3958 Research Article

IntroductionThe endoplasmic reticulum (ER) is a factory responsible forthe synthesis, initial post-translational modification, properfolding, and maturation of newly synthesized transmembraneand secretory proteins, as well as a regulator of intracellularcalcium homeostasis. Various stresses, including expression ofmutant proteins, viral infection, energy or nutrient deprivation,extreme environmental conditions, alterations in redox statusor glycosylation status, or calcium release from the lumen ofthe ER, disrupt proper function of ER and cause so-called ERstress. The prototypic response of ER stress is the congestionof misfolded proteins. ER stress leads cells to activate self-protective mechanisms: (1) transcriptional up-regulation of ERchaperones and folding enzymes; (2) translational attenuationto limit further accumulation of misfolded proteins; and(3) ER-associated degradation (ERAD) which eliminatesmisfolded proteins from the ER. The current understanding isthat three types of ER membrane receptors, ATF6, IRE1 andPERK may sense the stress in the ER, and eventually activatetranscription factors for induction of ER chaperones, such asGRP78, or phosphorylate eIF2� for inhibition of synthesis ofnew proteins. These processes are collectively called unfoldedprotein response (UPR) (Imaizumi et al., 2001). Excessive andprolonged stresses, however, lead cells to apoptosis (Schroderand Kaufman, 2005). ER stress-induced apoptosis is associatedwith a range of diseases, including ischemia/reperfusion injury,neurodegeneration and diabetes (Xu et al., 2005).

There are two major pathways that induce apoptotic celldeath. One is the extrinsic pathway, in which ligation of deathreceptors by death ligands is followed by recruitment ofadaptor molecules and activation of caspase-8 or caspase-10.The other is the intrinsic pathway, in which the release ofcytochrome c from mitochondria triggers the formation of theapoptosome composed of Apaf-1, pro-caspase-9, dATP, andcytochrome c. Apoptosome formation results in the activationof executioner caspases including caspase-3, -6, and -7. Theprecise molecular mechanisms of ER stress-induced apoptosis,however, have not been fully elucidated (Xu et al., 2005).CHOP, a transcription factor induced under ER stress, has beenreported to be involved in ER stress-induced apoptosis byreducing the expression of Bcl-2 (McCullough et al., 2001).Indeed, CHOP-deficiency causes resistance to ER stress-induced cell death both in vitro and in vivo (Zinszner et al.,1998). ER stress also leads to increase cytosolic calcium levels,which induces the activation of m-calpain. Activated m-calpaincleaves Bcl-XL and proteolytically activates caspase-12(Nakagawa and Yuan, 2000). Processed-caspase-12 reportedlyactivates caspase-9 independent of Apaf-1, followed byactivation of caspase-3 (Rao et al., 2002). Several groups alsoreported that ER stress increases reactive oxygen species(ROS), and antioxidant protects cells from ER stress-inducedcell death (Cullinan and Diehl, 2004; Harding et al., 2003;Haynes et al., 2004; Mauro et al., 2006). The stress kinasepathway is also associated with ER stress-induced apoptosis.

Accumulation of unfolded proteins induces endoplasmicreticulum (ER) stress. Excessive and prolonged stresseslead cells to apoptosis. However, the precise molecularmechanisms of ER stress-induced apoptosis have not beenfully elucidated. We investigated the involvement of theapoptosome in ER stress-induced cell death pathway usingmouse embryonic fibroblasts (MEFs) and mice deficient forApaf-1. Apaf-1-deficient MEFs showed more resistance toER stress-inducing reagents as compared with wild typecells. Despite comparable induction of ER stress in bothwild type and Apaf-1-deficient cells, activation of caspase-3 was only observed in wild type, but not Apaf-1-deficient,MEFs. Under ER stress conditions, BAX translocated to

mitochondria and cytochrome c was released frommitochondria. We also demonstrated that caspase-12 wasprocessed downstream of Apaf-1 and caspase-3, andneither overexpression nor knockdown of caspase-12affected susceptibility of the cells to ER stress-induced celldeath. Furthermore, in the kidneys of Apaf-1-deficientmice, apoptosis induced by in vivo administration oftunicamycin was remarkably suppressed as compared withwild type mice. These data collectively demonstrated thatApaf-1 and the mitochondrial pathway of apoptosis playsignificant roles in ER stress-induced apoptosis.

