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Phosphatidylinositol 3-Kinase Inhibitors Block Differentiation of Skeletal Muscle Cells*

  • Perla Kaliman
    Footnotes
    Affiliations
    Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
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  • Francesc Viñals
    Footnotes
    Affiliations
    Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
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  • Xavier Testar
    Affiliations
    Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
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  • Manuel Palacín
    Affiliations
    Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
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  • Antonio Zorzano
    Correspondence
    To whom correspondence should be addressed. Tel.: 34-3-4021519; Fax: 34-3-4021559;
    Affiliations
    Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
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  • Author Footnotes
    * This work was supported in part by research grants from the Dirección General de Investigación Científica y Técnica (PB92/0805) and Generalitat de Catalunya (GRQ 94-1040), Spain. 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 a post-doctoral fellowship from CIRIT, Generalitat de Catalunya.
    § Recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia, Spain.
Open AccessPublished:August 09, 1996DOI:https://doi.org/10.1074/jbc.271.32.19146
      Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells.

      INTRODUCTION

      The development of skeletal muscle is a multistep process that involves the determination of pluripotential mesodermal cells to give rise to myoblasts, withdrawal of the myoblasts from the cell cycle and differentiation into muscle cells, and finally growth and maturation of skeletal muscle fibers (
      • Olson E.N.
      ).
      At the molecular level, myogenic commitment and muscle-specific gene expression involve the skeletal muscle-specific helix-loop-helix (bHLH)
      The abbreviations used are: bHLH
      basic helix-loop-helix
      IGF
      insulin-like growth factor
      PI 3-kinase
      phosphatidylinositol 3-kinase
      FBS
      fetal bovine serum
      PBS
      phosphate-buffered saline
      bp
      base pair(s)
      PAGE
      polyacrylamide gel electrophoresis.
      MyoD family of proteins, which includes MyoD, myogenin, myf-5, and MRF4, and the myocyte enhancer-binding factor 2 (MEF2). Each of the MyoD-family proteins and also MEF2A indiscriminately direct non-muscle cells into the myogenic lineage by auto- and cross-activating bHLH genes (
      • Weintraub H.
      ,
      • Edmondson D.G.
      • Olson E.N.
      ,
      • Kaushal S.
      • Schneider J.W.
      • Nadal-Ginard B.
      • Mahdavi V.
      ). The myogenic activity of the MyoD family proteins requires heterodimerization with ubiquitously expressed E2a gene products to form functional transcription complexes (
      • Murre C.
      • McCaw P.S.
      • Baltimore D.
      ,
      • Chakraborty T.
      • Brennan T.J.
      • Li L.
      • Edmondson D.
      • Olson E.N.
      ). The DNA binding activity of MyoD family proteins is attenuated by Id, which forms complexes with E2a gene products in proliferating cells and is down-regulated when they are induced to differentiate (
      • Benezra R.
      • Davis R.L.
      • Lockshon D.
      • Turner D.L.
      • Weintraub H.
      ,
      • Sun X.H.
      • Copeland N.G.
      • Jenkins N.A.
      • Baltimore D.
      ).
      The decision to differentiate into myotubes is influenced negatively by several factors. Treatment of myoblasts with fetal bovine serum, basic fibroblast growth factor 2, or transforming growth factor β1 is known to inhibit differentiation of myoblasts (
      • Brennan T.J.
      • Edmondson D.G.
      • Li L.
      • Olson E.N.
      ,
      • Li L.
      • Zhou J.
      • James G.
      • Heller-Harrison R.
      • Czech M.P.
      • Olson E.N.
      ). Myogenesis is also regulated negatively by oncogenes such as c-myc, c-jun, c-fos, H-ras, and E1a (
      • Lassar A.B.
      • Thayer M.J.
      • Overell R.W.
      • Weintraub H.
      ,
      • Caruso M.
      • Martelli F.
