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Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Protects against Interleukin-3 Starvation but Not DNA Damage-induced Apoptosis*

  • Anne-Laure Mathieu
    Footnotes
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Sandrine Gonin
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Yann Leverrier
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Bariza Blanquier
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Joëlle Thomas
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Carole Dantin
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Guy Martin
    Affiliations
    Laboratoire de physiopathologie métabolique et rénale, INSERM U499, Faculté de médecine Laennec, 12 rue Guillaume Paradin, 69372 Lyon, cedex 08, France
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  • Gabriel Baverel
    Affiliations
    Laboratoire de physiopathologie métabolique et rénale, INSERM U499, Faculté de médecine Laennec, 12 rue Guillaume Paradin, 69372 Lyon, cedex 08, France
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  • Jacqueline Marvel
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the
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  • Author Footnotes
    * This work was supported in part by institutional grants from CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche and by additional support from the Association pour la Recherche sur le Cancer, the Région Rhône-Alpes and the Comité Départemental (Rhône and Saône et Loire) de la Ligue Nationale Française contre le Cancer.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 fellowship from CNRS.
Open AccessPublished:April 06, 2001DOI:https://doi.org/10.1074/jbc.M007147200
      Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against γ-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.
      IL-3
      interleukin-3
      PI3K
      phosphatidylinositol 3-kinase
      STAT
      signal transducers and activators of transcription
      DMEM
      Dulbecco's modified Eagle's medium
      PBS
      phosphate-buffed saline
      kbp
      kilobase pair(s)
      LDH
      lactate dehydrogenase
      PKB
      protein kinase B
      MAPK
      mitogen-activated protein kinase
      Growth factors are necessary to inhibit the intrinsic apoptotic machinery, which is constitutively expressed in all cells. Baf-3 cells are dependent on IL-31 for their proliferation and survival (
      • Rodriguez-Tarduchy G.
      • Collins M.
      • Lopez-Rivas A.
      ). A number of signaling pathways that are activated by IL-3 play a role in the inhibition of apoptosis (
      • Miyajima A.
      • Ito Y.
      • Kinoshita T.
      ,
      • Gotoh N.
      • Tojo A.
      • Shibuya M.
      ,
      • Ikushima S.
      • Inukai T.
      • Inaba T.
      • Nimer S.D.
      • Cleveland J.L.
      • Look A.T.
      ). One major signaling pathway involved in the control of cell death by growth factors is the PI3K/Akt pathway. Activation of PI3K leads to the generation of 3′-phosphorylated phosphatidylinositides that act by multiple mechanisms to activate Akt (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). Akt will in turn phosphorylate proteins that play a key role in the control of apoptosis. These proteins include Bad, a pro-apoptoticbcl-2 family member, which when phosphorylated by Akt, releases Bcl-x allowing it to perform its anti-apoptotic function (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ). Caspase 9, another effector protein of the intrinsic cell-death machinery can also be inactivated following phosphorylation by Akt (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). Finally, the transcription factor FKHRL1 that regulates the expression of genes encoding pro-apoptotic proteins such as Fas-ligand is located in the cytoplasm following its phosphorylation by Akt on serine 253 and threonine 32 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Kops G.J.
      • de Ruiter N.D.
      • De Vries-Smits A.M.
      • Powell D.R.
      • Bos J.L.
      • Burgering B.M.
      ).
      Growth factors can also delay DNA damage-induced death leading in some systems to an increased clonogenic survival (
      • Frasca D.
      • Pioli C.
      • Guidi F.
      • Pucci S.
      • Arbitrio M.
      • Leter G.
      • Doria G.
      ,
      • Mor F.
      • Cohen I.R.
      ). It has previously been shown that IL-3 protects Baf-3 cells from DNA damage-induced apoptosis (
      • Collins M.K.
      • Marvel J.
      • Malde P.
      • Lopez-Rivas A.
      ,
      • Canman C.E.
      • Gilmer T.M.
      • Coutts S.B.
      • Kastan M.B.
      ). Indeed, in the absence of IL-3, the kinetics of cell death is accelerated following DNA damage. In contrast, in the presence of IL-3, cells are resistant to high doses of DNA damage-inducing agents. The increased rate of death observed when cells are irradiated in the absence of IL-3 is dependent on functional p53, indicating that IL-3 acts by inhibiting a p53-dependent apoptotic pathway (
      • Silva A.