Key words: Apoptosis, Apaf-1, ER stress, Mitochondria

Summary

ER stress-induced apoptosis and caspase-12activation occurs downstream of mitochondrialapoptosis involving Apaf-1Hiroshi Shiraishi1, Hideaki Okamoto2, Akihiko Yoshimura1 and Hiroki Yoshida2,*1Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Japan2Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima,Saga, 849-8501, Japan*Author for correspondence (e-mail: yoshidah@med.saga-u.ac.jp)

Accepted 5 July 2006Journal of Cell Science 119, 3958-3966 Published by The Company of Biologists 2006doi:10.1242/jcs.03160

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3959Apaf-1 and ER stress-induced apoptosis

During ER stress, Ask1, one of mitogen-activated protein(MAP) kinase-kinase (MAPKK) kinases, is recruited tooligomerized IRE1 complex containing TRAF2, and isactivated for the downstream activation of JNK (Nishitoh et al.,2002). In addition, it is reported that caspase-8 or c-Abl isinvolved in ER stress-induced apoptosis (Ito et al., 2001; Jimboet al., 2003). Such complexity may be because the signalingpathways leading to ER stress-mediated apoptosis might varyin different cell types and in different stresses.

Murine caspase-12 is a member of the inflammatory groupof the caspase family, and study of the caspase-12-deficientmice revealed that it specifically involves in ER stress-inducedcell death (Martinon and Tschopp, 2004; Nakagawa et al.,2000). In humans, the caspase-12 gene has a single nucleotidepolymorphism that results in the production of either atruncated or full-length caspase-12 protein (Saleh et al., 2004).However, the full-length human caspase-12 appears to beenzymatically inactive and functions as a dominant-negativeregulator of proinflammatory signaling pathways rather than inER stress-induced cell death pathway. As an alternative tocaspase-12 in human, caspase-4 is involved in ER stress-induced cell death pathway (Hitomi et al., 2004a). Both murinecaspase-12 and human caspase-4 are localized to the ER andcleaved specifically by ER stress (Hitomi et al., 2004a; Hoppeand Hoppe, 2004; Nakagawa et al., 2000).

In the current study, we investigated the possible involvementof the apoptosome and mitochondria in ER stress-induced celldeath pathways using murine embryonic fibroblasts (MEFs)deficient for Apaf-1. Just like Bcl-XL-expressing MEFs, Apaf-1-deficient MEFs showed more resistance to ER stress-inducingreagents as compared with wild type cells. Although caspase-12 activation was observed in wild type, but not in Apaf-1-deficient, MEFs under ER stress, neither overexpression norknockdown of caspase-12 gene affected susceptibility of thecells to ER stress-induced cell death. In the kidneys of Apaf-1-deficient mice, apoptosis induced by in vivo administration oftunicamycin was remarkably suppressed as compared with thatof wild type mice. Our data suggest that ER stress-inducedapoptosis is dependent on the mitochondrial pathways ofapoptosis involving Apaf-1 and also that caspase-12, activateddownstream of Apaf-1, plays little, if any, role in ER stress-induced apoptosis.

ResultsApaf-1–/– MEFs are resistant to ER stress-induced celldeathApaf-1 is the essential adaptor protein in the intrinsic,mitochondrial pathways of apoptosis and, along withcytochrome c, dATP and caspase-9, forms to the apoptosome(Li et al., 1997; Zou et al., 1997). To investigate the role ofApaf-1 in ER stress-induced cell death, we examined survivalof Apaf-1+/– or Apaf-1–/– primary MEFs against tunicamycin,an inhibitor of N-linked glycosylation, or thapsigargin, aninhibitor of sarcoplasmic-endoplasmic reticulum Ca2+ ATPase.Both drugs are well characterized as inducers of ER stress andexcessive treatments lead to cell death (Kaufman, 1999). First,we checked the growth rate of Apaf-1+/– and Apaf-1–/– MEFsand found that they proliferated similarly (data not shown).Next, cell survival after stimulation with the ER stress-inducing drugs was examined. Apaf-1+/– MEFs died aftertreatment with these drugs (Fig. 1A). Contrary to the previous

report by Rao et al. that Apaf-1-deficient immortalized MEFs,Sak2 cells, died just like wild type NIH/3T3 cells (Rao et al.,2002), Apaf-1–/– MEFs showed significant resistance to ERstress-induced cell death over Apaf-1+/– MEFs (Fig. 1A). Thiswas checked in more than six different clones of MEFs frommore than three different heterozygous intercrosses. This wasalso the case for MEFs immortalized by SV40 large T antigen(data not shown).