      • Giordano A.
      • Felsani A.
      ,
      • Kong Y.
      • Johnson S.E.
      • Taparowsky E.J.
      • Konieczny S.F.
      ,
      • Alema S.
      • Tato F.
      ,
      • Bengal E.
      • Ransone L.
      • Scharfmann R.
      • Dwarki V.J.
      • Tapscott S.
      • Weintraub H.
      • Verma I.M.
      ,
      • Li L.
      • Chambard J.C.
      • Karin M.
      • Olson E.N.
      ).
      There is very little information regarding the signaling that is triggered in the myoblast upon serum withdrawal which leads to the induction of the MyoD family gene expression and to muscle differentiation. Myogenic differentiation seems to depend on the activation of integrins present on the plasma membrane of myoblasts (
      • Menko A.S.
      • Boettiger D.
      ,
      • Rosen G.D.
      • Sanes J.R.
      • LaChance R.
      • Cunningham J.M.
      • Roman J.
      • Dean D.C.
      ), suggesting the operation of an “outside-in” biochemical pathway in which integrin is the upstream molecular species. Interactions of insulin-like growth factor (IGF)-I and -II with their receptors are also positive regulators of skeletal muscle differentiation (
      • Florini J.R.
      • Ewton D.Z.
      • Magri K.A.
      ). However, the intracellular events occurring between receptor activation and terminal differentiation remain obscure.
      Phosphatidylinositol 3-kinases (PI 3-kinases) are involved in receptor signal transduction via tyrosine kinase receptors and in protein trafficking in eukaryotic cells. Multiple forms of PI 3-kinases have been reported. A well characterized group of PI 3-kinases are heterodimers composed of a regulatory p85 subunit and a catalytic p110 subunit (
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). In contrast, the recently cloned PI 3-kinase γ (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Justin Hsuan J.
      • Waterfield M.D.
      • Wetzker R.
      ) does not couple to p85 and is activated by Gβγ subunits (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ). A third group of PI 3-kinases includes the VPS34 gene product from Saccharomyces cerevisiae (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ,
      • Stack J.H.
      • Emr S.D.
      ) and its human homolog (
      • Volinia S.
      • Dhand R.
      • Vanhaesebroeck B.
      • MacDougall L.K.
      • Stein R.
      • Zvelebil M.J.
      • Domin J.
      • Panaretou C.
      • Waterfield M.D.
      ). The Vps34p only phosphorylates the 3′ position of PI and is required for vacuolar protein sorting (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). This last group of PI 3-kinases associate with a 150-160-kDa protein, which in S. cerevisiae is a serine/threonine kinase essential for Vps34p activity (
      • Stack J.H.
      • Dewald D.B.
      • Takegawa K.
      • Emr S.D.
      ). All PI 3-kinase isoforms so far described are potently inhibited in the nanomolar or low micromolar range by two structurally unrelated membrane permeant reagents: wortmannin (for review, see Ref.
      • Ui M.
      • Okada T.
      • Hazeki K.
      • Hazeki O.
      ) and LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ).
      While much information has been accumulated on the role of PI 3-kinase activities in tyrosine kinase receptor signal transduction or vesicle trafficking, little is known about the possible role of PI 3-kinases in cell differentiation. In this regard, it has been observed that PI 3-kinase might be involved in neurite outgrowth of PC12 cells (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ). In a previous report, we showed that PI 3-kinase activity and insulin-stimulated glucose transport in L6E9 muscle cells were inhibited in a dose-response manner by a 1-h treatment with wortmannin (
      • Kaliman P.
      • Viñals F.
      • Testar X.
      • Palacín M.
      • Zorzano A.
      ). Here we show that chronic treatment with LY294002 or wortmannin blocked morphological and biochemical changes associated with L6E9 myoblast terminal differentiation and that this inhibition is not bypassed by the overexpression of MyoD in 10T1/2 cells. Data presented here implicate PI 3-kinase as an essential positive regulator of terminal differentiation of skeletal muscle cells.

