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Hypoxia Induces the Activation of the Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway in PC12 Cells

PROTECTIVE ROLE IN APOPTOSIS*
  • Miguel Alvarez-Tejado
    Footnotes
    Affiliations
    From the Servicio de Inmunologı́a, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain and the
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  • Salvador Naranjo-Suárez
    Footnotes
    Affiliations
    From the Servicio de Inmunologı́a, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain and the
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  • Concepción Jiménez
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnologı́a, Consejo Superior de Investigaciones Cientificas, Cantoblanco, 28049 Madrid, Spain
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  • Ana C. Carrera
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnologı́a, Consejo Superior de Investigaciones Cientificas, Cantoblanco, 28049 Madrid, Spain
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  • Manuel O. Landázuri
    Footnotes
    Affiliations
    From the Servicio de Inmunologı́a, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain and the
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  • Luis del Peso
    Correspondence
    Recipient of a Contrato de Investigación from Fondo de Investigaciones Sanitarias. To whom correspondence should be addressed. Tel.: 34-91-520-2371; Fax: 34-91-520-2374; E-mail: [email protected]
    Footnotes
    Affiliations
    From the Servicio de Inmunologı́a, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain and the
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  • Author Footnotes
    * This work was supported in part by grants from Ministerio de Educación y Cultura (PM 98/1328 and PM 97/0132), from Fondo de Investigaciones Sanitarias (FIS 98/1328), and from Comunidad Autónoma de Madrid (CAM 08.3/0016/99).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 postdoctoral fellowship from the Comunidad de Madrid.
    ¶ Supported by a fellowship from Universidad Autónoma de Madrid.
    ** Both senior authors contributed equally to this work.
Open AccessPublished:June 22, 2001DOI:https://doi.org/10.1074/jbc.M011688200
      Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3–4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novoprotein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.
      EPAS
      endothelial PAS (Per-Arnt-Sim)
      NGF
      nerve growth factor
      PI3K
      phosphatidylinositol 3-kinase
      PBS
      phosphate-buffered saline
      PI
      propidium iodide
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      MAPK
      mitogen-activated protein kinase
      MEK
      MAPK/extracellular signal-regulated kinase kinase
      Mammalian cell function is critically dependent on a continuous supply of oxygen. Organisms respond to changes in oxygen tension with specific local and systemic adaptations aimed to restore a normal oxygen supply. Several tissues and cell types are responsible for the detection of blood pO2 and the induction of specific adaptive responses; among them, the chromaffin cells of the adrenal medulla play a critical role by releasing catecholamines in response to hypoxia (
      • Tian H.
      • Hammer R.E.
      • Matsumoto A.M.
      • Russell D.W.
      • McKnight S.L.
      ). PC12, a rat pheochromocytoma cell line derived from a tumor of adrenal medulla chromaffin tissue, is an oxygen-sensitive cell type that provides a useful system to study the effects of hypoxia on catecholamine gene expression (
      • Norris M.L.
      • Millhorn D.E.
      ). PC12 cells are exquisitely sensitive to hypoxia, because very small reductions in atmospheric oxygen dramatically induce tyrosine hydroxylase gene expression and mRNA stability (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ). In response to hypoxia several transcription factors are activated in PC12 cells. These include the cAMP response element-binding protein, the hypoxia-inducible factor (HIF-2 or EPAS),1 and c-Fos (
      • Norris M.L.
      • Millhorn D.E.
      ,
      • Beitner-Johnson D.
      • Millhorn D.E.
      ,
      • Erickson J.T.
      • Millhorn D.E.
      ). PC12 cells also express hypoxia-regulated ion channels, as shown by the finding that PC12 cells depolarize under hypoxia via an oxygen-regulated K+ current; as a consequence of depolarization, they secrete dopamine and norepinephrine.
      In addition, PC12 cells have been used extensively as a model to study programmed cell death. Programmed cell death, or apoptosis, is an evolutionary conserved mechanism of cellular demise developed by animals to delete damaged, misplaced, or redundant cells during development and tissue homeostasis. Apoptosis was first described as cell death with specific morphologic features (
      • Kerr J.
      • Wyllie A.
      • Currie A.
