3958 Research Article ER stress-induced apoptosis and -12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1

Hiroshi Shiraishi1, Hideaki Okamoto2, Akihiko Yoshimura1 and Hiroki Yoshida2,* 1Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Japan 2Division 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: [email protected])

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

Summary Accumulation of unfolded induces endoplasmic mitochondria and cytochrome c was released from reticulum (ER) stress. Excessive and prolonged stresses mitochondria. We also demonstrated that caspase-12 was lead cells to apoptosis. However, the precise molecular processed downstream of Apaf-1 and caspase-3, and mechanisms of ER stress-induced apoptosis have not been neither overexpression nor knockdown of caspase-12 fully elucidated. We investigated the involvement of the affected susceptibility of the cells to ER stress-induced cell apoptosome in ER stress-induced cell death pathway using death. Furthermore, in the kidneys of Apaf-1-deficient mouse embryonic fibroblasts (MEFs) and mice deficient for mice, apoptosis induced by in vivo administration of Apaf-1. Apaf-1-deficient MEFs showed more resistance to tunicamycin was remarkably suppressed as compared with ER stress-inducing reagents as compared with wild type wild type mice. These data collectively demonstrated that cells. Despite comparable induction of ER stress in both Apaf-1 and the mitochondrial pathway of apoptosis play wild type and Apaf-1-deficient cells, activation of caspase- significant roles in ER stress-induced apoptosis. 3 was only observed in wild type, but not Apaf-1-deficient, MEFs. Under ER stress conditions, BAX translocated to Key words: Apoptosis, Apaf-1, ER stress, Mitochondria

Journal of Cell Science Introduction There are two major pathways that induce apoptotic cell The endoplasmic reticulum (ER) is a factory responsible for death. One is the extrinsic pathway, in which ligation of death the synthesis, initial post-translational modification, proper receptors by death ligands is followed by recruitment of folding, and maturation of newly synthesized transmembrane adaptor molecules and activation of caspase-8 or caspase-10. and secretory proteins, as well as a regulator of intracellular The other is the intrinsic pathway, in which the release of calcium homeostasis. Various stresses, including expression of cytochrome c from mitochondria triggers the formation of the mutant proteins, viral infection, energy or nutrient deprivation, apoptosome composed of Apaf-1, pro-caspase-9, dATP, and extreme environmental conditions, alterations in redox status cytochrome c. Apoptosome formation results in the activation or glycosylation status, or calcium release from the lumen of of executioner including caspase-3, -6, and -7. The the ER, disrupt proper function of ER and cause so-called ER precise molecular mechanisms of ER stress-induced apoptosis, stress. The prototypic response of ER stress is the congestion however, have not been fully elucidated (Xu et al., 2005). of misfolded proteins. ER stress leads cells to activate self- CHOP, a transcription factor induced under ER stress, has been protective mechanisms: (1) transcriptional up-regulation of ER reported to be involved in ER stress-induced apoptosis by chaperones and folding ; (2) translational attenuation reducing the expression of Bcl-2 (McCullough et al., 2001). to limit further accumulation of misfolded proteins; and Indeed, CHOP-deficiency causes resistance to ER stress- (3) ER-associated degradation (ERAD) which eliminates induced cell death both in vitro and in vivo (Zinszner et al., misfolded proteins from the ER. The current understanding is 1998). ER stress also leads to increase cytosolic calcium levels, that three types of ER membrane receptors, ATF6, IRE1 and which induces the activation of m-. Activated m-calpain PERK may sense the stress in the ER, and eventually activate cleaves Bcl-XL and proteolytically activates caspase-12 transcription factors for induction of ER chaperones, such as (Nakagawa and Yuan, 2000). Processed-caspase-12 reportedly GRP78, or phosphorylate eIF2␣ for inhibition of synthesis of activates caspase-9 independent of Apaf-1, followed by new proteins. These processes are collectively called unfolded activation of caspase-3 (Rao et al., 2002). Several groups also response (UPR) (Imaizumi et al., 2001). Excessive and reported that ER stress increases reactive oxygen species prolonged stresses, however, lead cells to apoptosis (Schroder (ROS), and antioxidant protects cells from ER stress-induced and Kaufman, 2005). ER stress-induced apoptosis is associated cell death (Cullinan and Diehl, 2004; Harding et al., 2003; with a range of diseases, including ischemia/reperfusion injury, Haynes et al., 2004; Mauro et al., 2006). The stress kinase neurodegeneration and diabetes (Xu et al., 2005). pathway is also associated with ER stress-induced apoptosis. Apaf-1 and ER stress-induced apoptosis 3959

