 cells) by Bastien et al.,
      Bastien, J., Plassat, J. L., Chambow, P., and Rochette-Egly, C., manuscript submitted.
      who have shown that RA acts at two levels, phosphorylation of the phosphatase PTEN and inhibition of PI3K through its p85α subunit, both of them leading to Akt inhibition.
      Interestingly our present study has demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targets not only the phosphorylation of RARγ2 through the activation of the p38MAPK but also its transcriptional activity and its degradation by the proteasome. Thus, RARγ2 phosphorylation, RARγ2 turnover, and RARγ2-mediated transcription of RA target genes are interrelated events resulting from the RA-induced down-regulation of the PI3K/Akt pathway, which therefore plays an important role in RA signaling.
      It is interesting to note that Akt is a mediator of cell growth and survival, while RA has pronounced antiproliferative potential that is usually linked to its capacity to induce differentiation. In keeping with this activity, RA is used in the treatment of several cancers (
      • Altucci L.
      • Gronemeyer H.
      ,
      • Smith M.A.
      • Anderson B.
      ). As a number of tumoral processes have been correlated with constitutively high Akt activity (
      • Lawlor M.A.
      • Alessi D.R.
      ,
      • Brazil D.P.
      • Hemmings B.A.
      ,
      • Scheid M.P.
      • Woodgett J.R.
      ) and therefore to aberrant downstream kinase activities, one can speculate that inhibition of this pathway would improve the efficiency of RA therapy. In that respect it should be noted that STI571, which not only inhibits c-Abl tyrosine kinase but also other receptor tyrosine kinases that are often amplified in carcinoma (
      • Blume-Jensen P.
      • Hunter T.
      ) and lead to increased activation of the PI3K/Akt pathway, is currently used in cancer therapy (
      • Barthe C.
      • Cony-Makhoul P.
      • Melo J.V.
      • Mahon J.R.
      ,
      • Hochhaus A.
      • Kreil S.
      • Corbin A., La
      • Rosee P.
      • Lahaye T.
      • Berger U.
      • Cross N.C.
      • Linkesch W.
      • Druker B.J.
      • Hehlmann R.
      • Gambacorti-Passerini C.
      • Corneo G.
      • D'Incalci M.
      ,
      • Gambacorti-Passerini C.
      • Barni R.
      • Marchesi E.
      • Verga M.
      • Rossi F.
      • Pioltelli P.
      • Pogliani E.
      • Corneo G.M.
      ,
      • Gorre M.E.
      • Mohammed M.
      • Ellwood K.
      • Hsu N.
      • Paquette R.
      • Rao P.N.
      • Sawyers C.L.
      ). Moreover it synergizes with retinoids in terms of cytodifferentiation and growth inhibition (
      • Giannı̀ M.
      • Kalac Y.
      • Ponzanelli I.
      • Rambaldi A.
      • Terao M.
      • Garattini E.
      ) and is capable of partially reversing the RA resistance of some acute promyelocytic leukemia cells (
      • Giannı̀ M.
      • Kalac Y.
      • Ponzanelli I.
      • Rambaldi A.
      • Terao M.
      • Garattini E.
      ). Altogether these results strongly suggest that the combination of retinoids with agents that affect the PI3K/Akt pathway could be exploited at the clinical level to improve retinoid therapy and/or reverse RA resistance. In conclusion, this study will provide new insights not only into retinoid signaling but also into retinoid therapy.

      Acknowledgments

      We acknowledge Dr. Barbara Willi for the generous gift of STI571 and Dr. P. Cohen for providing the p38MAPK expression vector. We thank Jean-Luc Plassat and Annie Bauer for help. We also thank P. Eberling for preparation of the synthetic phosphopeptides, G. Duval for the rabbit injections, and A. Tarrade for critical reading of the manuscript.

