eIF2α Phosphorylation Tips the Balance to Apoptosis during Osmotic Stress

Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2α, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2α phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2α phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2α promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2α phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2α phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2α phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2α phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1. Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2α, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2α phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2α phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2α promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2α phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2α phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2α phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2α phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1. IntroductionThe regulation of cell volume in response to changes in extracellular osmolarity is an important function. Hypertonic conditions induce responses that protect cells by inducing transport of osmolytes referred to as compatible solutes. Intermediate levels of hypertonic stress cause cell cycle arrest followed by cell survival, but higher levels induce apoptosis (1Burg M.B. Ferraris J.D. Dmitrieva N.I. Physiol. Rev. 2007; 87: 1441-1474Crossref PubMed Scopus (557) Google Scholar).The master regulator of this response is the tonicity-responsive enhancer-binding protein, TonEBP, which translocates to the nucleus and induces transcription of genes encoding osmolyte transporters and heat shock proteins (1Burg M.B. Ferraris J.D. Dmitrieva N.I. Physiol. Rev. 2007; 87: 1441-1474Crossref PubMed Scopus (557) Google Scholar). A remarkable example of adaptation is seen in kidney medulla, which is exposed to hypertonic urine. Mice deficient in TonEBP show atrophy of the renal medulla and deficiencies in immune system function (2Go W.Y. Liu X. Roti M.A. Liu F. Ho S.N. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10673-10678Crossref PubMed Scopus (226) Google Scholar). In addition, brain cells have adaptive mechanisms for cell volume recovery that involve the TonEBP target gene SNAT2 (3Maallem S. Mutin M. González-González I.M. Zafra F. Tappaz M.L. Neuroscience. 2008; 153: 95-107Crossref PubMed Scopus (19) Google Scholar). This is further supported by the induction of TonEBP in neurons during systemic hypertonicity (4Loyher M.L. Mutin M. Woo S.K. Kwon H.M. Tappaz M.L. Neuroscience. 2004; 124: 89-104Crossref PubMed Scopus (54) Google Scholar). Neurological disorders have also been associated with hypertonic blood plasma, as in diseases of water imbalance (5Lin M. Liu S.J. Lim I.T. Emerg. Med. Clin. North Am. 2005; 23 (ix): 749-770Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).The cellular response to stress involves regulation of mRNA translation (6Graber T.E. Holcik M. Mol. Biosyst. 2007; 3: 825-834Crossref PubMed Scopus (57) Google Scholar, 7Komar A.A. Hatzoglou M. J. Biol. Chem. 2005; 280: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 8Silvera D. Arju R. Darvishian F. Levine P.H. Zolfaghari L. Goldberg J. Hochman T. Formenti S.C. Schneider R.J. Nat. Cell Biol. 2009; 11: 903-908Crossref PubMed Scopus (184) Google Scholar). Diverse stresses limit protein synthesis, mostly via inhibition of translation initiation. Much of this regulation involves eIF2, 5The abbreviations used are: eIF2eukaryotic initiation factor 2hnRNP A1heterogeneous nuclear ribonucleoprotein A1IRESinternal ribosome entry siteSGstress granuleMEFmouse embryonic fibroblastUTRuntranslated regionRT-qPCRreverse transcription-quantitative PCRTUNELterminal dUTP nick end-labelingHAhemagglutininGFPgreen fluorescent proteinDAPI4′,6-diamidino-2-phenylindoleWTwild typeXIAPX-linked inhibitor of apoptosisUPRunfolded protein