      ). In addition to the characteristic morphology, apoptotic cells present specific biochemical alterations including exposure of phosphatidylserine to the extracellular side of the plasma membrane and activation of specific proteases (caspases). Upon activation, caspases cleave several intracellular molecules, leading to the functional and morphological changes observed during apoptosis. Caspase processing also leads to the activation of specific nucleases, which in turn cleave genomic DNA, giving rise to a characteristic pattern of DNA degradation that is considered a hallmark of apoptosis (
      • Saraste A.
      • Pulkki K.
      ).
      Cell fate is largely dependent upon extracellular survival signals that prevent the activation of the apoptotic machinery (
      • Raff M.C.
      ). Studies on the survival effect of nerve growth factor (NGF) on PC12 cells provided the first evidence that activation of the enzyme PI3K was critical for its protective effect (
      • Yao R.
      • Cooper G.M.
      ). Upon activation, PI3K phosphorylates membrane phosphoinositides at the D-3 position. These 3′-phosphorylated phospholipids act as second messengers that mediate the diverse cellular functions of PI3K. One of the targets of these lipid second messengers is the serine/threonine kinase Akt/protein kinase B (
      • Toker A.
      • Cantley L.C.
      ). The amino terminus of Akt contains a pleckstrin homology domain that is thought to directly bind the phospholipid products of PI3K activation. This binding recruits Akt to the plasma membrane and induces a conformational change that allows the phosphorylation of Akt by the phosphoinositide-dependent kinases I and II at the residues Thr-308 and Ser-473, respectively (
      • Downward J.
      ). Phosphorylation of Akt results in the full activation of its kinase activity and the subsequent regulation of multiple cellular processes, including the transmission of growth factor-dependent survival signals. The effects of PI3K are controlled by the product of the tumor suppressor gene pten, which encodes a phosphatase that dephosphorylates 3′-phosphorylated phosphoinositides (
      • Maehama T.
      • Dixon J.E.
      ).
      The PI3K/Akt pathway is activated in response to a large number of stimuli (
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ). In addition to many different agonists, it has been described that Akt is also activated in response to several types of stress including oxidative stress (
      • Wang X.
      • McCullough K.D.
      • Franke T.F.
      • Holbrook N.J.
      ). Importantly, the activation of this pathway, even by stress signals, results in an antiapoptotic effect. Finally, it has recently been shown that hypoxia activates Akt in pten-deficient glioma cells (
      • Zundel W.
      • Schindler C.
      • Haas-Kogan D.
      • Koong A.
      • Kaper F.
      • Chen E.
      • Gottschalk A.R.
      • Ryan H.E.
      • Johnson R.S.
      • Jefferson A.B.
      • Stokoe D.
      • Giaccia A.J.
      ).
      In the present study we show that hypoxia results in the activation of the PI3K/Akt pathway in PC12 cells by a novel mechanism that involvesde novo protein synthesis. The activation of this pathway by hypoxia results in the protection against apoptosis induced by different stimuli.

      DISCUSSION

      Reduction of oxygen supply has deleterious effects on many tissues. Neurons and cardiac muscle cells are particularly sensitive because they suffer both necrotic and apoptotic cell death when deprived of oxygen, which occurs during ischemia (
      • Williams R.S.
      • Benjamin I.J.
      ,
      • Lee J.-M.
      • Grabb M.C.
      • Zipfel G.J.
      • Choi D.W.
      ). However, hypoxia is not always lethal. Faced with this stress, some cell types undergo cell cycle arrest but remain viable (
      • Schmaltz C.
      • Hardenbergh P.H.
      • Wells A.
      • Fisher D.E.
      ), and others remain unaltered. Moreover, the induction of apoptosis observed after long periods of hypoxia could be due to acidosis rather than to a direct effect of hypoxia (
      • Schmaltz C.
      • Hardenbergh P.H.
      • Wells A.
      • Fisher D.E.