During ER stress, Ask1, one of mitogen-activated protein report by Rao et al. that Apaf-1-deficient immortalized MEFs, (MAP) kinase-kinase (MAPKK) kinases, is recruited to Sak2 cells, died just like wild type NIH/3T3 cells (Rao et al., oligomerized IRE1 complex containing TRAF2, and is 2002), Apaf-1–/– MEFs showed significant resistance to ER activated for the downstream activation of JNK (Nishitoh et al., stress-induced cell death over Apaf-1+/– MEFs (Fig. 1A). This 2002). In addition, it is reported that caspase-8 or c-Abl is was checked in more than six different clones of MEFs from involved in ER stress-induced apoptosis (Ito et al., 2001; Jimbo more than three different heterozygous intercrosses. This was et al., 2003). Such complexity may be because the signaling also the case for MEFs immortalized by SV40 large T antigen pathways leading to ER stress-mediated apoptosis might vary (data not shown). in different cell types and in different stresses. Murine caspase-12 is a member of the inflammatory group Apaf-1 is required for cleavage of caspase-3 during ER of the caspase family, and study of the caspase-12-deficient stress-induced cell death mice revealed that it specifically involves in ER stress-induced To examine further whether Apaf-1 plays a role of induction cell death (Martinon and Tschopp, 2004; Nakagawa et al., of ER stress, we detected caspase-3 activation by western 2000). In humans, the caspase-12 has a single nucleotide blotting. First, we evaluated induction of GRP78, an indicator polymorphism that results in the production of either a of ER stress, by western blotting. Induction of GRP78 by truncated or full-length caspase-12 protein (Saleh et al., 2004). treatment of either tunicamycin or thapsigargin in Apaf-1–/– However, the full-length human caspase-12 appears to be MEFs was comparable to that of Apaf-1+/– MEFs (Fig. 1B), enzymatically inactive and functions as a dominant-negative confirming that ER stresses were similarly induced both in regulator of proinflammatory signaling pathways rather than in Apaf-1+/– MEFs and Apaf-1–/– MEFs. For caspase-3 activation, ER stress-induced cell death pathway. As an alternative to although treatment with tunicamycin or thapsigargin induced caspase-12 in human, caspase-4 is involved in ER stress- the cleavage of caspase-3 to p17 fragments in Apaf-1+/– MEFs induced cell death pathway (Hitomi et al., 2004a). Both murine at 12-24 hours and thereafter, caspase-3 activation was not caspase-12 and human caspase-4 are localized to the ER and observed in Apaf-1–/– MEFs under the same condition (Fig. cleaved specifically by ER stress (Hitomi et al., 2004a; Hoppe 1B). It was thus demonstrated that ER stress-induced cleavage and Hoppe, 2004; Nakagawa et al., 2000). of caspase-3 is dependent on Apaf-1. In the current study, we investigated the possible involvement While showing significant resistance to ER stress-induced of the apoptosome and mitochondria in ER stress-induced cell cell death, Apaf1–/– MEFs gradually died during long culture death pathways using murine embryonic fibroblasts (MEFs) periods (Fig. 1C). To characterize the cell death induced by ER –/– deficient for Apaf-1. Just like Bcl-XL-expressing MEFs, Apaf- stress in Apaf-1 MEFs, we observed the morphological 1-deficient MEFs showed more resistance to ER stress-inducing features of cell death using transmission electron microscopy reagents as compared with wild type cells. Although caspase- (TEM) (Fig. 1D). Apaf-1+/– MEFs showed typical apoptotic 12 activation was observed in wild type, but not in Apaf-1- changes, such as chromatin condensation, nuclear deficient, MEFs under ER stress, neither overexpression nor fragmentation, and plasma membrane blebbing at 48 hours knockdown of caspase-12 gene affected susceptibility of the after stimulation. Although Apaf-1–/– MEFs showed marginal