response. which delivers the initiator tRNA (Met-tRNAiMet) to cellular mRNAs that use an AUG initiation codon. Phosphorylation of the α subunit on Ser51 sequesters eIF2 in an inactive complex, thus decreasing translation initiation (9Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar).The outcome of the stress response depends on the intensity and duration of the stress. Events during the early response promote the recovery of translation and the synthesis of proteins essential for adaptation (10Rutkowski D.T. Arnold S.M. Miller C.N. Wu J. Li J. Gunnison K.M. Mori K. Sadighi Akha A.A. Raden D. Kaufman R.J. PLoS Biol. 2006; 4: e374Crossref PubMed Scopus (616) Google Scholar). However, prolonged and severe stress promotes apoptosis. The regulated synthesis of pro-survival and pro-death proteins plays an important role in determining cell fate during stress. It has been suggested that persistent inhibition of protein synthesis can lead to apoptosis by inhibiting translation of mRNAs encoding anti-apoptotic proteins (11Fritsch R.M. Schneider G. Saur D. Scheibel M. Schmid R.M. J. Biol. Chem. 2007; 282: 22551-22562Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Scheuner D. Patel R. Wang F. Lee K. Kumar K. Wu J. Nilsson A. Karin M. Kaufman R.J. J. Biol. Chem. 2006; 281: 21458-21468Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Translation of some pro-apoptotic and anti-apoptotic mRNAs is mediated by internal ribosome entry sites (IRESs) in the mRNA leaders. IRES-mediated translation initiation involves recruitment of the ribosome to the mRNA independently of the 5′-cap. In most cases, this requires IRES trans-acting factors that mediate ribosome recruitment. The activity of many IRESs can be positively or negatively regulated by IRES trans-acting factors depending on the type of stress (6Graber T.E. Holcik M. Mol. Biosyst. 2007; 3: 825-834Crossref PubMed Scopus (57) Google Scholar, 13Spriggs K.A. Bushell M. Mitchell S.A. Willis A.E. Cell Death Differ. 2005; 12: 585-591Crossref PubMed Scopus (134) Google Scholar).We previously showed that mild osmotic stress (400 mosmol/liter) causes a pro-survival response without induction of eIF2α phosphorylation (14Gaccioli F. Huang C.C. Wang C. Bevilacqua E. Franchi-Gazzola R. Gazzola G.C. Bussolati O. Snider M.D. Hatzoglou M. J. Biol. Chem. 2006; 281: 17929-17940Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study, we show that severe osmotic stress induces apoptosis in a manner dependent on eIF2α phosphorylation. In contrast to the current view, decreased protein synthesis in the absence of eIF2α phosphorylation does not induce apoptosis. Rather, eIF2α phosphorylation is required for the translocation of the RNA-binding protein hnRNP A1 from the nucleus to the cytoplasm, which blocks translation of mRNAs encoding inhibitors of apoptosis. The pro-apoptotic function of eIF2α phosphorylation during osmotic stress is also demonstrated in primary cortical neurons. This mechanism of translational control may have direct implications in many diseases that involve undesirable apoptosis such as heart failure and neurodegeneration.DISCUSSIONAcute hypertonic conditions cause cell shrinkage and induce an adaptive response that leads to volume recovery. However, severe and prolonged hypertonic stress induces apoptosis. We show here that the balance between survival and apoptosis involves signaling mediated by phosphorylation of the translation initiation factor eIF2α (summarized in Fig. 7B). A critical factor that shifts the balance from survival to apoptosis is the eIF2α phosphorylation-dependent accumulation of hnRNP A1 in cytoplasmic SGs. hnRNP A1 is likely to sequester anti-apoptotic mRNAs in SGs, leading to their translational attenuation. This will shift the balance toward activation of the execution caspases and cleavage of survival factors, among them members of the eIF4G family (data not shown). These findings challenge the current view (11Fritsch R.M. Schneider G. Saur D. Scheibel M. Schmid R.M. J. Biol. Chem. 2007; 282: 