      ). In addition, organisms are able to respond to both acute and chronic reductions in oxygen tension with specific adaptive responses aimed to restore appropriate oxygen supply. Some of these responses, such as induction of angiogenesis, are local, whereas others are systemic. The systemic responses include hyperventilation, increase in heart output, increase in erythropoiesis, pulmonary vasoconstriction, and carotid body hypertrophy, among others. The induction of these responses is under the control of specific cell types that have evolved to sense oxygen levels. These cell types include the glomus cells of the carotid body, the cells of the neuroepithelial bodies in the lung, and the chromaffin cells of the medulla of the adrenal gland, among others. In particular, the chromaffin cells are critical players in the response to oxygen deprivation in fetuses and neonates (
      • Tian H.
      • Hammer R.E.
      • Matsumoto A.M.
      • Russell D.W.
      • McKnight S.L.
      ). Thus, it is feasible that these cell types have developed specific mechanisms ensuring their viability and functionality during hypoxia.
      In this study, we have shown that under hypoxic conditions PC12 cells are protected against apoptosis triggered by different stimuli. The protection against apoptosis is observed at pO2levels within the physiopathological range, and it can be induced by pharmacological agents that mimic hypoxia. This effect is paralleled by activation of the pro-survival PI3K/Akt pathway through a mechanism that requires de novo protein synthesis. Moreover, inhibition of PI3K not only prevents Akt activation but also the antiapoptotic effect of hypoxia. Thus, it is likely that the activation of the PI3K/Akt pathway by hypoxia is the mechanism responsible for its antiapoptotic effect.
      To our knowledge, this is the first report showing that, at least in specific cell types, hypoxia is able to promote survival. Our data concur with a previous report describing deferoxamine treatment as being able to prevent apoptosis (
      • Zaman K.
      • Ryu H.
      • Hall D.
      • O'Donovan K.
      • Lin K.I.
      • Miller M.P.
      • Marquis J.C.
      • Baraban J.M.
      • Semenza G.L.
      • Ratan R.R.
      ). Whether the phenomenon of ischemic preconditioning (
      • Semenza G.L.
      ), the partial resistance to ischemia-induced damage found after a previous episode of moderate ischemia, is due to a mechanism similar to the one described here will require further work. However, it is intriguing that preconditioning, similar to activation of Akt, requires de novo protein synthesis (
      • Williams R.S.
      • Benjamin I.J.
      ). In addition, we have shown that hypoxia renders cells resistant to apoptosis induced by the chemotherapeutic drugs taxol and fluorouracil, an effect that might contribute to the partial resistance to therapy observed in the hypoxic regions of tumors as compared with the normoxic areas of the same tumors (
      • Teicher B.A.
      ). Nevertheless, this is not the only example in which proapoptotic stimuli induce an antiapoptotic response. Exposure of cells to H2O2 results in the activation of the PI3K/Akt pathway and resistance to induction of apoptosis (
      • Wang X.
      • McCullough K.D.
      • Franke T.F.
      • Holbrook N.J.
      ). Hence, activation of the PI3K/Akt survival pathway could be a general cellular response to cell and tissue injury.
      The activation of Akt by hypoxia in cells derived frompten−/− tumors has recently been described (
      • Zundel W.
      • Schindler C.
      • Haas-Kogan D.
      • Koong A.
      • Kaper F.
      • Chen E.
      • Gottschalk A.R.
      • Ryan H.E.
      • Johnson R.S.
      • Jefferson A.B.
      • Stokoe D.
      • Giaccia A.J.
      ). Here we provide evidence that the activation of the PI3K/Akt pathway by hypoxia also occurs in some specific cells that are apparently normal forpten (
      • Lachyankar M.B.
      • Sultana N.
      • Schonhoff C.M.
      • Mitra P.
      • Poluha W.
      • Lambert S.
      • Quesenberry P.J.
      • Litofsky N.S.
      • Recht L.D.
      • Nabi R.
      • Miller S.J.
      • Ohta S.
      • Neel B.G.
      • Ross A.H.