Apaf-1 is required for cleavage of caspase-3 during ERstress-induced cell deathTo examine further whether Apaf-1 plays a role of inductionof ER stress, we detected caspase-3 activation by westernblotting. First, we evaluated induction of GRP78, an indicatorof ER stress, by western blotting. Induction of GRP78 bytreatment of either tunicamycin or thapsigargin in Apaf-1–/–

MEFs was comparable to that of Apaf-1+/– MEFs (Fig. 1B),confirming that ER stresses were similarly induced both inApaf-1+/– MEFs and Apaf-1–/– MEFs. For caspase-3 activation,although treatment with tunicamycin or thapsigargin inducedthe cleavage of caspase-3 to p17 fragments in Apaf-1+/– MEFsat 12-24 hours and thereafter, caspase-3 activation was notobserved in Apaf-1–/– MEFs under the same condition (Fig.1B). It was thus demonstrated that ER stress-induced cleavageof caspase-3 is dependent on Apaf-1.

While showing significant resistance to ER stress-inducedcell death, Apaf1–/– MEFs gradually died during long cultureperiods (Fig. 1C). To characterize the cell death induced by ERstress in Apaf-1–/– MEFs, we observed the morphologicalfeatures of cell death using transmission electron microscopy(TEM) (Fig. 1D). Apaf-1+/– MEFs showed typical apoptoticchanges, such as chromatin condensation, nuclearfragmentation, and plasma membrane blebbing at 48 hoursafter stimulation. Although Apaf-1–/– MEFs showed marginalperinuclear chromatin condensation and cytoplasmicvacuolization, typical apoptotic changes were not observed by72 hours after stimulation. Nuclear swelling and massivevacuolization observed in Apaf-1–/– MEFs rather resemblednecrotic cell death.

Bcl-XL protects MEFs from ER stress-induced cell deathBcl-XL is one of the most potent anti-apoptotic members of theBcl-2 family proteins. Reportedly, it protects cells from ERstress-induced cell death in various types of cells (Morishimaet al., 2004; Obeng and Boise, 2005; Srivastava et al., 1999).Unlike Bcl-2, which may localize ER and affect its function,Bcl-XL specifically localizes to mitochondria outer membranewith its C-terminal transmembrane domain (Kaufmann et al.,2003). To examine the effect of Bcl-XL expression on ERstress-induced cell death, both Apaf-1+/– and Apaf-1–/– MEFswere transduced with bcl-xl gene by a retrovirus vector. Weconfirmed that overexpressed-Bcl-XL localized preferentiallyto mitochondria (data not shown). The retrovirus-mediatedexpression of Bcl-XL partially protected Apaf-1+/– MEFs fromER stress-induced cell death at approximately the same levelwith Apaf-1–/– MEFs (Fig. 2A). Consistent with this Bcl-XL-mediated resistance to ER stress-induced cell death, cleavageof caspase-3 to active form was significantly reduced in MEFsoverexpressing Bcl-XL against tunicamycin and thapsigargin(Fig. 2B and data not shown). These data, along with theresistance of Apaf-1–/– cells against ER stress-induced cell

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death, clearly indicate that ER stress-induced cell death isdependent on the mitochondrial pathway of apoptosis.However, similar to Apaf-1–/– MEFs, Bcl-XL-overexpressingMEFs showed cell death at later time points (Fig. 2C).Interestingly, Bcl-XL conferred Apaf-1–/– MEFs additionalresistance against ER stress-induced cell death (Fig. 2C).

Cytochrome c is released from mitochondria during ERstress-induced cell deathNext, to further substantiate the relationship between ER

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stress-induced cell death and mitochondria, we investigated therelease of cytochrome c from mitochondria under ER stress.At the 12 and 24 hours after stimulation, we observed thatcytochrome c was released from mitochondria to cytosolin both Apaf-1+/– and Apaf-1–/– MEFs stimulated withthapsigargin or tunicamycin (Fig. 3 and data not shown).Simultaneously, translocation of BAX, a pro-apoptotic memberof the Bcl-2 family, was also detected, indicating theinvolvement of BAX in ER stress-induced cell death (Martinouand Green, 2001; Ruiz-Vela et al., 2005).

