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Loss of c/EBP-β activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons

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Loss of c/EBP-β activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons
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  Loss of c/EBP- β  activity promotes the adaptive to apoptotic switchin hypoxic cortical neurons Marc W. Halterman a,b,c,*, Christopher De Jesus b, David A. Rempe a,c, Nina F. Schor  b,c, and Howard J. Federoff  da  Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642,USA b  Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642,USA c Center for Neural Development and Disease, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA d  Department of Neurology, Georgetown University Medical Center, Washington, D.C. 20007, USA  Abstract Understanding the mechanisms governing the switch between hypoxia-induced adaptive and  pathological transcription may reveal novel therapeutic targets for stroke. Using an in vitro  hypoxiamodel that temporally separates these divergent responses, we found apoptotic signaling was preceded by a decline in c/EBP- β  activity and was associated with markers of ER-stress includingtransient eIF2 α  phosphorylation, and the delayed induction of the bZIP proteins ATF4 and CHOP-10.Pretreatment with the eIF2 α  phosphatase inhibitor salubrinal blocked the activation of caspase-3,indicating that ER-related stress responses are integral to this transition. Delivery of either full-length,or a transcriptionally inactive form of c/EBP- β  protected cultures from hypoxic challenge, in part byinducing levels of the anti-apoptotic protein Bcl-2. These data indicate that the pathologic responsein cortical neurons induced by hypoxia involves both the loss of c/EBP- β -mediated survival signalsand activation of pro-death pathways srcinating from the endoplasmic reticulum. Keywords c/EBP- β ; bZIP; Hypoxia; Neuron; Delayed death; Apoptosis * Corresponding author. Center for Neural Development and Disease, University of Rochester School of Medicine and Dentistry, 601Elmwood Avenue, Box 645, Rochester, NY 14642, USA. Fax: +1 585 276 1947. E-mail address: marc_halterman@urmc.rochester.edu,(M.W. Halterman).The authors report no commercial affiliations or conflicts of interest pertaining to this manuscript. Publisher's Disclaimer: This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internalnon-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutionalrepository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit:http://www.elsevier.com/copyright  NIH Public Access Author Manuscript  Mol Cell Neurosci . Author manuscript; available in PMC 2009 March 6. Published in final edited form as:  Mol Cell Neurosci . 2008 June ; 38(2): 125–137. doi:10.1016/j.mcn.2008.01.014. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Introduction  Neuroprotective trials for stroke have focused mainly on molecular targets activated early inthe pathological signaling cascade. However, evidence suggest that cell-autonomous delayed apoptotic signaling plays a critical role in determining the ultimate burden of neuron loss after injury (Abe et al., 1995; Schwamm et al., 1998). Hypoxia is a potent stimulus for de novo  geneexpression, and sub-lethal hypoxic stress can enhance cell survival through the regulated expression of factors such as erythropoietin and vascular endothelial growth factor (Dirnagl etal., 2003; Jones and Bergeron, 2001). This process, also referred to as ischemic preconditioning, is supported in part by the activation of the hypoxia-inducible factor (HIF-1 α ) and an array of immediate early transcription factors with diverse biological functionsincluding c-Jun and Egr-1/Krox-24 (Collaco-Moraes et al., 1994; Herdegen and Leah, 1998;Hsu et al., 1993). Stroke-induced gene expression also plays a critical role in promoting delayed neuron loss after ischemia (Honkaniemi et al., 1996). In this regard, pre-treatment with themacromolecular synthesis inhibitor cycloheximide confers neuroprotection (Du et al., 1996;Gwag et al., 1995). Data from in vitro  studies and models of