      REFERENCES

        • Chambon P.
        FASEB J. 1996; 10: 940-954
        • Mangelsdorf D.J.
        • Thummel C.
        • Beato M.
        • Herrlich P.
        • Schutz G.
        • Umesono K.
        • Blumberg B.
        • Kastner P.
        • Mark M.
        • Chambon P.
        • Evans R.M.
        Cell. 1995; 83: 835-839
        • Kastner P.
        • Mark M.
        • Chambon P.
        Cell. 1995; 83: 859-869
        • Rochette-Egly C.
        • Adam S.
        • Rossignol M.
        • Egly J.M.
        • Chambon P.
        Cell. 1997; 90: 97-107
        • Bastien J.
        • Adam-Stitah S.
        • Riedl T.
        • Egly J.M.
        • Chambon P.
        • Rochette-Egly C.
        J. Biol. Chem. 2000; 275: 21896-21904
        • Pearson G.
        • Robinson F.
        • Beers Gibson T., Xu, B.E.
        • Karandikar M.
        • Berman K.
        • Cobb M.H.
        Endocr. Rev. 2001; 22: 153-183
        • Chang L.
        • Karin M.
        Nature. 2001; 410: 37-40
        • Martin-Blanco E.
        Bioessays. 2000; 22: 637-645
        • Nebreda A.R.
        • Porras A.
        Trends Biochem. Sci. 2000; 25: 257-260
        • Kim A.H.
        • Khursigara G.
        • Sun X.
        • Franke T.F.
        • Chao M.V.
        Mol. Cell. Biol. 2001; 21: 893-901
        • Lawlor M.A.
        • Alessi D.R.
        J. Cell Sci. 2001; 114: 2903-2910
        • Park H.S.
        • Kim M.S.
        • Huh S.H.
        • Park J.
        • Chung J.
        • Kang S.S.
        • Choi E.J.
        J. Biol. Chem. 2002; 277: 2573-2578
        • Gratton J.P.
        • Morales-Ruiz M.
        • Kureishi Y.
        • Fulton D.
        • Walsh K.
        • Sessa W.C.
        J. Biol. Chem. 2001; 276: 30359-30365
        • Nagpal S.
        • Saunders M.
        • Kastner P.
        • Durand B.
        • Nakshatri H.
        • Chambon P.
        Cell. 1992; 70: 1007-1019
        • Ghyselinck N.B.
        • Dupe V.
        • Dierich A.
        • Messaddeq N.
        • Garnier J.M.
        • Rochette-Egly C.
        • Chambon P.
        • Mark M.
        Int. J. Dev. Biol. 1997; 41: 425-447
        • Boylan J.F.
        • Lohnes D.
        • Taneja R.
        • Chambon P.
        • Gudas L.J.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9601-9605
        • Alsayed Y.
        • Uddin S.
        • Mahmud N.
        • Lekmine F.
        • Kalvakolanu D.V.
        • Minucci S.
        • Bokoch G.
        • Platanias L.C.
        J. Biol. Chem. 2001; 276: 4012-4019
        • Schindler T.
        • Bornmann W.
        • Pellicena P.
        • Miller W.T.
        • Clarkson B.
        • Kuriyan J.
        Science. 2000; 289: 1938-1942
        • Yuan Z.M.
        • Utsugisawa T.
        • Huang Y.
        • Ishiko T.
        • Nakada S.
        • Kharbanda S.
        • Weichselbaum R.
        • Kufe D.
        J. Biol. Chem. 1997; 272: 23485-23488
        • Pandey P.
        • Raingeaud J.
        • Kaneki M.
        • Weichselbaum R.
        • Davis R.J.
        • Kufe D.
        • Kharbanda S.
        J. Biol. Chem. 1996; 271: 23775-23779
        • Cohen P.
        Adv. Pharmacol. 1996; 36: 15-27
        • Kopf E.
        • Plassat J.L.
        • Vivat V.
        • de The H.
        • Chambon P.
        • Rochette-Egly C.
        J. Biol. Chem. 2000; 275: 33280-33288
        • Altucci L.
        • Gronemeyer H.
        Nat. Rev. Cancer. 2001; 1: 181-193
        • Smith M.A.
        • Anderson B.
        Clin. Cancer Res. 2001; 7: 2955-2957
        • Brazil D.P.
        • Hemmings B.A.
        Trends Biochem. Sci. 2001; 26: 657-664
        • Scheid M.P.
        • Woodgett J.R.
        Nat. Rev. Mol. Cell. Biol. 2001; 2: 760-768
        • Blume-Jensen P.
        • Hunter T.
        Nature. 2001; 411: 355-365
        • Barthe C.
        • Cony-Makhoul P.
        • Melo J.V.
        • Mahon J.R.
        Science. 2001; 293: 2163
        • Hochhaus A.
        • Kreil S.
        • Corbin A., La
        • Rosee P.
        • Lahaye T.
        • Berger U.
        • Cross N.C.
        • Linkesch W.
        • Druker B.J.
        • Hehlmann R.
        • Gambacorti-Passerini C.
        • Corneo G.
        • D'Incalci M.
        Science. 2001; 293: 2163
        • Gambacorti-Passerini C.
        • Barni R.
        • Marchesi E.
        • Verga M.
        • Rossi F.
        • Pioltelli P.
        • Pogliani E.
        • Corneo G.M.
        Br. J. Haematol. 2001; 112: 972-974
        • Gorre M.E.
        • Mohammed M.
        • Ellwood K.
        • Hsu N.
        • Paquette R.
        • Rao P.N.
        • Sawyers C.L.
        Science. 2001; 293: 876-880
        • Giannı̀ M.
        • Kalac Y.
        • Ponzanelli I.
        • Rambaldi A.
        • Terao M.
        • Garattini E.
        Blood. 2001; 97: 3234-3243