Fig. 1. Apaf-1–/– MEFs showresistance to ER stress-inducedcell death. (A) Cell survival ofApaf-1+/– (WT) and Apaf-1–/–

(KO) MEFs in response to ERstress. MEFs were treated withthe indicated doses oftunicamycin or thapsigargin for24 hours and cell survival wasdetermined by Annexin V andpropidium iodide (PI) stainingwith flow cytometry: AnnexinV and PI negative populationwas regarded as viable. Shownare mean + s.d. (B) caspase-3activation during ER stress.Apaf-1+/– and Apaf-1–/– MEFstreated for indicated hours with1 �g/ml tunicamycin or 0.1�M thapsigargin were lysedand analyzed for caspase-3cleavage. (C) Time course ofER stress-induced cell death ofMEFs. MEFs were treated witheither 1 �g/ml tunicamycin or0.1 �M thapsigargin forindicated hours, collected, andanalyzed for cell viability asdescribed in A. Shown aremean ± s.d. (D) Transmissionelectron microscopic analysisof Apaf-1+/– and Apaf-1–/–

MEFs. MEFs were treated with1 �g/ml of tunicamycin for 0,48 or 72 hours. Experimentswere repeated three times withsimilar results.

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3961Apaf-1 and ER stress-induced apoptosis

Caspase-12 is downstream of Apaf-1 in ER stress-induced apoptosis and does not affect cell viability inMEFsCaspase-12 is phylogenetically a member of the inflammatorygroup of the caspase family, and proteolytically activated underER stress-induced cell death specifically (Martinon andTschopp, 2004; Nakagawa et al., 2000). Given the dependenceof ER stress-induced cell death on Apaf-1 and mitochondrialpathways demonstrated above, we investigated whethercaspase-12 cleavage during ER stress-induced cell deathrequires Apaf-1. As has been shown previously (Rao et al.,2001), caspase-12 was cleaved into a size of 42 kDa in ERstress-induced cell death in wild type MEFs (Fig. 4A). Thiscleavage, however, was not observed in Apaf-1–/– MEFs.Furthermore, a caspase-3 specific inhibitor, Ac-DNLD-CHO,inhibited the activation of caspase-12 during ER stress. Takentogether, these data indicate that caspase-12 activation isdependent on Apaf-1 and also on caspase-3 activation. Asexpected, etoposide, a topoisomerase II inhibitor that causesgenostress but not ER stress, did not induce caspase-12activation.

Previous reports showed that caspase-12 is processed atamino acids T132/A133 and K158/T159 by m-Calpain(Nakagawa and Yuan, 2000), at D94 by caspase-7, and at D318or D341 by autoprocessing of caspase-12 (Fujita et al., 2002;Rao et al., 2001). As caspase-12 was cleaved downstream ofApaf-1/caspase-3 in our experimental condition, we testedwhether a mutant form of caspase-12, in which amino acid D94was substituted with A, was cleaved during ER stress. To thisend, we constructed a plasmid expressing caspase-12 with

Fig. 2. Bcl-XL protects MEFs from ER stress-induced cell death. (A) Cell survival of MEFs overexpressing Bcl-XL. bcl-xl was introducedinto MEFs with retrovirus-mediated transduction system. Apaf-1+/– (WT) or Apaf-1–/– (KO) MEFs with or without bcl-xl transduction weretreated with tunicamycin or thapsigargin for 24 hours. Cell survival was analyzed as described in Fig. 1A. (B) Caspase-3 activation inMEFs overexpressing Bcl-XL. Cells as in A were analyzed for expression of GRP78 and for proteolytic activation of caspase-3. (C) Timecourse of ER stress-induced cell death of in Bcl-XL-overexpressing MEFs. Cells as in A were analyzed for cell viability after treatment witheither 1 �g/ml tunicamycin or 1 �M thapsigargin for indicated hours. Shown are mean ± s.d. Experiments were repeated three times withsimilar results. Note that the difference of kinetics of cell death in Apaf-1–/– MEFs from Fig. 1C may be due to the infection of retrovirus.

Fig. 3. Cytochrome c is released from mitochondria in MEFs inresponse to ER stress. Apaf-1+/– MEFs were treated with eitherthapsigargin or tunicamycin for 24 hours. Cells were fixed andanalyzed for intracellular localization of cytochrome c and BAX byimmunofluorescence. Arrowheads show cells in which cytochrome cwas released from mitochondria and BAX was translocated intomitochondria.