      DISCUSSION

      In this study we have shown that two structurally different inhibitors of phosphatidylinositol 3-kinase, i.e. LY294002 and wortmannin, blocked both the morphological and the biochemical differentiation of two skeletal muscle models, L6E9 myoblasts and 10T1/2 cells stably transfected with MyoD (10T1/2-MyoD).
      In a previous report, we showed that PI 3-kinase activity and insulin-stimulated glucose transport in L6E9 muscle cells were inhibited by 1 h treatment with wortmannin. The inhibition of insulin action was dose-dependent with an ED50 around 10-20 nM (
      • Kaliman P.
      • Viñals F.
      • Testar X.
      • Palacín M.
      • Zorzano A.
      ). Here we show that chronic treatment with LY294002 or wortmannin blocked both the morphological and the biochemical differentiation of L6E9 cells. PI 3-kinase inhibitors did not interfere with myoblast proliferation at the doses used (data not shown) and they did not cause any alteration in the elongation or alignment of myoblast observed during the first 24 h after serum deprivation. However, whereas untreated cells began to fuse a few hours after alignment, wortmannin- or LY294002-treated cells remained aligned but did not fuse into myotubes. PI 3-kinase also appears to be essential for signaling leading to the onset of muscle-specific gene expression. Thus, both wortmannin and LY294002 blocked the induction of myogenin mRNA levels and the exit from the cell cycle as measured by the lack of p21 mRNA expression. The induction of the skeletal muscle marker GLUT4 was also impaired without changes in the expression of two non-muscle-specific proteins: β1-integrin, an integral plasma membrane protein and the p85 regulatory subunit of PI 3-kinase. This suggests that PI 3-kinase inhibitors only affected differentiation-associated events.
      Similar results were obtained when 10T1/2-MyoD cells were allowed to differentiate in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002: cells elongated but they were unable to fuse. Upon serum deprivation, untreated 10T1/2-MyoD cells formed multinucleate myotubes and induced the expression of myogenin and p21 mRNAs. In contrast, none of these myogenic characteristics was observed when 10T1/2-MyoD myoblasts were allowed to differentiate in the presence of LY294002.
      Our results seem to indicate that PI 3-kinase inhibitors prevented the activation of other muscle regulatory factors such as myf5 or MyoD, already expressed during the myoblast phenotype (
      • Muthuchamy M.
      • Pajak L.
      • Wieczorek D.F.
      ). Indeed, this was the case in 10T1/2-MyoD cells where MyoD overexpression in undifferentiated cells did not bypass the inhibitory action of LY294002. It has been reported that the activity of muscle regulatory factors can be prevented by increasing the levels of Id protein or by silencing their transcriptional activity as a result of phosphorylation catalyzed by different kinases (
      • Bengal E.
      • Ransone L.
      • Scharfmann R.
      • Dwarki V.J.
      • Tapscott S.
      • Weintraub H.
      • Verma I.M.
      ,
      • Li L.
      • Chambard J.C.
      • Karin M.
      • Olson E.N.
      ). In this study, we focused our attention on the dominant negative bHLH protein Id, which is normally down-regulated after serum deprivation but which remained expressed at high levels in LY294002-treated 10T1/2-MyoD cells. This blockade of Id down-regulation could explain, at least in part, the inability of overexpressed MyoD to induce differentiation.
      In all, our data implicate PI 3-kinase at an early step in the terminal differentiation of muscle cells and rule it out as an essential component in the control of proliferation of myoblast cells or the initial morphological changes associated with differentiation such as alignment and elongation of myoblasts. A schematic summary of our results is presented in Fig. 8.
      Figure thumbnail gr8
      Fig. 8Model for a role of PI 3-kinase in differentiation of muscle cells. Upon serum withdrawal, myoblasts differentiate. At the morphological level, myoblasts initially elongate and align. A few hours later, cells begin to fuse to each other to give rise to multinucleate myotubes. When PI 3-kinase inhibitors are added together with the differentiation medium and maintained throughout the differentiation period, myoblasts are able to proceed through the first differentiation events, i.e. elongation and alignment, but the rest of the differentiation program appeared to be blocked: (i) Id down-regulation is inhibited and it is known that high levels of Id inactivate MyoD; (ii) MyoD remains inactive; (iii) myogenin gene expression is not induced and (iv) the gene expression p21 cdk inhibitor is blocked, suggesting that myoblasts are unable to exit the cell cycle, an essential step for full differentiation. These events finally lead to the inability of myoblasts to fuse into multinucleate myotubes and to express muscle-specific proteins such as GLUT4.
      PI 3-kinases have been associated with many signal transduction pathways, indicating that this family of enzymes plays a variety of roles in response to different stimuli. Here we present results that strongly implicate PI 3-kinase activity in myogenesis. However, the precise myogenic mechanism(s) that are blocked by the PI 3-kinase inhibitors remain to be defined. At the moment, there is very scarce information regarding the signaling pathways by which the microenvironment influences myogenesis. Among the non-exclusive positive regulators of myogenesis that have been described are the insulin growth factors IGF-I, IGF-II, and their specific receptors, and also, the occupation of the extracellular matrix receptors integrins. The following observations allow us to postulate the involvement of PI 3-kinase(s) in myogenic signaling pathways.