      ), including PC12 cells and primary chromaffin cells from cow adrenal gland medulla. This seems to be a specific response, restricted to at least cells of chromaffin lineage, rather than a general effect of hypoxia, because we did not detect any significant phosphorylation of Akt in other cell types tested. The lack of Akt activation in these cell lines was not the result of defective responses to hypoxia, because in all cases hypoxia-inducible factors were stabilized in response to hypoxia.2
      In addition, the mechanism, involving de novo protein synthesis, and the sustained activation of Akt have not been described previously. One remaining question about this novel mechanism of Akt activation is the nature of the protein whose synthesis is required for the activation of Akt. Most likely it is not a soluble factor, at least not a stable one, released by hypoxic cells, because conditioned medium from hypoxia-treated cells failed to induce Akt activation in PC12 cells grown in normoxia.2 It is still possible that the effect is mediated by the induction of a nonsecreted membrane-bound ligand or by the induction of both a soluble factor and its receptor. One further possibility is that the synthesized molecule acts in a cell-autonomous manner by direct activation of the PI3K/Akt pathway from inside the cell. Further work will be required to differentiate among these possibilities. The identification of such a molecule could explain why the effect of hypoxia on Akt is restricted to some cell types.
      Hypoxia treatment results in changes in gene expression that are mediated by the activation of different transcription factors. Hypoxia-inducible factors are the best characterized; others include cAMP response element-binding protein (
      • Beitner-Johnson D.
      • Millhorn D.E.
      ), NF-κB (
      • Koong A.C.
      • Chen E.Y.
      • Giaccia A.J.
      ), and c-Fos (
      • Norris M.L.
      • Millhorn D.E.
      ). Although we cannot formally rule out that hypoxia-inducible transcription factors are involved in Akt activation, indirect evidence suggests that it is not the case, because both calmodulin antagonists and the MEK inhibitor PD98059 prevent EPAS activity in PC12 cells (
      • Conrad P.W.
      • Freeman T.L.
      • Beitner-Johnson D.
      • Millhorn D.E.
      ) without affecting hypoxia-induced Akt activation.
      We suggest that the activation of survival pathways by hypoxia, at least in chromaffin cells, ensures that the cells remain viable and able to trigger the responses required for the adaptation to varying oxygen tension. Finally, activation of Akt has many other effects, in addition to promoting survival. Among these effects are changes in gene expression, induction of cell proliferation, and increasing glucose uptake (
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ). It is thus possible that many other effects of hypoxia in cell biology, in this cell type, could be mediated by activation of the PI3K/Akt pathway.

      Acknowledgments

      We thank M. Vitón for valuable assistance with flow cytometry, M. C. Castellanos for help with the calmodulin inhibitors, A. Vara for valuable technical assistance, A. Alfranca, J. Aragones, E. Temes, S. Garcia, F. Vidal, and A. Garcia for critically reviewing the manuscript, and V. Alvarez for suggestions. We also thank D. Jones for valuable help.

      REFERENCES

        • Tian H.
        • Hammer R.E.
        • Matsumoto A.M.
        • Russell D.W.
        • McKnight S.L.
        Genes Dev. 1998; 12: 3320-3324
        • Norris M.L.
        • Millhorn D.E.
        J. Biol. Chem. 1995; 270: 23774-23779
        • Czyzyk-Krzeska M.F.
        • Furnari B.A.
        • Lawson E.E.
        • Millhorn D.E.
        J. Biol. Chem. 1994; 269: 760-764
        • Beitner-Johnson D.
        • Millhorn D.E.
        J. Biol. Chem. 1998; 273: 19834-19839
        • Erickson J.T.
        • Millhorn D.E.
        J. Comp. Neurol. 1994; 348: 161-182
        • Kerr J.
        • Wyllie A.
        • Currie A.
        Br. J. Cancer. 1972; 26: 239-257
        • Saraste A.
        • Pulkki K.
        Cardiovasc. Res. 2000; 45: 528-537
        • Raff M.C.
        Nature. 1992; 356: 397-400
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Toker A.
        • Cantley L.C.
        Nature. 1997; 387: 673-676
        • Downward J.
        Science. 1998; 279: 673-674
        • Maehama T.
        • Dixon J.E.
        J. Biol. Chem. 1998; 273: 13375-13378
        • Chan T.O.
        • Rittenhouse S.E.
        • Tsichlis P.N.
        Annu. Rev. Biochem. 1999; 68: 965-1014
        • Wang X.
        • McCullough K.D.
        • Franke T.F.
        • Holbrook N.J.
        J. Biol. Chem. 2000; 19: 14624-14631
        • Zundel W.