      • Wyllie A.
      • Collins M.K.
      ). There are multiple pathways downstream of p53 that are potentially involved in the induction of apoptosis (
      • Amundson S.A.
      • Myers T.G.
      • Fornace Jr., A.J.
      ,
      • Lakin N.D.
      • Jackson S.P.
      ,
      • Sionov R.V.
      • Haupt Y.
      ). p53 regulates the transcription of pro-apoptotic genes such asBax, Fas, or PERP (
      • Miyashita T.
      • Krajewski S.
      • Krajewska M.
      • Wang H.G.
      • Lin H.K.
      • Liebermann D.A.
      • Hoffman B.
      • Reed J.C.
      ,
      • Owen-Schaub L.B.
      • Zhang W.
      • Cusack J.C.
      • Angelo L.S.
      • Santee S.M.
      • Fujiwara T.
      • Roth J.A.
      • Deisseroth A.B.
      • Zhang W.W.
      • Kruzel E.
      • Radinsky R.
      ,
      • Attardi L.D.
      • Reczek E.E.
      • Cosmas C.
      • Demicco E.G.
      • McCurrach M.E.
      • Lowe S.W.
      • Jacks T.
      ), which could play a role in this process, although some of them such asBax could have a redundant function because p53-induced death is not affected by its absence (
      • Brady H.J.
      • Salomons G.S.
      • Bobeldijk R.C.
      • Berns A.J.
      ). Recently it was shown that, in irradiation or myc-induced p53-dependent death, APAF-1 and Caspase 9 were essential down-stream targets of p53 (
      • Soengas M.S.
      • Alarcon R.M.
      • Yoshida H.
      • Giaccia A.J.
      • Hakem R.
      • Mak T.W.
      • Lowe S.W.
      ).
      The signaling pathways involved in the inhibition of p53-dependent apoptosis by growth factors have been studied in a number of systems. In erythropoietin-dependent myeloid cell lines, Jak2 kinase activation by erythropoietin receptor mutants was shown to be necessary and sufficient to inhibit p53-dependent apoptosis induced by γ-irradiation (
      • Quelle F.W.
      • Wang J.
      • Feng J.
      • Wang D.
      • Cleveland J.L.
      • Ihle J.N.
      • Zambetti G.P.
      ). In the same system, activation of other signaling pathways, including PI3K, STATs, and Ras was not required to inhibit death. In contrast, it has recently been shown that death induced by p53 expression could be inhibited by activating the PI3K/Akt pathway (
      • Sabbatini P.
      • McCormick F.
      ). Finally, it has been suggested that IL-3 could delay p53-dependent apoptosis induced by γ-irradiation by regulating the levels of p21 and Rb, two proteins involved in the regulation of the G1/S transition (
      • Canman C.E.
      • Gilmer T.M.
      • Coutts S.B.
      • Kastan M.B.
      ,
      • Gottlieb E.
      • Oren M.
      ). Although in one report, the expression of v-Src or activated c-Raf could mimic the effect of IL-3 on p21 levels, the signaling pathway activated by IL-3, involved in the control of G1 arrest or apoptosis following DNA damage, has not been identified.
      We have performed a retroviral insertion mutagenesis to obtain mutants that are resistant to apoptosis following growth factors starvation. We describe the characterization of one mutant that proliferates in the absence of growth factors and that, in contrast to previously described mutants, does not overexpress bcl-x (
      • Thomas J.
      • Leverrier Y.
      • Marvel J.
      ). This mutant (the S4 mutant) is able to survive for prolonged periods of time in the absence of IL-3 but shows no resistance to γ-irradiation-induced cell death. This mutant shows a constitutive IL-3-independent, 3′-phosphorylated phosphatidylinositides-dependent, Akt kinase activation. In this mutant, we could demonstrate that Akt activation leads to FKHRL1 phosphorylation and constitutive glycolytic activity. In Baf-3 cells, glycolysis is regulated by IL-3 and is the main ATP-generating source (
      • Garland J.M.