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D94A mutation and a FLAG-tag at its Nterminus, and introduced into Apaf-1+/–

MEFs. While wild type caspase-12, eitherendogenous or transduced, was cleaved inMEFs in response to ER stress, the mutatedcaspase-12 was not properly cleaved butgave a faint band with slower gel mobility(see * in Fig. 4B). We did not detect thecleaved band in the mutant caspase-12sample with antibody against the FLAG tagflanking the cleaved fragment (data notshown). These data suggested that caspase-12 is cleaved at D94 by caspase-3 ordownstream of it. The mutant caspase-12was degraded presumably by calpain orother proteases during ER stress-inducedcell death process, resulting in the reductionin the amount of unprocessed form.

Next we investigated if alteredexpression of caspase-12 would affect cellsurvival of MEFs during ER stress. First,Apaf-1+/– or Apaf-1–/– MEFs weretransduced with wild type casp-12 byretrovirus for overexpression of the gene. Although Kalai et al.reported caspase-12 overexpression induced cell death inHEK293T cells (Kalai et al., 2003), sensitivity to ER stress-induced cell death was not altered at 24 hours after treatmentwith tunicamycin (Fig. 4C). Furthermore, proteolyticactivation of caspase-3 was not induced by the overexpressionof caspase-12 in MEFs regardless of Apaf-1 deficiency (Fig.4D). Next, we conducted a knockdown experiment for caspase-12. Among different three RNAi constructs for targetingcaspase-12 different sequences, C12 siRNA-3 clearly reducedthe expression of caspase-12 in MEFs (Fig. 4E). However, inthese MEFs with reduced expression of caspase-12, thesensitivity to ER stress-induced cell death was not altered (Fig.

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4F). These indicate that, in MEFs, caspase-12 is downstreamof Apaf-1, cleaved by caspase-3 and possibly caspase-7, butnot involved in ER stress-induced cell death.

Apaf-1-deficiency protects cells from ER stress-inducedapoptosis in vivoWe finally investigated the impact of Apaf-1-deficiency in anin vivo model. Although most Apaf-1–/– mice die perinatally,some mutant mice normally grow up to adulthood with fertility(Honarpour et al., 2000; Okamoto et al., 2006). It waspreviously reported that injection of a single dose oftunicamycin induces a characteristic renal lesion in mice,which is mainly caused by apoptosis of the renal tubular

Fig. 4. Caspase-12 is processed downstream ofthe apoptosome in MEFs in response to ERstress. (A) Caspase-12 processing during ERstress-induced cell death was analyzed bywestern blotting. MEFs (Apaf-1+/–; WT or Apaf-1–/–; KO) were treated with tunicamycin (1�g/ml) with or without Caspase-3 specificinhibitor Ac-DNLD-CHO (100 mM) for theindicated time. Caspase-12 (C12) activation wasdetected by western blotting. C3, caspase-3.(B) MEFs were introduced with wild ormutated caspase-12 and analyzed for caspase-12 processing by western blotting. (C) Effect ofcaspase-12 overexpression to ER stress-inducedcell death was analyzed by flow cytometry(Annexin V and PI staining). (D) Effect ofcaspase-12 overexpression on caspase-3activation was determined by western blotting.(E) Reduced expression of caspase-12 bysiRNA was confirmed by western blotting.(F) Apaf-1+/– (WT) or Apaf-1–/– (KO) MEFswith reduced caspase-12 expression (by C12si-3 in E) were analyzed for their cell survivalby flow cytometry. Shown are means ± s.d.Experiments were repeated twice with similarresults.

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epithelium (Chae et al., 2004; Marciniak et al., 2004;Nakagawa et al., 2000; Zinszner et al., 1998). We tested thesensitivity of age- and sex-matched Apaf-1+/– and Apaf-1–/–

mice to tunicamycin. Tunicamycin injection resulted inincrease in GRP78 protein level in both Apaf-1+/– and Apaf-1–/–

mice (Fig. 5A). Gross inspection of the kidneys oftunicamycin-injected Apaf-1+/– mice revealed conspicuouszonal drop-out of epithelial cells and accumulation of a largenumber of TUNEL-positive cells (Fig. 5B,C). By contrast,Apaf-1–/– mice exhibited much milder damages and no

TUNEL-positive cells in the kidney. These data indicate thatApaf-1 plays an important role in ER stress-induced apoptosisin vivo.

