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

Abstract

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. 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. endothelial PAS (Per-Arnt-Sim) nerve growth factor phosphatidylinositol 3-kinase phosphate-buffered saline propidium iodide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide mitogen-activated protein kinase 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 (1Tian H. Hammer R.E. Matsumoto A.M. Russell D.W. McKnight S.L. Genes Dev. 1998; 12: 3320-3324Crossref PubMed Scopus (520) Google Scholar). 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 (2Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). PC12 cells are exquisitely sensitive to hypoxia, because very small reductions in atmospheric oxygen dramatically induce tyrosine hydroxylase gene expression and mRNA stability (3Czyzyk-Krzeska M.F. Furnari B.A. Lawson E.E. Millhorn D.E. J. Biol. Chem. 1994; 269: 760-764Abstract Full Text PDF PubMed Google Scholar). 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 (2Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 4Beitner-Johnson D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Erickson J.T. Millhorn D.E. J. Comp. Neurol. 1994; 348: 161-182Crossref PubMed Scopus (247) Google Scholar). 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 (6Kerr J. Wyllie A. Currie A. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12874) Google Scholar). 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 (7Saraste A. Pulkki K. Cardiovasc. Res. 2000; 45: 528-537Crossref PubMed Scopus (1029) Google Scholar). Cell fate is largely dependent upon extracellular survival signals that prevent the activation of the apoptotic machinery (8Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2497) Google Scholar). 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 (9Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1293) Google Scholar). 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 (10Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1226) Google Scholar). 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 (11Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). 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 (12Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar). The PI3K/Akt pathway is activated in response to a large number of stimuli (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). 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 (14Wang X. McCullough K.D. Franke T.F. Holbrook N.J. J. Biol. Chem. 2000; 19: 14624-14631Abstract Full Text Full Text PDF Scopus (403) Google Scholar). 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 (15Zundel 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: 391-396Crossref PubMed Google Scholar). 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. Undifferentiated PC12 cells were maintained in RPMI 1640 medium with GLUTAMAX-I (Life Technologies, Inc.) supplemented with 10% horse serum (Life Technologies, Inc.), 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Hypoxia (1% O2) was induced by culture of cells inside an air-tight chamber with inflow and outflow valves that was infused with a mixture of 1% O2, 5% CO2, 94% N2 (S.E. Carburos Metalicos S.A., Madrid, Spain); in those experiments using mild hypoxia (5% O2) the mixture used was 5% O2, 5% CO2, 90% N2 (S.E. Carburos Metalicos S.A.). Prior to transferring to the hypoxia chamber, the medium was removed from the plates, cells were washed, and fresh (complete or serum-free) medium was added to the plates. In experiments in which drugs were used, the compounds were added immediately prior to transfer to hypoxia/normoxia conditions. For experiments using kinase inhibitors, cells were pretreated during 1 h with the different inhibitors or vehicle and then placed in hypoxia chambers. After treatments, cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested in 70 or 200 μl (P35 or P60mm dishes, respectively) of a lysis buffer containing 2% SDS, 10% glycerol, 10 mm dithiothreitol, 62 mm Tris, pH 6.8, and 0.004% bromphenol blue. Lysates were sonicated for 4 s in a Sonicator 150 (MSE) and then centrifuged at 4 °C for 2 min at 14,000 × g. Lysates were resolved on 8% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad) using electroblotting procedures. Nitrocellulose membranes were blocked with 5% nonfat dry milk in TBS-T (50 mm Tris, pH 7.6, 150 mm NaCl, 0.1% Tween 20) and incubated overnight at 4 °C with the indicated antibodies (anti-Akt (number 9272) and anti-phospho-Akt serine 473 (number 9271) from Cell Signaling, Beverly, MA; anti-HIF-2α/EPAS from was detected by to the Apoptosis was by of the cell after DNA with propidium iodide as described A. A. in Scholar). were in they was then with or medium specific compounds were in culture and cells were placed in or hypoxic conditions for the indicated of After cells that to were harvested in mm in and with those cells. were then centrifuged for min at × the was and the cell was in of by to cell The cell was transferred to of and in this for a of 2 cells were centrifuged for min at and washed with 10 of Finally, the cell was in 1 of μg/ml in and incubated for min at 37 °C. were by The of a first in which with the and were were to using a Finally, that were in 1 and 2 were in a the number of containing a specific of Apoptosis was as the of cells with a DNA in the A. A. in Scholar). was by the as described D. J. 2000; PubMed Scopus Google Scholar). cells were on and in medium they medium was with or and cells were placed under O2) or hypoxic conditions (1% O2) for After in was added to the culture medium to a of and were incubated at 37 °C for h under conditions. Finally, medium was and were in in was by at using a were in culture and in medium they culture medium was with and cells were placed under O2) or hypoxic conditions (1% O2) for cells were with for 10 After treatments, cells were with 1% lysis buffer (1% 10% glycerol, mm NaCl, mm pH mm 1 1 1 μg/ml 1 μg/ml and 1 mm for PI3K 1 mm was 10% of was to for in the and the was with μl of number 9271) or of number for 2 h at 4 °C. were washed, and were to an in kinase using or phosphatidylinositol as A. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. Science. 1997; PubMed Scopus Google Scholar). were using the were by the of by the the in this are indicated in the and results were using the or after or of by the the effect of hypoxia on apoptosis, PC12 cells were with different of or medium and O2) or hypoxic conditions (1% O2) for of 1 After treatments, apoptosis was as the of cells with a of DNA by of the cell by A. A. in Scholar). 1 that hypoxia can induce apoptosis in PC12 cells, as K. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google its is after h in oxygen conditions 1 In serum which is a apoptotic for PC12 cells R. E. R. Res. 1995; Google results in a of cells apoptosis as as h the induction of apoptosis upon serum withdrawal was reduced cells were under hypoxic conditions 1 The of apoptosis was at and h after serum and B the cell of PC12 cells h after the indicated in a The effect of hypoxia on cellular was using the D. J. 2000; PubMed Scopus Google Scholar, A.R. D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). hypoxia (1% for effect on cell in as with cells in serum for h reduced to as with cells In with the of cell by serum withdrawal was under hypoxic conditions of cell as with cells in 1 The cellular responses to hypoxia can be observed at of with a in apoptosis, by serum was also observed at 5% 2 which to a pO2 the in blood in treatment with deferoxamine or and two compounds used to many of the effects of hypoxia PubMed Google in a of apoptosis 2 from apoptosis. PC12 cells were in and they the culture medium was with or culture medium and cells were incubated at the indicated of oxygen. deferoxamine was in the culture medium at a of or 150 and cells were incubated in Apoptosis was by h after PC12 cells were as in A. the culture medium was with fresh culture medium with 1 μg/ml or μg/ml were incubated for h under or hypoxic conditions (1% O2, and apoptosis was by from experiments are the of apoptosis in from a The the of the experiments The of the results is hypoxia was to against apoptotic stimuli serum of PC12 cells with the drugs or for h induced a of apoptosis 2 for serum under hypoxic conditions (1% O2) PC12 cells were against apoptosis for and for induced by the drugs 2 Thus, hypoxic conditions induced by oxygen or the deferoxamine are to prevent apoptosis induced by different stimuli in PC12 cells. the mechanism responsible for the effect of hypoxia, we to study the effect of hypoxia on the serine/threonine kinase a in the of antiapoptotic signals (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar, 1998; PubMed Scopus Google Scholar). of Akt can be detected by with antibodies that Akt at serine because this phosphorylation correlates with Akt activity (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). Hypoxia treatment in phosphorylation of Akt on serine 473 The phosphorylation of Akt was observed of the or of serum or the type of culture used Phosphorylation of Akt on serine 473 its activation as by kinase activity of Akt from cells the activation of PI3K/Akt to be a cellular response to hypoxia, because Akt phosphorylation was observed in cell including endothelial cells, cells, cells, cells, cells, cells, and cells. it to be a response to specific cell types, in addition to the PC12 cell Akt phosphorylation was observed in chromaffin cells from adrenal and cells under hypoxic Akt phosphorylation was also observed in PC12 cells that were to a by treatment with for and it with protection against that Akt phosphorylation was detected h after of hypoxic treatment The of Akt activation in response to hypoxia was from that observed after with hypoxia several to induce Akt activation, with activates Akt Akt phosphorylation was in with the effect after 4 The activation of signaling pathways was by the of the hypoxia-inducible of the intracellular responses to oxygen 4 by Akt activation The activation of Akt by deferoxamine, a that mimics hypoxia, a to that observed under hypoxia 4 The in the of and Akt activation was after deferoxamine treatment 4 In addition, activation of Akt by deferoxamine was observed at of signaling as by 4 hypoxia (5% O2) also in Akt Finally, Akt phosphorylation was also observed by treatment with the 4 we the mechanism by which hypoxia induced Akt activation. Akt activation, in is dependent on PI3K activation (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google we first PI3K activity was by hypoxia. shown in PI3K was activated by hypoxia in PC12 cells. the PI3K activity shown in was detected in from hypoxic cells, suggesting that hypoxia in the activation of a tyrosine kinase of the of we the effect of two PI3K inhibitors, and inhibitors prevented Akt phosphorylation induced by hypoxia The of Akt activation with the that it after the induction of transcription factors as that Akt activation by hypoxia gene Thus, treatment of cells with inhibitors of or protein synthesis prevented Akt activation The effect of actinomycin or cycloheximide was to a or because with these compounds for several Akt Finally, we signaling pathways to Akt The and mitogen-activated protein kinase are activated by hypoxia and mediate of its effects in PC12 cells D. Millhorn D.