      ). The activation of Akt was necessary for the survival observed in the absence of IL-3. In contrast we show in the S4 mutant and in Baf-3 cells that Akt activation is neither sufficient nor necessary to inhibit p53-dependent DNA damage-induced cell death. These results indicate that IL-3 activates multiple signaling pathways, which can inhibit growth factor starvation-induced apoptosis (called intrinsic apoptosis here after); however, only some of these can delay DNA damage-induced p53-dependent apoptosis.

      DISCUSSION

      In this study we report that, in a Baf-3 mutant cell line, the S4 mutant, which was obtained after retroviral-insertion mutagenesis, shows an IL-3 independent Akt phosphorylation on serine 473, which leads to FKHRL1 phosphorylation on threonine 32 and constitutive glycolysis. Akt activation was necessary for the inhibition of the intrinsic death pathway and long term survival of these cells in the absence of growth factor. The gene modified upstream of Akt responsible for the constitutive activation of Akt has not yet been identified. Several pathways downstream of Akt could be responsible for the survival of the S4 mutant in the absence of growth factor. In neurones, inactivation of the FKHRL1 transcription factor following phosphorylation by Akt is potentially involved in the inhibition of apoptosis by growth factors (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). Indeed, FKHRL1 mutants where the three residues phosphorylated by Akt have been converted to alanine are strong transactivators and trigger apoptosis when overexpressed in a number of cell types. Conversely, replacement of these residues by aspartic acid, which mimics the presence of a phosphate group, disrupts the transactivation function of FKHRL1 (
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ). FKHRL1-mediated death could result from the expression of death genes, such as Fas-ligand, which expression is regulated by FKHRL1 and which are strongly up-regulated following growth factor withdrawal in neurones (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). In Baf-3 cells we have found that FKHRL1 threonine 32 phosphorylation is controlled by IL-3. In contrast, FKHRL1 serine 253 remains phosphorylated in Baf-3 cells grown in the absence of IL-3 up to a stage when cells are starting to enter apoptosis. Because FKHRL1 phosphorylation on serine 253 should maintain FKHRL1 in the cytoplasm (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ), these results suggest that IL-3 starvation-induced apoptosis is not mediated by FKHRL1-dependent gene transactivation. This is in agreement with data showing that IL-3 starvation-induced apoptosis proceeds with similar kinetics in the presence of protein or RNA synthesis inhibitors (
      • Leverrier Y.
      • Thomas J.
      • Perkins G.R.
      • Mangeney M.
      • Collins M.K.
      • Marvel J.
      ). In the Baf-3 cells or the S4 mutant we have been unable to detect a phosphorylation of the Bad protein. Even if we cannot completely exclude a role for Bad in the inhibition of death, we have shown that in the S4 mutant the Bcl-x mRNA and protein are down-regulated following IL-3 starvation, suggesting that the Bad-Bcl-x pathway is not involved in the survival of these cells in the absence of IL-3. In contrast, there is a strong correlation between expression of Bcl-x and protection against DNA damage-induced death. Hence, pathways such as Jak/Stat5 or MAPK, which are involved in the up-regulation of Bcl-x by IL-3 could play a role in the inhibition of DNA damage-induced death (
      • Leverrier Y.
      • Thomas J.
      • Perkins G.R.
      • Mangeney M.
      • Collins M.K.
      • Marvel J.
      ,
      • Socolovsky M.
      • Fallon A.E.
      • Wang S.
      • Brugnara C.
      • Lodish H.F.
      ,
      • Dumon S.
      • Santos S.C.
      • Debierre-Grockiego F.
      • Gouilleux-Gruart V.
      • Cocault L.
      • Boucheron C.
      • Mollat P.
      • Gisselbrecht S.
      • Gouilleux F.
      ). The PI3K/Akt pathway has also been involved in the regulation of bcl-x expression (
      • Leverrier Y.
      • Thomas J.
      • Mathieu A.L.
      • Low W.
      • Blanquier B.
      • Marvel J.
      ,
      • Yang F.C.
      • Kapur R.
      • King A.J.
      • Tao W.
      • Kim C.
      • Borneo J.
      • Breese R.
      • Marshall M.
      • Dinauer M.C.
      • Williams D.A.