        • Schindler C.
        • Haas-Kogan D.
        • Koong A.
        • Kaper F.
        • Chen E.
        • Gottschalk A.R.
        • Ryan H.E.
        • Johnson R.S.
        • Jefferson A.B.
        • Stokoe D.
        • Giaccia A.J.
        Genes Dev. 2000; 14: 391-396
        • J. P. Robinson
        • Z. Darzynkiewicz
        • P. N. Dean
        • A. Orfao
        • Rabinovitch A.
        • Stewart P.S.
        • Tanke C.C.
        • WeelessL. L. H.J.
        Current Protocols in Cytometry.
        John Wiley & Sons Inc., New York1997
        • Lobner D.
        J. Neurosci. Methods. 2000; 96: 147-152
        • Gonzalez-Garcia A.
        • Merida I.
        • Martinez-Alonso C.
        • Carrera A.C.
        J. Biol. Chem. 1997; 272: 10220-10226
        • del Peso L.
        • Gonzalez-Garcia M.
        • Page C.
        • Herrera R.
        • Nunez G.
        Science. 1997; 278: 687-689
        • Yoshimura S.
        • Banno Y.
        • Nakashima S.
        • Takenaka K.
        • Sakai H.
        • Nishimura Y.
        • Sakai N.
        • Shimizu S.
        • Eguchi Y.
        • Tsujimoto Y.
        • Nozawa Y.
        J. Biol. Chem. 1998; 273: 6921-6927
        • Lindenboim L.
        • Diamond R.
        • Rothenberg E.
        • Stein R.
        Cancer Res. 1995; 55: 1242-1247
        • Parrizas M.
        • Saltiel A.R.
        • LeRoith D.
        J. Biol. Chem. 1997; 272: 154-161
        • Wang G.L.
        • Semenza G.L.
        Blood. 1993; 82: 3610-3615
        • Nunez G.
        • del Peso L.
        Curr. Opin. Neurobiol. 1998; 8: 613-618
        • Conrad P.W.
        • Freeman T.L.
        • Beitner-Johnson D.
        • Millhorn D.E.
        J. Biol. Chem. 1999; 274: 33709-33713
        • Conrad P.W.
        • Millhorn D.E.
        • Beitner-Johnson D.
        Adv. Exp. Med. Biol. 2000; 475: 293-302
        • Beitner-Johnson D.
        • Leibold J.
        • Millhorn D.E.
        Biochem. Biophys. Res. Commun. 1998; 242: 61-66
        • Xia Z.
        • Dickens M.
        • Raingeaud J.
        • Davis R.J.
        • Greenberg M.E.
        Science. 1995; 270: 1326-1331
        • Williams R.S.
        • Benjamin I.J.
        J. Clin. Invest. 2000; 106: 813-818
        • Lee J.-M.
        • Grabb M.C.
        • Zipfel G.J.
        • Choi D.W.
        J. Clin. Invest. 2000; 106: 723-731
        • Schmaltz C.
        • Hardenbergh P.H.
        • Wells A.
        • Fisher D.E.
        Mol. Cell. Biol. 1998; 18: 2845-2854
        • Zaman K.
        • Ryu H.
        • Hall D.
        • O'Donovan K.
        • Lin K.I.
        • Miller M.P.
        • Marquis J.C.
        • Baraban J.M.
        • Semenza G.L.
        • Ratan R.R.
        J. Neurosci. 1999; 19: 9821-9830
        • Semenza G.L.
        J. Clin. Invest. 2000; 106: 809-812
        • Teicher B.A.
        Cancer Metastasis Rev. 1994; 13: 139-168
        • Lachyankar M.B.
        • Sultana N.
        • Schonhoff C.M.
        • Mitra P.
        • Poluha W.
        • Lambert S.
        • Quesenberry P.J.
        • Litofsky N.S.
        • Recht L.D.
        • Nabi R.
        • Miller S.J.
        • Ohta S.
        • Neel B.G.
        • Ross A.H.
        J. Neurosci. 2000; 20: 1404-1413
        • Koong A.C.
        • Chen E.Y.
        • Giaccia A.J.
        Cancer Res. 1994; 54: 1425-1430