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Millhorn D.E. D. Biol. 2000; PubMed Google Scholar, D. J. Millhorn D.E. Biochem. Res. 1998; PubMed Scopus Google Scholar). of MAPK/extracellular signal-regulated kinase kinase of the kinases in the with or treatment with of signaling and effect on Akt phosphorylation induced by hypoxia The of effect of is because the protein kinase was recently shown to be to and Akt (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). activation of Akt by hypoxia de of and was dependent on PI3K activity or Importantly, a medium from PC12 cells was to induce Akt activation in suggesting that the effect of hypoxia was to the synthesis of a factor to the PI3K/Akt pathway in an or have shown that hypoxia apoptosis in response to several stimuli and that this effect correlates with the activation of the PI3K/Akt pathway. was a functional link between these two we the effect of PI3K inhibitors on the protective effect of hypoxia. shown in the of apoptosis, upon serum observed under hypoxia was prevented by treatment of cells with the PI3K inhibitors or These results that the protection against apoptosis induced by hypoxia on the activation of the PI3K/Akt survival pathway. In addition to the PI3K/Akt pathway, the has an role in survival of PC12 cells J. Science. 1995; 270: PubMed Scopus Google Scholar). the effect on the protective effect of hypoxia the of cells apoptosis upon serum withdrawal under conditions In with these the protective effects of deferoxamine and on apoptosis upon serum withdrawal 2 were also by inhibition of of oxygen supply has effects on many and cells are sensitive because they both and apoptotic cell death of which during R.S. J. 2000; PubMed Scopus Google Scholar, M.C. D.W. J. 2000; PubMed Scopus Google Scholar). hypoxia is with this cell types cell C. A. D.E. Biol. 1998; PubMed Scopus Google and the induction of apoptosis observed after of hypoxia be to to a effect of hypoxia C. A. D.E. Biol. 1998; PubMed Scopus Google Scholar). In addition, are to respond to both and reductions in oxygen tension with specific adaptive responses aimed to restore oxygen supply. of these as induction of are are The systemic responses include in in and among The induction of these responses is under the of specific cell types that have evolved to sense oxygen These cell types include the cells of the the cells of the in the and the chromaffin cells of the medulla of the adrenal among In the chromaffin cells are critical in the response to oxygen in and (1Tian H. Hammer R.E. Matsumoto A.M. Russell D.W. McKnight S.L. Genes Dev. 1998; 12: 3320-3324Crossref PubMed Scopus (520) Google Scholar). Thus, it is that these cell types have developed specific and during hypoxia. In this we have shown that under hypoxic conditions PC12 cells are against apoptosis by different stimuli. The protection against apoptosis is observed at the and it can be induced by that hypoxia. This effect is by activation of the PI3K/Akt pathway through a mechanism that de novo protein synthesis. inhibition of PI3K Akt activation also the antiapoptotic effect of hypoxia. Thus, it is that the activation of the PI3K/Akt pathway by hypoxia is the mechanism responsible for its antiapoptotic effect. this is the first report at in specific cell types, hypoxia is to with a report deferoxamine treatment as to prevent apoptosis K. H. D. K. J. 1999; 19: PubMed Google Scholar). the of J. 2000; PubMed Scopus Google the to after a of is to a mechanism to the described it is that to activation of de novo protein synthesis R.S. J. 2000; PubMed Scopus Google Scholar). In addition, we have shown that hypoxia cells resistant to apoptosis induced by the drugs and an effect that to the to observed in the hypoxic of as with the of the B.A. Rev. 1994; PubMed Scopus Google Scholar). this is the in which stimuli induce an antiapoptotic of cells to results in the activation of the PI3K/Akt pathway and to induction of apoptosis (14Wang X. McCullough K.D. Franke T.F. Holbrook N.J. J. Biol. Chem. 2000; 19: 14624-14631Abstract Full Text Full Text PDF Scopus (403) Google Scholar). activation of the PI3K/Akt survival pathway be a cellular response to cell and tissue The activation of Akt by hypoxia in cells derived has recently been described (15Zundel 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: 391-396Crossref PubMed Google Scholar). Here we evidence that the activation of the PI3K/Akt pathway by hypoxia also in specific cells that are normal W. R. J. 2000; PubMed Google including PC12 cells and chromaffin cells from adrenal This to be a specific to at cells of chromaffin a effect of hypoxia, because we phosphorylation of Akt in cell types The of Akt activation in these cell was the of responses to hypoxia, because in hypoxia-inducible factors were in response to In addition, the de novo protein and the sustained activation of Akt have been described One this novel mechanism of Akt activation is the of the protein synthesis is for the activation of it is a at a by hypoxic cells, because medium from cells to induce Akt activation in PC12 cells in is that the effect is by the induction of a or by the induction of both a factor and its One is that the in a by activation of the PI3K/Akt pathway from inside the be to among these The of a the effect of hypoxia on Akt is to cell Hypoxia treatment results in changes in gene expression that are by the activation of different transcription factors are the include cAMP response element-binding protein D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Chen Giaccia A.J. Res. 1994; Google and c-Fos (2Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). we that hypoxia-inducible transcription factors are in Akt activation, evidence that it is the because both and the prevent activity in PC12 cells D. Millhorn D.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Akt activation. that the activation of survival pathways by hypoxia, at in chromaffin cells, that the cells and to the responses for the to oxygen Finally, activation of Akt has many in addition to these effects are changes in gene induction of cell and (13Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). is that many effects of hypoxia in cell in this cell be by activation of the PI3K/Akt pathway. for with C. for with the inhibitors, A. for A. J. E. F. and A. for critically the and for also D. for