DiscussionDespite the importance and possible involvement of the ERstress in various diseases (Xu et al., 2005), the pathway of ERstress-induced cell death has not been fully elucidated. In thepresent paper, we examined the role of Apaf-1 andmitochondria in ER stress-induced cell death both in vitro and

in vivo. Under our experimental conditions usingprimary MEFs, Apaf-1–/– MEFs showed no caspase-3 activation and were resistant to ER stress-inducedcell death. Furthermore, in response to ER stress, therelease of cytochrome c from mitochondriaaccompanied by the translocation of BAX tomitochondria was observed. Bcl-XL expressionprotected Apaf-1+/– MEFs from ER stress-inducedcell death to similar extent to Apaf-1–/– MEFs. Whilecaspase-12 was activated downstream of Apaf-1 inresponse to ER stress, it played little, if any, role inthe ER stress-induced cell death. Apaf-1-deficiencyalso protected epithelial cells from ER stress-inducedcell death in the kidney in vivo. These data showedthe importance of Apaf-1 and the mitochondrialpathway in ER stress-induced cell death. Contrary tothis, Rao et al. have reported that Sak-2 cells, Apaf-1-deficient immortalized MEFs, died comparablywith NIH/3T3 cells and induced the processing ofcaspase-3, -7, -9 and -12 under their ER stressconditions (Rao et al., 2002). Although Apaf-1-deficient MEFs died at a later time point, we couldnot observe the processing of caspase-3 and -12in our experimental conditions even in MEFsimmortalized by the SV40 large T antigen. Thediscrepancy may be caused by the type oftransformation factor, possible involvement ofm-calpain activation (Nakagawa and Yuan, 2000),or the difference of the genetic background ofMEFs. Phenotypes of knockout mice mayoccasionally depend on their genetic background.Recently, Di Sano et al. have reported the similarfindings that ER stress-induced apoptosis isdependent on Apaf-1 and independent of caspase-12(Di Sano et al., 2005).

We demonstrated that caspase-12 is cleaveddownstream of Apaf-1 at the site of D94 by caspase-3 in MEFs. Although Nakagawa and Yuan previouslyreported that m-calpain cleaves caspase-12 under ERstress (Nakagawa and Yuan, 2000), we did not detectactivity of m-calapain using fluorescent substrates(data not shown). These are consistent with the dataobserved in in vitro assay using recombinant caspase-12 and -3 (Hoppe and Hoppe, 2004). Furthermore,knockdown or overexpression experiments showedthat caspase-12 plays little role in ER stress-inducedapoptosis of MEFs. Recent papers by Obeng et al. andby Di Sano et al. also demonstrated similar findingsthat ER stress-induced cell death is independent ofcaspase-12 but depends on the apoptosome (Di Sanoet al., 2005; Obeng and Boise, 2005). Caspase-12 is,

Fig. 5. Loss of Apaf-1 protects the renal epithelium from tunicamycin. (A)Apaf-1+/– (WT) and Apaf-1–/– (KO) adult mice were injected intraperitoneallywith 3 �g/gram tunicamycin. After 3 days, mice were sacrificed and kidneyswere lysed or fixed for GRP78 expression or histological analyses,respectively. The induction of GRP78 in the kidneys was analyzed by westernblotting. (B) Photomicrographs (original magnification, �50; upper panels and�200; lower panels) of kidney sections from Apaf-1+/– or Apaf-1–/– mice aftertunicamycin injection. Thin sections were stained with hematoxylin and eosin.(C) TUNEL assay in kidneys from mice 48 hours after injection. Thin sectionswere stained for TUNEL assay or with DAPI. Experiments were repeatedtwice with similar results.

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however, localized on the cytoplasmic side of the ER membrane(Nakagawa et al., 2000; Rao et al., 2001), and is actuallycleaved in our ER stress condition, too. These indicate thepossible involvement of caspase-12 in ER stress. Caspase-12 isphylogenetically one of the inflammatory group caspases(Martinon and Tschopp, 2004). It has been reported that murinecaspase-12 expression is induced by IFN-� (Kalai et al., 2003)and the expected NF-�B- and AP1-binding sites are present inits promoter region (Oubrahim et al., 2005). In humans, acaspase-12 isozyme functions as dominant-negative regulator ofpro-inflammatory signaling pathway (Saleh et al., 2004). Inaddition, ER stress also induces NF-�B activation mediated byTRAF2 (Mauro et al., 2006). These data indicate a role ofcaspase-12 in inflammation but not apoptosis. Furthermore,Saleh et al. have recently reported that murine caspase-12 alsofunctions the regulator of inflammation in vivo (Saleh et al.,2006). It is possible that ER stress activates some of the pro-inflammatory signal transduction pathway associated with theinnate immunity in situations such as viral infection.