global ischemia indicate that thisdeath mechanism is cell-autonomous. And from a therapeutic perspective, identification of thekey regulatory nodes in hypoxia signaling networks that discriminate between these divergenttranscriptional programs would be advantageous.One  potential sensor capable of triggering both adaptive and pathologic signaling after stroke is the endoplasmic reticulum (ER), shown previously to influence other diseases affecting thecentral nervous system (Kaufman, 2002; Rao et al., 2004). The physiologic changes associated with ischemia also activate stress-sensing proteins resident in the ER, which in turn stimulateadaptive transcription via the unfolded protein response (UPR) (Harding et al., 2002). For exam ple, translational arrest induced by PERK-mediated phosphorylation of the translation initiation factor eIF2 α  at Ser  51  is associated with cell survival and occurs in neurons within theischemic penumbra (Kumar et al., 2001; Liu et al., 2006; Mengesdorf et al., 2002). Similarly,the bZIP transcription factor ATF6 and the inositol-requiring transmembrane kinase and endonuclease-1 α  (IRE-1 α ) regulate the expression of BiP/GRP78 and other factors thatenhance the folding capacity of the ER. However, hyper-activation of ER-stress pathways canhave negative consequences. Prolonged eIF2 α  inactivation induces the protein phosphataseregulatory subunit GADD34, which reverses eIF2 α -mediated translational inhibition promoting programmed cell death (Brush et al., 2003). As such, phosphatase inhibitors like salubrinal that prolong translational arrest are protective against ER-stress (Boyce et al.,2005; Sokka et al., 2007). And while activation of CHOP-10 may enhance mitochondrialfunction through the direct regulation of heat shock proteins including mtDnaJ and ClpP,deletion of CHOP-10 is neuroprotective after stroke (Tajiri et al., 2004; Zhao et al., 2002).Lastly, activated caspase-3, caspase-12 and several BH3 proteins (i.e., Bcl-2, Bax, PUMA and others) associate with, and link the ER to the cellular apoptotic signaling machinery (Masud et al., 2007; Rao et al., 2004; Reimertz et al., 2003). A better understanding of the interplay between hypoxia, ER-stress signaling and the factors controlling downstream transcriptionalresponses to hypoxia could have significant implications for the treatment of stroke.In the current study we characterized a translation-dependent in vitro  model of hypoxia-induced neur onal apoptosis. By defining the temporal boundaries separating adaptation from thecommitment to cell death, we sought to identify the factors required to activate neuronal deathfollowing prolonged hypoxic stress. In the current work, we report a novel cell survival functionfor the bZIP factor c/EBP- β , and show that the loss of c/EBP- β  activity precedes the onset of cell death promoted in part by stress signals emanating from the endo plasmic reticulum. Furthermore, based on the observed delayed induction of the heterodimeric factors ATF4 and CHOP-10, we propose a model in which hypoxia-induced ER-stress responses shift the activity Halterman et al.Page 2  Mol Cell Neurosci . Author manuscript; available in PMC 2009 March 6. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    of the bZIP protein network from an initial adaptive response, towards a pro-apoptotictranscriptional program. Results Chronic hypoxia induces delayed neuronal death in cortical neurons Classical oxygen-glucose deprivation (OGD) produces acute necrosis in mature corticalcultures, however sub-lethal challenge or OGD performed in the presence of glutamateantagonists can trigger a delayed form of neuron death that is dependent on gene expression(Gwag et al., 1995). To study the role of gene expression in the delayed loss of neurons after stroke, we developed an in vitro  model of hypoxia-induced neuronal apoptosis usingdissociated embryonic cortical cultures. By DIV7, most cells in culture express NeuN and havedeveloped a dense network of β -III tubulin positive axonal projections (data not shown).Exposure to hypoxia (0.5% O 2 ) induces procaspase-3 cleavage and nuclear pyknosis in up to40% of