22551-22562Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Scheuner D. Patel R. Wang F. Lee K. Kumar K. Wu J. Nilsson A. Karin M. Kaufman R.J. J. Biol. Chem. 2006; 281: 21458-21468Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) that induction of apoptosis during osmotic stress is due to depletion of short lived anti-apoptotic proteins caused by the inhibition of global protein synthesis that is triggered by eIF2α phosphorylation (11Fritsch R.M. Schneider G. Saur D. Scheibel M. Schmid R.M. J. Biol. Chem. 2007; 282: 22551-22562Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).The phosphorylation of eIF2α on Ser51 is an important regulator of the decision between survival and apoptosis in response to diverse stress conditions (44Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1004) Google Scholar). This phosphorylation is carried out by four stress-regulated kinases (GCN2, PERK, PKR, and HRI), suggesting that eIF2α is a master regulator of the stress response (44Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1004) Google Scholar). The initial response to eIF2α phosphorylation is inhibition of global protein synthesis that may have protective (47Jiang H.Y. Wek S.A. McGrath B.C. Scheuner D. Kaufman R.J. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2003; 23: 5651-5663Crossref PubMed Scopus (350) Google Scholar) or proapoptotic functions. In most stress conditions that activate PKR (viral infection, UV irradiation, exposure to interferon, or tumor necrosis factor-α), eIF2α phosphorylation has a proapoptotic role (48Gil J. Esteban M. Apoptosis. 2000; 5: 107-114Crossref PubMed Scopus (325) Google Scholar). This is also supported by several studies showing that dephosphorylation of eIF2α during diverse stress conditions correlates with better survival (49Boyce M. Bryant K.F. Jousse C. Long K. Harding H.P. Scheuner D. Kaufman R.J. Ma D. Coen D.M. Ron D. Yuan J. Science. 2005; 307: 935-939Crossref PubMed Scopus (1170) Google Scholar).The translation inhibition induced by eIF2α phosphorylation has been suggested to induce apoptosis by inhibiting the expression of anti-apoptotic proteins such as the Bcl-2 family member, Mcl-1, which inhibits the proapoptotic proteins Bax and Bak (50Kim H. Rafiuddin-Shah M. Tu H.C. Jeffers J.R. Zambetti G.P. Hsieh J.J. Cheng E.H. Nat. Cell Biol. 2006; 8: 1348-1358Crossref PubMed Scopus (697) Google Scholar). It was suggested that down-regulation of Mcl-1 enables but does not cause apoptosis and that eIF2α phosphorylation induced by osmotic stress inhibits Mcl-1 mRNA translation and causes mitochondrial apoptosis (11Fritsch R.M. Schneider G. Saur D. Scheibel M. Schmid R.M. J. Biol. Chem. 2007; 282: 22551-22562Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, we have shown that Mcl-1 mRNA translation was repressed in both S/S and A/A cells, suggesting that decreased Mcl-1 protein levels do not induce apoptosis. We show here that the eIF2α phosphorylation-mediated shift to apoptosis involves reduced translation of the IRES-containing Bcl-xL mRNA.Regulation of IRES-mediated translation can play a critical role in cell fate during severe stress by controlling the synthesis of pro-apoptotic or anti-apoptotic proteins. IRES-mediated translation of these mRNAs is regulated by positive and negative factors. The mRNA-binding protein hnRNP A1 has been shown to inhibit translation of the prototypic member of the inhibitors of apoptosis proteins, XIAP, by binding and decreasing the activity of the XIAP IRES. Osmotic stress was shown to increase the cytoplasmic accumulation of hnRNP A1, thus inhibiting translation of the XIAP mRNA (16Lewis S.M. Veyrier A. Hosszu Ungureanu N. Bonnal S. Vagner S. Holcik M. Mol. Biol. Cell. 2007; 18: 1302-1311Crossref PubMed Scopus (91) Google Scholar). We show here that hnRNP A1 also the activity of the Bcl-xL in with the decreased translation of this mRNA during osmotic stress in S/S The eIF4G member has also been shown to positively XIAP S. G. T. L. G. A. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) and Bcl-xL N. L. A. Cell 2009; 8: PubMed Scopus Google Scholar) IRES is during osmotic stress and hnRNP A1 cytoplasmic levels the balance between positive and negative may of These the regulation of IRES-mediated translation and show the balance between positive and negative IRES trans-acting factors leads to regulation during studies the important does eIF2α phosphorylation hnRNP A1 eIF2α phosphorylation nuclear or nuclear by phosphorylation in the Phosphorylation of hnRNP A1 during osmotic stress is carried out by the J. J. J. Cell Biol. 2000; PubMed Scopus Google Scholar). both kinases in S/S and A/A cells during osmotic stress Fig. factors to cytoplasmic The involve such as and that on hnRNP A1. of hnRNP proteins has been associated with their nuclear S. Mol. Cell. 2005; 18: Full Text Full Text PDF PubMed Scopus Google and several the of hnRNP A1 are E. S. M. J. Proc. Natl. Acad. Sci. U.S.A. 2005; PubMed Scopus Google Scholar). We are the that a of hnRNP A1 in the nucleus nuclear phosphorylation in the of studies was that cytoplasmic hnRNP A1 was required for the induction of apoptosis by osmotic stress. This was shown by the of protein, which is in the cytoplasm, to hypertonic apoptosis in A/A This is with a showing that cytoplasmic but not nuclear hnRNP A1 protein causes translational inhibition of IRES-mediated translation A. F. Bonnal S. S.M. N. Holcik M. Vagner S. Mol. Biol. Cell. 2007; 18: PubMed Scopus Google Scholar). We that of IRES-mediated translation by hnRNP A1 during osmotic stress cell In to role in mRNA hnRNP A1 of some anti-apoptotic mRNAs during stress via the in their T.E. M. S.M. J. Holcik M. Cell Death Differ. 2009; PubMed Scopus Google Scholar). we not this is of control that the cellular response to stress. in S/S cells is likely to be controlled by several proteins with mRNAs by hnRNP A1, including the anti-apoptotic proteins XIAP (16Lewis S.M. Veyrier A. Hosszu Ungureanu N. Bonnal S. Vagner S. Holcik M. Mol. Biol. Cell. 2007; 18: 1302-1311Crossref PubMed Scopus (91) Google Scholar) and Bcl-xL studied here. The importance of Bcl-xL in cells from apoptosis by inhibiting mitochondrial and has been previously L. D.R. Cell Death Differ. 2006; PubMed Scopus Google Scholar). Bcl-xL was shown to be important for of A/A cells from apoptosis during osmotic regulation of Bcl-xL levels cell fate during osmotic stress novel of this is the absence of hnRNP A1 in cytoplasmic SGs in A/A cells during hypertonic stress. hnRNP A1 has been shown to in SGs during osmotic stress S. Long Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). We show here that the absence of translational of hnRNP A1 target mRNAs in A/A cells was not due to their to SGs. However, hnRNP A1 was from SGs in A/A cells, suggesting that eIF2α phosphorylation is required for this This that a of mRNAs are translationally repressed during osmotic stress by sequestration in SGs. XIAP (16Lewis S.M. Veyrier A. Hosszu Ungureanu N. Bonnal S. Vagner S. Holcik M. Mol. Biol. Cell. 2007; 18: 1302-1311Crossref PubMed Scopus (91) Google Scholar) and Bcl-xL are mRNAs for this contrast to the proapoptotic function of hnRNP A1 and S. Long Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar) demonstrated a protective role of this protein during osmotic stress by mechanisms that involve both nuclear and cytoplasmic functions. hnRNP A1 has in protein synthesis and is likely that of these promotes We that hnRNP A1 promotes apoptosis by inhibiting the IRES-mediated translation of a of anti-apoptotic This a system for osmotic stress the diverse of hnRNP of to hypertonic or conditions is the recovery of cell volume (1Burg M.B. Ferraris J.D. Dmitrieva N.I. Physiol. Rev. 2007; 87: 1441-1474Crossref PubMed Scopus (557) Google Scholar). hypertonic stress induces transcription of genes via levels and nuclear of TonEBP and translation of the induced this response does not involve eIF2α phosphorylation (14Gaccioli F. Huang C.C. Wang C. Bevilacqua E. Franchi-Gazzola R. Gazzola G.C. Bussolati O. Snider M.D. Hatzoglou M. J. Biol. Chem. 2006; 281: 