Although showing resistance to ER stress-induced celldeath, Apaf-1–/– MEFs also gradually died. The cells showednon-apoptotic features, such as many vacuoles. Non-apoptotic,necrosis-like cell death of Apaf-1–/– cells at later stages ofapoptotic stimulation was also reported previously (Chautan etal., 1999; Miyazaki et al., 2001). These vacuoles might be ERs,because ER stress is known to induce swelling of the ER lumenand dissociation of ribosomes from rough ER (Hitomi et al.,2004b). In some neurodegenerative diseases where ER stresshas been allegedly involved, morphological changes includingformation of cytoplasmic vacuoles have been reported whileaccumulation of abnormal proteins and cell death playsignificant roles in the development of the diseases (Kobayashiet al., 2002; Mizuno et al., 2003). In this view, the cell deathwith vacuoles observed in Apaf-1–/– MEFs may havephysiological relevance.

Bcl-XL conferred resistance to wild type cells against ERstress-induced cell death just like Apaf-1-deficient cells,confirming the involvement of Apaf-1/mitochondrial pathwaysof apoptosis in ER stress-induced cell death. Interestingly, Bcl-XL bestowed additional resistance to Apaf-1–/– MEFs againstER stress-induced cell death. Lines of evidence have shownthat various pro-apoptotic factors are released frommitochondria including Smac/DIABLO (Du et al., 2000),Omi/HtrA2 (Suzuki et al., 2001), AIF (Joza et al., 2001; Susinet al., 1999), and Endo G (Li et al., 2001) as well as cytochromec. Smac/DIABLO and Omi/HtrA2 block IAP-family proteins,which inhibit caspases at post-mitochondrial phases. AIF andEndo G cause large fragmentation of chromosomal DNAleading cell death either dependent on or independent ofcaspases (Arnoult et al., 2003). Considering the lack ofcaspase-3 activation in Apaf-1–/– MEFs, the additional effectsof Bcl-XL may be due to blocking the release of AIF and EndoG from mitochondria by mitochondrial membranestabilization. Another possibility is that Bcl-XL may functionat the ER membrane. Bcl-XL blocks the release of pro-apoptotic factors by blocking functions of Bax and Bakchannels on mitochondria. Therefore, Bcl-XL may regulate thefunction of Bax and Bak at the ER (Zong et al., 2003).However, neither Bcl-XL overexpression nor Apaf-1-deficiency protected cells from ER stress-induced cell deathobserved at later phases of stimulation. Consistent with the in

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vitro finding, in vivo administration of tunicamycin stillinduced epithelial cell death in Apaf-1–/– mice, albeit reducedin degree. These data thus indicate that the ER stress inducesApaf-1-independent, non-apoptotic cell death, as well as Apaf-1/mitochondria-dependent apoptosis. The non-apoptotic celldeath observed in the absence of Apaf-1 (Chautan et al., 1999;Miyazaki et al., 2001) may be physiologically veiled by Apaf-1- and caspase-3-dependent canonical apoptosis, which occursfaster. The presence of non-apoptotic cell death induced bygenostresses and ER stresses indicates that targeting ofapoptotic pathway may not be enough to suppress cell death indiseases such as neurodegenerative disorders.

In conclusion, in the present study we provide evidence thatApaf-1 and mitochondria are critically involved in ER stress-induced cell death in MEFs as well as in renal epithelial cells.Further elucidation of the molecular mechanisms linking ERstress to mitochondrial damages will no doubt shed light to theunderstandings of and therapeutic intervention of variousdiseases in which ER stress play pathological roles.

Materials and MethodsCell culture and reagentsApaf-1+/– or Apaf-1–/– mouse embryonic fibroblasts were obtained from E14.5embryos born to heterozygous intercrosses and cultured in DMEM supplementedwith 10% FCS, L-glutamine and �-mercaptoethanol. All experiments were donewith cells at between three and five population doublings level (PDL). Tunicamycin,thapsigargin and etoposide were purchased from Sigma. The Caspase-3 specificinhibitor Ac-DNLD-CHO was purchased from the Peptide Institute.

Plasmids and constructsA human bcl-xl cDNA was subcloned into the retroviral expression vector, pMX-IRES-PURO (Nosaka et al., 1999). Murine casp-12 cDNA obtained from MEFs wascloned into pFLAG-CMV2 (Sigma). The substitution mutant casp-12 D94A cDNAwas generated using standard polymerase chain reaction method. Wild type andmutant cDNAs tagged with FLAG-epitope were subcloned into pMX-IRES-PURO.