neurons (Fig. 1A). Time course analyses indicate that both phenotypes depend on theduration of hypoxia (Fig. 1B) and not on media glucose concentrations provided cultures aremaintained at or above 10 mM prior to the onset of hypoxia (Fig. S1). Nuclear pyknosis plateaued 24 h after the onset of hypoxia and was followed by the delayed uptake of trypan blue beginning 48 h post-exposure consistent with the phenomenon of secondary necrosis (Fig.S2) (Ankarcrona et al., 1995). And while pre-treatment with a combination of the glutamatereceptor antagonists MK-801 (10 μ M) and CNQX (100 μ M) had no effect on neuron survival,addition 1 μ g/ml of the translation inhibitor cycloheximide conferred complete protectionagainst hypoxia-induced pyknosis (Fig. 1C). These data suggest that hypoxia alone is sufficientto induce a cell-autonomous death program in a subset of dissociated cortical neurons thatrequires de novo  gene expression. Defining adaptive and pathologic phases of gene expression in hypoxic cultures Ischemic tolerance induced by sub-lethal challenge confers delayed neuroprotection through the expression of mRNA species with adaptive properties (Dirnagl et al., 2003). To test whether the onset of cell loss in our model was associated with the sequential expression of adaptiveand pathologic genes, we performed a time course analysis of gene expression usingquantitative RT-PCR against transcriptional targets previously validated using in vivo  strokemodels (Kim et al., 2004; Pirianov et al., 2007; Zhang et al., 2007). Selected genes included those associated with either ischemic preconditioning and survival (VEGF, Hexokinase II and the type-1 glucose transporter), or apoptosis (BNIP3, PUMA and NOXA). Results demonstratethat the adaptive expression began early (3–6 h) and remained elevated, while apoptotictranscription was delayed in onset (9–12 h) in keeping with the observed kinetics of cell death(12–18 h) (Fig. 2). If the observed transcriptional profiles are representative of the populationresponse to hypoxia, these results indicate neurons are capable of mounting separable adaptiveand pathological responses. Alternatively, if embryonic cortical cultures recapitulate patternsof selective vulnerability observed in vivo , it is possible that adaptive and pro-apoptotic geneexpressions occur in distinct cellular populations. Either way, the existence of qualitativelyand temporally ditinct transcriptional responses to hypoxic stress provide a framework to studythe genetic networks regulating their activation. Defining the role of ER-stress responses in hypoxia-induced delayed neuronal loss The endoplasmic reticulum has emerged as an important sensor of cellular stress in diseasesaffecting the central nervous system including stroke (Rao et al., 2004). Shared, as well as ER-stress specific pathways have evolved to respond to diverse stimuli including hypoxia, aminoacid starvation, hypoglycemia and perturbations in calcium homeostasis (Fig. 3A). The proximate sensors of ER-stress include PERK, ATF6 and IRE-1 α , which exist in an inactiveform bound to the ER chaperonin GRP78/BiP (Sommer and Jarosch, 2002). Exposure to Halterman et al.Page 3  Mol Cell Neurosci . Author manuscript; available in PMC 2009 March 6. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t     physiologic stress enhances the release and homo-dimerization of these factors activating their latent signaling capacity. For example, PERK-mediated phosphorylation of eIF2 α  at Ser51triggers the arrest of cap-dependent translation, an event that is both cytoprotective under conditions of ER-stress and is associated with adaptive responses in the ischemic penumbra(Harding et al., 2000; Mengesdorf et al., 2002). This and other strategies, including theregulated proteolysis and release of the transcriptionally active amino terminal fragment of ATF6, have evolved to regulate the expression of nuclear transcriptional targets with bothadaptive and pathological effects on the cell.The observation that expression of the pro-apoptotic Bcl-2 family members NOXA and PUMAwere induced in our cultures suggested that ER-stress responses might be involved