17929-17940Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) or of global protein synthesis M. D. M.A. Biochem. J. 2003; PubMed Scopus Google with the translation of the induced stress response In we show that severe osmotic stress induces eIF2α phosphorylation and inhibits by inhibiting global protein synthesis and the anti-apoptotic This response of It is that A/A cells apoptosis during severe osmotic stress. global protein synthesis is in these cells, is likely that survival requires IRES-containing mRNAs that are in A/A but not S/S The of mRNAs that are in S/S and A/A cells during severe osmotic stress will on the role of eIF2α phosphorylation in the induction of used primary cortical neurons from S/S and A/A mouse to the importance of eIF2α phosphorylation and hnRNP A1 in the response to severe osmotic stress. as a because the of apoptosis is important for apoptosis in embryonic (4Loyher M.L. Mutin M. Woo S.K. Kwon H.M. Tappaz M.L. Neuroscience. 2004; 124: 89-104Crossref PubMed Scopus (54) Google Scholar, Huang A.E. K.M. T. R.J. S. M. Proc. Natl. Acad. Sci. U.S.A. 2007; PubMed Scopus Google Scholar). We show here that cells do not eIF2α phosphorylation for in However, severe hypertonic which also causes mitochondrial required eIF2α phosphorylation for the induction of apoptosis. It will be to the importance of eIF2α phosphorylation in apoptosis during findings in this may have in the of including and In with with causes cytoplasmic accumulation of hnRNP A1, leading to induction of apoptosis J. 2009; PubMed Scopus Google Scholar). In addition, hnRNP A1 levels and cytoplasmic accumulation of hnRNP A1 increase in several but the subcellular distribution of the protein has not been with survival of the signaling during osmotic stress that cytoplasmic hnRNP A1 phosphorylation is also important for induction of apoptosis in cells with It is likely that phosphorylation of eIF2α plays an important role in the balance between survival and apoptosis in a of physiological stresses via of of hnRNP A1. IntroductionThe regulation of cell volume in response to changes in extracellular osmolarity is an important function. Hypertonic conditions induce responses that protect cells by inducing transport of osmolytes referred to as compatible solutes. Intermediate levels of hypertonic stress cause cell cycle arrest followed by cell survival, but higher levels induce apoptosis (1Burg M.B. Ferraris J.D. Dmitrieva N.I. Physiol. Rev. 2007; 87: 1441-1474Crossref PubMed Scopus (557) Google Scholar).The master regulator of this response is the tonicity-responsive enhancer-binding protein, TonEBP, which translocates to the nucleus and induces transcription of genes encoding osmolyte transporters and heat shock proteins (1Burg M.B. Ferraris J.D. Dmitrieva N.I. Physiol. Rev. 2007; 87: 1441-1474Crossref PubMed Scopus (557) Google Scholar). A remarkable example of adaptation is seen in kidney medulla, which is exposed to hypertonic urine. Mice deficient in TonEBP show atrophy of the renal medulla and deficiencies in immune system function (2Go W.Y. Liu X. Roti M.A. Liu F. Ho S.N. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10673-10678Crossref PubMed Scopus (226) Google Scholar). In addition, brain cells have adaptive mechanisms for cell volume recovery that involve the TonEBP target gene SNAT2 (3Maallem S. Mutin M. González-González I.M. Zafra F. Tappaz M.L. Neuroscience. 2008; 153: 95-107Crossref PubMed Scopus (19) Google Scholar). This is further supported by the induction of TonEBP in neurons during systemic hypertonicity (4Loyher M.L. Mutin M. Woo S.K. Kwon H.M. Tappaz M.L. Neuroscience. 2004; 124: 89-104Crossref PubMed Scopus (54) Google Scholar). Neurological disorders have also been associated with hypertonic blood plasma, as in diseases of water imbalance (5Lin M. Liu S.J. Lim I.T. Emerg. Med. Clin. North Am. 2005; 23 (ix): 749-770Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).The cellular response to stress involves regulation of mRNA translation (6Graber T.E. Holcik M. Mol. Biosyst. 