Detection of cell deathMEFs were plated in a 24-well dish at 5�104 cells per well 1 day prior to treatment.The cells were left untreated or treated with tunicamycin or thapsigargin at indicatedconcentration. At various time points after treatment, cells were trypsinized andcollected with the supernatants, and cell viability was determined by propidiumiodide (PI, Sigma) and Annexin V (Sigma) staining using flow cytometry.

Western blotting analysisMEFs were plated at 5�105 cells per dish in 100 mm dish one day prior to treatment.After treatment, cells were scraped and collected with the supernatants, and lysedwith NP40 lysis buffer. Proteins were separated by SDS-PAGE (10% for GRP78,12% for caspase-12, 15% for caspase-3 and Bcl-XL), transferred onto nitrocellulosemembranes, and incubated with antibodies reactive to GRP78 (Santa Cruz), Bcl-XL

(BD Transduction Laboratories), Caspase-3 mixed with Cleaved caspase-3 orcaspase-12 (Cell Signaling). Optimal antibody concentrations were determined inpilot assays.

Electron microscopyBefore or after apoptotic treatment, MEFs were fixed on ice with 1� fixing buffer(2.5% glutaraldehyde, 0.1 M sucrose, and 3 mM CaCl2 in 0.1 M cacodylate buffer[pH 7.4]). The cells were processed through 1% OsO4 for 1 hour at 4°C, dehydratedin graded ethanol and in propylene oxide, and embedded in Epon 812 resin. Thinsections were stained with 5-uranyl acetate for 30 minutes and lead acetate for 25minutes, then examined under a JEM-2000-EX (JEOL, Tokyo) electron microscope.

Immunocytochemistry for cytochrome cMEFs were grown on 1mm glass coverslips for 24 hours and stimulated withtunicamycin (1 �g/ml) or thapsigargin (0.1 �M). Twenty-four hours afterstimulation, cells were fixed with 4% paraformaldehyde, and then permeabilizedwith 0.1% Triton X-100 in phosphate buffered saline at room temperature for 5minutes. MEFs were immunostained with the antibody reactive to cytochrome c(BD PharMingen) and BAX (Upstate).

Retrovirus-mediated gene transductionHuman bcl-xl, murine casp-12, FLAG-casp-12, or FLAG-casp-12(D94A)subcloned into pMX-IRES-PURO were transfected into the PLAT-E packaging cell

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3965Apaf-1 and ER stress-induced apoptosis

line using the calcium phosphate method to obtain viruses. MEFs were infected withthe virus, treated with polybrene (Sigma) a day after infection, and then culturemedium was replaced with the selection medium containing puromycin. Twenty-four hours after selection, surviving cells were used for experiments.

Knockdown of casp-12The mammalian expression vector, pSUPER.retro.puro (Oligoengine) was used forexpression of siRNA targeting murine caspase-12 in MEFs. Sense and anti-senseoligonucleotides corresponding to 522-542 (C12 si-1), 838-858 (C12 si-2) and1075-1095 (C12 si-3) of murine casp-12 cDNA (GenBank accession no.NM_009808) were annealed and subcloned into pSUPER.retro.puro vector,respectively.

In vivo analysis of ER stress-induced cell deathAge- and sex-matched Apaf-1+/– and Apaf-1–/– mice (6- to 8-week-old) were givena single injection of tunicamycin (3 �g/gram body weight, intraperitonealy (i.p.)).After 2 or 3 days, mice were sacrificed, and their kidneys were removed. One kidneywas lysed by NP40 lysis buffer for western blotting analysis, and the other was fixedin 4% paraformaldehyde for hematoxylin and eosin staining and terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and4�,6-diamideino-2-phenylindole (DAPI) staining. Fixed samples were paraffin-embedded and 5 �m sections were mounted on glass slides. TUNEL assay wasperformed using Apop Tag In Situ Apoptosis detection kit (CHEMICON) accordingto the manufacturer’s instructions.

We thank Dr Hiroshi Nishina for reagents; Masafumi Sasaki,Masumi Ohtsu, and Norihiko Kinoshita for technical help; HidenoriOtera and members of ‘Project W’ for helpful discussion. This workwas supported in part by grants from the Ministry of Education,Science, Technology, Sports and Culture of Japan (H.Y. and A.Y.).

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