      • (i)
        Growth factors are generally considered to inhibit myogenesis (reviewed in Ref.
        • Olson E.N.
        ). However, it has been reported that myoblasts in low serum medium initiate the expression of IGF-II which is secreted in significant amounts to the medium (
        • Florini J.R.
        • Magri K.A.
        • Ewton D.Z.
        • James P.L.
        • Grindstaff K.
        • Rotwein P.S.
        ). It has been proposed that the insulin-like growth factors IGF-I and -II act in an autocrine and/or paracrine manner on myoblasts to promote myogenic differentiation by interacting primarily with the IGF-I receptor (
        • Ewton D.Z.
        • Falen S.L.
        • Florini J.R.
        ). In this regard, IGF-binding proteins from muscle cells inhibit IGF-I-induced differentiation of L6E9 myoblasts (
        • Silverman L.A.
        • Cheng Z.-Q.
        • Hsiao D.
        • Rosenthal S.M.
        ).
        The intracellular myogenic signaling process dependent on IGFs is poorly understood but it is known that IGF-I receptors associate through IRS-I with the p85 regulatory subunit of PI 3-kinase. This interaction results in the activation of the enzyme (
        • Giorgetti S.
        • Ballotti R.
        • Kowalski-Chauvel A.
        • Tartare S.
        • Van Obberghen E.
        ,
        • Myers Jr., M.G.
        • Grammer T.C.
        • Wang L.-M.
        • Sun X.J.
        • Pierce J.H.
        • Blenis J.
        • White M.F.
        ) and the phosphorylation of IRS-1 by the serine kinase activity of PI 3-kinase (
        • Tanti J.-F.
        • Grémaux T.
        • Van Obberghen E.
        • Le Marchand-Brustel Y.
        ,
        • Lam K.
        • Carpenter C.L.
        • Ruderman N.B.
        • Friel J.C.
        • Kelly K.L.
        ). If such an association were also essential for myogenic signaling, PI 3-kinase inhibitors may block terminal differentiation at this level. On the other hand, IGF-II receptors have also been implicated in myogenesis by using the IGF-II receptor-selective (Leu-27) IGF-II analog (
        • Rosenthal S.M.
        • Hsiao D.
        • Silverman L.A.
        ) and, interestingly, wortmannin has been shown to block exocytosis of IGF-II receptors in 3T3-L1 adipocytes (
        • Shepherd P.R.
        • Soos M.A.
        • Siddle K.
        ).
      • (ii)
        Integrins seem to play an important role in mediating signals from the extracellular matrix to influence myogenesis. Indeed, in the presence of a monoclonal antibody which blocks the function of the integrin β-subunit, myoblasts continue to replicate but do not fuse or express muscle-specific markers (
        • Menko A.S.
        • Boettiger D.
        ). Myotube formation is also inhibited by antibodies against the integrin VLA-4 and its counter-receptor VCAM-1 (
        • Rosen G.D.
        • Sanes J.R.
        • LaChance R.
        • Cunningham J.M.
        • Roman J.
        • Dean D.C.
        ). Interestingly, an integrin-dependent translocation of PI 3-kinase to the cytoskeleton which involves specific interactions of p85α with actin filaments and focal adhesion kinase has been recently demonstrated in thrombin-stimulated platelets (
        • Guinebault C.
        • Payrastre B.
        • Racaud-Sultan C.
        • Mazarguil H.
        • Breton M.
        • Mauco G.
        • Plantavid M.
        • Chap H.
        ). Moreover, integrin and platelet-derived growth factor receptor regulate the association of PI 3-kinase with focal adhesion kinase in NIH3T3 cells (
        • Chen H.-C.
        • Guan J.-L.
        ). An analogous mechanism for PI 3-kinase activation via integrins in muscle cells would provide an explanation for the effect of PI 3-kinase inhibitors on myogenic differentiation.
      • (iii)
        Many reports have recently proposed a role for a mammalian PI 3-kinase in membrane trafficking. Indeed, wortmannin and/or LY294002 have been shown to disrupt GLUT1 trafficking in L6E9 myoblasts (
        • Kaliman P.
        • Viñals F.
        • Testar X.
        • Palacín M.
        • Zorzano A.
        ), to block exocytosis of IGF-II receptors in 3T3-L1 adipocytes (
        • Shepherd P.R.
        • Soos M.A.
        • Siddle K.
        ), and to inhibit the sorting and transport of lysosomal enzymes (
        • Brown W.J.
        • DeWald D.B.
        • Emr S.C.
        • Plutner H.
        • Balch W.E.
        ,
        • Matsuoka K.
        • Bassham D.C.
        • Raikhel N.V.
        • Nakamura K.
        ). Moreover, a mammalian homolog of the yeast PI 3-kinase vacuolar protein sorting Vps34p has been recently cloned (
        • Volinia S.
        • Dhand R.
        • Vanhaesebroeck B.
        • MacDougall L.K.
        • Stein R.
        • Zvelebil M.J.
        • Domin J.
        • Panaretou C.
        • Waterfield M.D.
        ). Thus, PI 3-kinase inhibitors could be blocking myogenesis by disrupting the intracellular trafficking of proteins involved in the myogenic signaling through IGFs and/or integrins although other regulatory mechanisms cannot be ruled out.
      In conclusion, these data suggest that one or more PI 3-kinase isoforms might be involved in the myogenic signaling through different positive effectors. We are now attempting to define the myogenic pathway(s) that depend on PI 3-kinase activity and to identify the isoform(s) of PI 3-kinase involved in myogenesis.

      Acknowledgments

      We thank Robin Rycroft for editorial support. We thank Dr. Ricardo Casaroli and Dr. Manuel Reina for expert advice in microscopy techniques. We also thank Dr. Vicente Andrés for access to in press data.

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