Journal of Cell Science cells to ER stress-induced cell death. In the kidneys of Apaf-1- perinuclear chromatin condensation and cytoplasmic deficient mice, apoptosis induced by in vivo administration of vacuolization, typical apoptotic changes were not observed by tunicamycin was remarkably suppressed as compared with that 72 hours after stimulation. Nuclear swelling and massive of wild type mice. Our data suggest that ER stress-induced vacuolization observed in Apaf-1–/– MEFs rather resembled apoptosis is dependent on the mitochondrial pathways of necrotic cell death. apoptosis involving Apaf-1 and also that caspase-12, activated downstream of Apaf-1, plays little, if any, role in ER stress- Bcl-XL protects MEFs from ER stress-induced cell death induced apoptosis. Bcl-XL is one of the most potent anti-apoptotic members of the Bcl-2 family proteins. Reportedly, it protects cells from ER Results stress-induced cell death in various types of cells (Morishima Apaf-1–/– MEFs are resistant to ER stress-induced cell et al., 2004; Obeng and Boise, 2005; Srivastava et al., 1999). death Unlike Bcl-2, which may localize ER and affect its function, Apaf-1 is the essential adaptor protein in the intrinsic, Bcl-XL specifically localizes to mitochondria outer membrane mitochondrial pathways of apoptosis and, along with with its C-terminal transmembrane domain (Kaufmann et al., cytochrome c, dATP and caspase-9, forms to the apoptosome 2003). To examine the effect of Bcl-XL expression on ER (Li et al., 1997; Zou et al., 1997). To investigate the role of stress-induced cell death, both Apaf-1+/– and Apaf-1–/– MEFs Apaf-1 in ER stress-induced cell death, we examined survival were transduced with bcl-xl gene by a retrovirus vector. We +/– –/– of Apaf-1 or Apaf-1 primary MEFs against tunicamycin, confirmed that overexpressed-Bcl-XL localized preferentially an inhibitor of N-linked glycosylation, or thapsigargin, an to mitochondria (data not shown). The retrovirus-mediated 2+ +/– inhibitor of sarcoplasmic-endoplasmic reticulum Ca ATPase. expression of Bcl-XL partially protected Apaf-1 MEFs from Both drugs are well characterized as inducers of ER stress and ER stress-induced cell death at approximately the same level –/– excessive treatments lead to cell death (Kaufman, 1999). First, with Apaf-1 MEFs (Fig. 2A). Consistent with this Bcl-XL- we checked the growth rate of Apaf-1+/– and Apaf-1–/– MEFs mediated resistance to ER stress-induced cell death, cleavage and found that they proliferated similarly (data not shown). of caspase-3 to active form was significantly reduced in MEFs Next, cell survival after stimulation with the ER stress- overexpressing Bcl-XL against tunicamycin and thapsigargin inducing drugs was examined. Apaf-1+/– MEFs died after (Fig. 2B and data not shown). These data, along with the treatment with these drugs (Fig. 1A). Contrary to the previous resistance of Apaf-1–/– cells against ER stress-induced cell 3960 Journal of Cell Science 119 (19)

Fig. 1. Apaf-1–/– MEFs show resistance to ER stress-induced cell death. (A) Cell survival of Apaf-1+/– (WT) and Apaf-1–/– (KO) MEFs in response to ER stress. MEFs were treated with the indicated doses of tunicamycin or thapsigargin for 24 hours and cell survival was determined by Annexin V and propidium iodide (PI) staining with flow cytometry: Annexin V and PI negative population was regarded as viable. Shown are mean + s.d. (B) caspase-3 activation during ER stress. Apaf-1+/– and Apaf-1–/– MEFs treated for indicated hours with 1 ␮g/ml tunicamycin or 0.1 ␮M thapsigargin were lysed and analyzed for caspase-3 Journal of Cell Science cleavage. (C) Time course of ER stress-induced cell death of MEFs. MEFs were treated with either 1 ␮g/ml tunicamycin or 0.1 ␮M thapsigargin for indicated hours, collected, and analyzed for cell viability as described in A. Shown are mean ± s.d. (D) Transmission electron microscopic analysis of Apaf-1+/– and Apaf-1–/– MEFs. MEFs were treated with 1 ␮g/ml of tunicamycin for 0, 48 or 72 hours. Experiments were repeated three times with similar results.