2007; 3: 825-834Crossref PubMed Scopus (57) Google Scholar, 7Komar A.A. Hatzoglou M. J. Biol. Chem. 2005; 280: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 8Silvera D. Arju R. Darvishian F. Levine P.H. Zolfaghari L. Goldberg J. Hochman T. Formenti S.C. Schneider R.J. Nat. Cell Biol. 2009; 11: 903-908Crossref PubMed Scopus (184) Google Scholar). Diverse stresses limit protein synthesis, mostly via inhibition of translation initiation. Much of this regulation involves eIF2, 5The abbreviations used are: eIF2eukaryotic initiation factor 2hnRNP A1heterogeneous nuclear ribonucleoprotein A1IRESinternal ribosome entry siteSGstress granuleMEFmouse embryonic fibroblastUTRuntranslated regionRT-qPCRreverse transcription-quantitative PCRTUNELterminal dUTP nick end-labelingHAhemagglutininGFPgreen fluorescent proteinDAPI4′,6-diamidino-2-phenylindoleWTwild typeXIAPX-linked inhibitor of apoptosisUPRunfolded protein response. which delivers the initiator tRNA (Met-tRNAiMet) to cellular mRNAs that use an AUG initiation codon. Phosphorylation of the α subunit on Ser51 sequesters eIF2 in an inactive complex, thus decreasing translation initiation (9Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar).The outcome of the stress response depends on the intensity and duration of the stress. Events during the early response promote the recovery of translation and the synthesis of proteins essential for adaptation (10Rutkowski D.T. Arnold S.M. Miller C.N. Wu J. Li J. Gunnison K.M. Mori K. Sadighi Akha A.A. Raden D. Kaufman R.J. PLoS Biol. 2006; 4: e374Crossref PubMed Scopus (616) Google Scholar). However, prolonged and severe stress promotes apoptosis. The regulated synthesis of pro-survival and pro-death proteins plays an important role in determining cell fate during stress. It has been suggested that persistent inhibition of protein synthesis can lead to apoptosis by inhibiting translation of mRNAs encoding anti-apoptotic proteins (11Fritsch R.M. Schneider G. Saur D. Scheibel M. Schmid R.M. J. Biol. Chem. 2007; 282: 22551-22562Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Scheuner D. Patel R. Wang F. Lee K. Kumar K. Wu J. Nilsson A. Karin M. Kaufman R.J. J. Biol. Chem. 2006; 281: 21458-21468Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Translation of some pro-apoptotic and anti-apoptotic mRNAs is mediated by internal ribosome entry sites (IRESs) in the mRNA leaders. IRES-mediated translation initiation involves recruitment of the ribosome to the mRNA independently of the 5′-cap. In most cases, this requires IRES trans-acting factors that mediate ribosome recruitment. The activity of many IRESs can be positively or negatively regulated by IRES trans-acting factors depending on the type of stress (6Graber T.E. Holcik M. Mol. Biosyst. 2007; 3: 825-834Crossref PubMed Scopus (57) Google Scholar, 13Spriggs K.A. Bushell M. Mitchell S.A. Willis A.E. Cell Death Differ. 2005; 12: 585-591Crossref PubMed Scopus (134) Google Scholar).We previously showed that mild osmotic stress (400 mosmol/liter) causes a pro-survival response without induction of eIF2α phosphorylation (14Gaccioli F. Huang C.C. Wang C. Bevilacqua E. Franchi-Gazzola R. Gazzola G.C. Bussolati O. Snider M.D. Hatzoglou M. J. Biol. Chem. 2006; 281: 17929-17940Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study, we show that severe osmotic stress induces apoptosis in a manner dependent on eIF2α phosphorylation. In contrast to the current view, decreased protein synthesis in the absence of eIF2α phosphorylation does not induce apoptosis. Rather, eIF2α phosphorylation is required for the translocation of the RNA-binding protein hnRNP A1 from the nucleus to the cytoplasm, which blocks translation of mRNAs encoding inhibitors of apoptosis. The pro-apoptotic function of eIF2α phosphorylation during osmotic stress is also demonstrated in primary cortical neurons. This mechanism of translational control may have direct implications in many diseases that involve undesirable apoptosis such as heart failure and

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