      ). Indeed, PI3K inhibition delays bcl-x mRNA up-regulation induced by IL-3 restimulation of growth factor-starved cells. However, the addition of the PI3K inhibitor does not affect the steady-state level of Bcl-x protein in the presence of IL-3 (Fig. 7 C), and constitutive activation of the PI3K/Akt pathway does not inducebcl-x overexpression (Fig. 6, B andC), suggesting that the activation of the PI3K/Akt pathway is not sufficient for bcl-x expression.
      The maintenance of a glycolytic activity in the absence of IL-3 could contribute to the survival of the S4 mutant in these conditions. Indeed, glucose deprivation or inhibition of glycolysis by 2-deoxyglucose can lead to growth arrest or to apoptosis, suggesting that some as yet undefined checkpoint able to induce apoptosis in these conditions exists (
      • Shim H.
      • Chun Y.S.
      • Lewis B.C.
      • Dang C.V.
      ). In IC.DP IL-3-dependent cells, survival in the absence of IL-3 following v-abl transfection has been shown to be dependent on glucose transport activation. Indeed v-ABL activation in these cells induced an increased survival in the absence of growth factor which correlated with a stimulation of glucose uptake. Hence the control of glycolytic activity by growth factors could be an essential step in the inhibition of the intrinsic apoptotic pathway (
      • Rathmell J.C.
      • Vander Heiden M.G.
      • Harris M.H.
      • Frauwirth K.A.
      • Thompson C.B.
      ). Acquisition of a growth factor-independent glycolytic activity could delay the onset of apoptosis and contribute to the process of transformation. Indeed, tumor cells frequently exhibit a high rate of anaerobic glycolysis even under aerobic conditions (
      • Dang C.V.
      • Semenza G.L.
      ). In Baf-3 and S4 cells, we confirmed that anaerobic glycolysis is the main glucose-derived ATP-generating source, because these cells do not undergo aerobic glycolysis. The S4 mutant shows constitutive glucose transport and glycolytic metabolism in the absence of IL-3 as measured by glucose uptake and lactate production. In the presence or absence of IL-3, glycolytic activity was dependent on the PI3K/Akt pathway. In the absence of IL-3, inhibition of the glycolytic pathway using PI3K inhibitors, glucose deprivation, or 2-deoxy-glucose all resulted in rapid apoptotic death of the S4 mutant but not of Bcl-x-overexpressing cells (data not shown). These results indicate that the maintenance of the glycolytic activity of these cells was essential for their survival in the absence of growth factor.
      We also show that the inhibition of p53-dependent DNA damage-induced death is not inhibited by Akt activation. Indeed, the activation of Akt in the S4 mutant in the absence of growth factor is unable to protect these cells against DNA damage-induced death. Similarly, in parental Baf-3 cells inhibition of the PI3K/Akt pathway does not abrogate the protection conferred by IL-3. These results are in contradiction with data showing that overexpression of PI3K or activated Akt can protect against apoptosis following p53 transfection (
      • Sabbatini P.
      • McCormick F.
      ). This discrepancy could be due to difference in the level of Akt activation obtained by overexpressing an activated form of Akt compared with the level of Akt activation obtained in the S4 mutant. Indeed, the level of Akt phosphorylation in these cell in the absence of growth factors is lower than the level obtained when growth factor-starved cells are synchronously restimulated with IL-3. However, it is similar to the physiological level observed in Baf-3 or S4 cells continuously maintained in the presence of IL-3 and might mimic more accurately the functions fulfilled by Akt in IL-3-dependent cell lines. Alternatively, one could imagine that the apoptotic pathway induced by p53 following DNA damage-induced death is different from the pathway induced by p53 overexpression. p53 has multiple functions that are regulated by post-translational modifications such as protein phosphorylation or protein/protein interaction. A number of these functions are involved in the induction of apoptosis by p53 (
      • Amundson S.A.
      • Myers T.G.
      • Fornace Jr., A.J.
      ,
      • Sionov R.V.
      • Haupt Y.
      ). Therefore increasing the level of p53 protein by overexpression or by DNA damage-induced phosphorylation and stabilization might not result in the activation of the same function and apoptotic pathway. A better understanding of the apoptotic pathways activated by p53 in these different experimental conditions might help to resolve this issue.

      Acknowledgments

      We thank A. Brunet and J. Ham for kindly providing us with the phospho-specific FKHRL1 and the Bcl-x antibodies, respectively. We also thank E. Goillot for critical reading of the manuscript.

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