death, clearly indicate that ER stress-induced cell death is stress-induced cell death and mitochondria, we investigated the dependent on the mitochondrial pathway of apoptosis. release of cytochrome c from mitochondria under ER stress. –/– However, similar to Apaf-1 MEFs, Bcl-XL-overexpressing At the 12 and 24 hours after stimulation, we observed that MEFs showed cell death at later time points (Fig. 2C). cytochrome c was released from mitochondria to cytosol –/– +/– –/– Interestingly, Bcl-XL conferred Apaf-1 MEFs additional in both Apaf-1 and Apaf-1 MEFs stimulated with resistance against ER stress-induced cell death (Fig. 2C). thapsigargin or tunicamycin (Fig. 3 and data not shown). Simultaneously, translocation of BAX, a pro-apoptotic member Cytochrome c is released from mitochondria during ER of the Bcl-2 family, was also detected, indicating the stress-induced cell death involvement of BAX in ER stress-induced cell death (Martinou Next, to further substantiate the relationship between ER and Green, 2001; Ruiz-Vela et al., 2005). Apaf-1 and ER stress-induced apoptosis 3961

Fig. 2. Bcl-XL protects MEFs from ER stress-induced cell death. (A) Cell survival of MEFs overexpressing Bcl-XL. bcl-xl was introduced into MEFs with retrovirus-mediated transduction system. Apaf-1+/– (WT) or Apaf-1–/– (KO) MEFs with or without bcl-xl transduction were treated with tunicamycin or thapsigargin for 24 hours. Cell survival was analyzed as described in Fig. 1A. (B) Caspase-3 activation in MEFs overexpressing Bcl-XL. Cells as in A were analyzed for expression of GRP78 and for proteolytic activation of caspase-3. (C) Time course of ER stress-induced cell death of in Bcl-XL-overexpressing MEFs. Cells as in A were analyzed for cell viability after treatment with either 1 ␮g/ml tunicamycin or 1 ␮M thapsigargin for indicated hours. Shown are mean ± s.d. Experiments were repeated three times with similar 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.

Caspase-12 is downstream of Apaf-1 in ER stress- induced apoptosis and does not affect cell viability in MEFs

Journal of Cell Science Caspase-12 is phylogenetically a member of the inflammatory group of the caspase family, and proteolytically activated under ER stress-induced cell death specifically (Martinon and Tschopp, 2004; Nakagawa et al., 2000). Given the dependence of ER stress-induced cell death on Apaf-1 and mitochondrial pathways demonstrated above, we investigated whether caspase-12 cleavage during ER stress-induced cell death requires Apaf-1. As has been shown previously (Rao et al., 2001), caspase-12 was cleaved into a size of 42 kDa in ER stress-induced cell death in wild type MEFs (Fig. 4A). This cleavage, 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. Taken together, these data indicate that caspase-12 activation is dependent on Apaf-1 and also on caspase-3 activation. As expected, etoposide, a topoisomerase II inhibitor that causes genostress but not ER stress, did not induce caspase-12 activation. Previous reports showed that caspase-12 is processed at amino acids T132/A133 and K158/T159 by m-Calpain (Nakagawa and Yuan, 2000), at D94 by caspase-7, and at D318 Fig. 3. Cytochrome c is released from mitochondria in MEFs in response to ER stress. Apaf-1+/– MEFs were treated with either or D341 by autoprocessing of caspase-12 (Fujita et al., 2002; thapsigargin or tunicamycin for 24 hours. Cells were fixed and Rao et al., 2001). As caspase-12 was cleaved downstream of analyzed for intracellular localization of cytochrome c and BAX by Apaf-1/caspase-3 in our experimental condition, we tested immunofluorescence. Arrowheads show cells in which cytochrome c whether a mutant form of caspase-12, in which amino acid D94 was released from mitochondria and BAX was translocated into was substituted with A, was cleaved during ER stress. To this mitochondria. end, we constructed a plasmid expressing caspase-12 with 3962 Journal of Cell Science 119 (19)