in propagating delayed apoptotic signals after hypoxic challenge (Li et al., 2006). To assess this,we analyzed hypoxic neuronal lysates by western blotting for markers related to signalingthrough the shared and ER-stress specific pathways. Hypoxia induced a transient rise in BiP/GRP78 protein, transient phosphorylation of the elongation factor eIF2 α  at Ser  51 , a decline intotal eIF2 α  levels, and a relative increase in the amount of the low molecular weight hypo- phosphorylated eIF2 α  species (Fig. 3B). While levels of actin also declined over time consistentwith global translational arrest, neurofilament levels remained stable. The kinetics of eIF2 α  phosphorylation supported a causal link between the loss of eIF2 α  inactivation and delayed cell death after hypoxia. To test this, we pretreated cultures with the eIF2 α  phosphataseinhibitor salubrinal, and compared eIF2 α  post-translational modification with post-hypoxicneuron survival (Figs. 3C and D). Results indicate that 10 μ M salubrinal extended eIF2 α inactivation by 3 h and conferred a modest survival benefit compared to DMSO treated controlcultures (22±2% vs  31±1%, P <0.01). Interestingly, 10 μ M salubrinal did not extend hypoxia-induced eIF2 α  phosphorylation beyond 6 h (Fig. 4A). Although not directly tested, thisobservation suggests that hypoxia induced the expression of GADD34, which promoteseIF2 α  dephosphorylation through direct interactions with protein phosphatase 1c (PP1c)(Brush et al., 2003; Novoa et al., 2001).To better understand the relationship between hypoxia and ER-dependent signaling, we nextanalyzed hypoxia's effects on the shared (eIF2 α ) and ER-specific signaling involving ATF6,and the downstream factors ATF4 and CHOP-10. To demonstrate these pathways could beactivated in vitro , normoxic cultures were treated with the Ca 2+ -ATPase pump inhibitor thapsigargin (Tg). Tg (1  μ g/ml) induced eIF2 α  dephosphorylation, ATF6 cleavage (detected  by release of the amino terminal domain ATF6 α (n)), expression of the pro-apoptotic bZIPfactor CHOP-10, and the robust activation of caspase-3 (Fig. 4A). While hypoxia alone induced transient eIF2 α  phosphorylation, triggered the delayed induction of CHOP and ATF4, and stimulated cleavage of caspase-3, it had only a minor effect on ATF6 proteolysis and releaseof the transcriptionally active amino terminal fragment (Fig. 4B, right). Consistent with our  prior results, salubrinal blocked caspase-3 cleavage, enhanced ATF4 expression, and reduced the basal expression and cleavage of ATF6 without influencing levels of CHOP-10. In contrast,the serine protease inhibitor AEBSF disrupted ATF6 processing and CHOP-10 expression, ithad only moderate effects on caspase-3 cleavage (Ma et al., 2002;Okada et al., 2003). Thesedata argue that the shared unfolded protein response involving eIF2 α  is a key intermediate inthe activation of delayed apoptotic signaling in this model, while ATF6-mediated responsesappear less important in this regard. Moreover, the data demonstrating persistent CHOP-10expression despite salubrinal treatment suggest that CHOP-10 expression alone is not sufficientto stimulate apoptotic signaling after hypoxic stress.To identify additional transcription factors that could interface with the ER-signalingapparatus, we queried functional protein association networks using the STRING (SearchTool for Recurring Instances of Neighboring Genes) algorithm. This application draws uponempirically derived public data sets and constructs a map of interacting factors with greatest Halterman et al.Page 4  Mol Cell Neurosci . Author manuscript; available in PMC 2009 March 6. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    statistical relevance based on an input set of query proteins (von Mering et al., 2007). In additionto ATF4, CHOP-10 and eIF2 α , we included GADD34 in our search given its suspected activityin our system. Results confirmed several expected relationships including ATF4-mediated GADD34 expression, the association of p38MAPK with CHOP-10, and protein–proteininteractions between the immediate early genes c-Jun and Fos (Fig. 5A). What was mostintriguing was the set of interactions uncovered among the bZIP transcription factors ATF4,CHOP-10, c-Jun and c/EBP- β . And while c-Jun, ATF4 and CHOP have been linked withcellular responses to hypoxia, c/EBP- β 's role in hypoxic signaling has not been established (Blais et al., 2004; Ron and Habener, 1992). To investigate this further, we analyzed c-Jun and c/EBP- β  expression by western blotting and found that hypoxia stimulated the transientinduction of Jun and its downstream transcriptional target c/EBP- β  before any appreciableinduction of ATF4 was seen (Lin et al., 2002). We also observed that hypoxia induced therobust and transient phosphorylation of c/EBP- β  at Thr  188  (Fig. 5B), which is required for efficient function of the transcription activation domain (Piwien-Pilipuk et al., 2002). Thedecline in c/EBP- β  protein levels was insensitive to both salubrinal and AEBSF treatment,suggesting that the mechanisms regulating c/EBP- β  expression and/or stability are likely PERK and ATF6 independent. This complex pattern of c-Jun, c/EBP- β , CHOP-10 and ATF4regulation suggested that perhaps shifts in bZIP stoichiometry could drive the formation of  pathogenic heterodimers triggering delayed apoptosis in hypoxic neurons (Ohoka et al.,2005; Vinson et al., 2002). Reports describing CHOP and ATF4 driven expression of the Aktinhibitor Trib3 and its role in apoptosis support this idea (Ohoka et al., 2005).To further explore the relationship between hypoxia and the regulation of c/EBP- β  activity,we analyzed c/EBP- β  expression patterns in cortical neurons by immunocytochemistry usingan antibody recognizing the transcriptionally active species phosphorylated at Thr  188  (p-c/EBP- β ). When compared to the marker NeuN, we found that p-c/EBP- β  was ubiquitouslyexpressed and localized to the nucleus of neurons under basal conditions (Fig. 6A). We alsodetermined whether hypoxia-induced nuclear export was involved in the mechanism of c/EBP- β  downregulation as described previously in primary mouse hepatocytes exposed to TNF α (Buck et al., 2001). Our analysis indicated that while hypoxia increased immunoreactivity inthe neuritic processes, the majority of p-c/EBP- β  remained confined to the nucleus. We alsoobser ved differences in the relative levels of p-c/EBP- β  across individual neurons exposed tohypoxia and hypothesized this could be mechanistically related to the phenomenon of selectivevulnerability. To address this possibility, hypoxic cultures were analyzed for the accumulationof pyknotic nuclei relative to either the high or low levels of p-c/EBP- β  expression observed.While indirect, the data indicate that the maintenance of c/EBP- β  phosphorylation at Thr  188 was associated with greater post-hypoxic survival (72±12% vs  15±17%, P <0.05; Fig. 6B),suggesting the early induction and persistence of activated c/EBP- β  in culture is an adaptiveresponse to hypoxic challenge. Expression of c/EBP- β  protects cortical neurons from delayed apoptosis If the stoichiometry of bZIP heterodimerization governs neuron survival after hypoxicchallenge, we hypothesized that the apoptotic switch could be blocked by upregulating c/EBP- β  expression. To this end, we generated a bi-cistronic herpes amplicon vectors expressingeGFP, and one of three different cDNAs including full-length c/EBP- β , a dominant-negativeform of c/EBP- β  ( Δ TAD) lacking the transcriptional transactivation domain, or a control vector expressing luciferase (LUC). After confirming equivalent transduction across samples (Fig.7A), cultures were screened for their relative susceptibility to hypoxia-induced nuclear  pyknosis. Results show that transduction with wild-type c/EBP- β  virus reduced nuclear  pyknosis roughly 4-fold relative to cultures receiving control virus (6.7±1.6% vs  29.7±1.3%, P <0.01; Fig. 7B). Transduction with the Δ TAD construct was also protective albeit to a lesser degree (20.2±4.1% vs  29.7±1.3%, P <0.05). Western blot analysis confirmed the expression of  Halterman et al.Page 5  Mol Cell Neurosci . Author manuscript; available in PMC 2009 March 6. 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