Fig. 4. Caspase-12 is processed downstream of the apoptosome in MEFs in response to ER stress. (A) Caspase-12 processing during ER stress-induced cell death was analyzed by western blotting. MEFs (Apaf-1+/–; WT or Apaf- 1–/–; KO) were treated with tunicamycin (1 ␮g/ml) with or without Caspase-3 specific inhibitor Ac-DNLD-CHO (100 mM) for the indicated time. Caspase-12 (C12) activation was detected by western blotting. C3, caspase-3. (B) MEFs were introduced with wild or mutated caspase-12 and analyzed for caspase- 12 processing by western blotting. (C) Effect of caspase-12 overexpression to ER stress-induced cell death was analyzed by flow cytometry (Annexin V and PI staining). (D) Effect of caspase-12 overexpression on caspase-3 activation was determined by western blotting. (E) Reduced expression of caspase-12 by siRNA was confirmed by western blotting. (F) Apaf-1+/– (WT) or Apaf-1–/– (KO) MEFs with reduced caspase-12 expression (by C12 si-3 in E) were analyzed for their cell survival by flow cytometry. Shown are means ± s.d. Experiments were repeated twice with similar results.

D94A mutation and a FLAG-tag at its N terminus, and introduced into Apaf-1+/– MEFs. While wild type caspase-12, either endogenous or transduced, was cleaved in MEFs in response to ER stress, the mutated caspase-12 was not properly cleaved but gave a faint band with slower gel mobility (see * in Fig. 4B). We did not detect the cleaved band in the mutant caspase-12

Journal of Cell Science sample with antibody against the FLAG tag flanking the cleaved fragment (data not shown). These data suggested that caspase- 12 is cleaved at D94 by caspase-3 or downstream of it. The mutant caspase-12 was degraded presumably by calpain or other during ER stress-induced cell death process, resulting in the reduction in the amount of unprocessed form. Next we investigated if altered expression of caspase-12 would affect cell survival of MEFs during ER stress. First, Apaf-1+/– or Apaf-1–/– MEFs were transduced with wild type casp-12 by retrovirus for overexpression of the gene. Although Kalai et al. 4F). These indicate that, in MEFs, caspase-12 is downstream reported caspase-12 overexpression induced cell death in of Apaf-1, cleaved by caspase-3 and possibly caspase-7, but HEK293T cells (Kalai et al., 2003), sensitivity to ER stress- not involved in ER stress-induced cell death. induced cell death was not altered at 24 hours after treatment with tunicamycin (Fig. 4C). Furthermore, proteolytic Apaf-1-deficiency protects cells from ER stress-induced activation of caspase-3 was not induced by the overexpression apoptosis in vivo of caspase-12 in MEFs regardless of Apaf-1 deficiency (Fig. We finally investigated the impact of Apaf-1-deficiency in an 4D). Next, we conducted a knockdown experiment for caspase- in vivo model. Although most Apaf-1–/– mice die perinatally, 12. Among different three RNAi constructs for targeting some mutant mice normally grow up to adulthood with fertility caspase-12 different sequences, C12 siRNA-3 clearly reduced (Honarpour et al., 2000; Okamoto et al., 2006). It was the expression of caspase-12 in MEFs (Fig. 4E). However, in previously reported that injection of a single dose of these MEFs with reduced expression of caspase-12, the tunicamycin induces a characteristic renal lesion in mice, sensitivity to ER stress-induced cell death was not altered (Fig. which is mainly caused by apoptosis of the renal tubular Apaf-1 and ER stress-induced apoptosis 3963

epithelium (Chae et al., 2004; Marciniak et al., 2004; TUNEL-positive cells in the kidney. These data indicate that Nakagawa et al., 2000; Zinszner et al., 1998). We tested the Apaf-1 plays an important role in ER stress-induced apoptosis sensitivity of age- and sex-matched Apaf-1+/– and Apaf-1–/– in vivo. mice to tunicamycin. Tunicamycin injection resulted in increase in GRP78 protein level in both Apaf-1+/– and Apaf-1–/– Discussion mice (Fig. 5A). Gross inspection of the kidneys of Despite the importance and possible involvement of the ER tunicamycin-injected Apaf-1+/– mice revealed conspicuous stress in various diseases (Xu et al., 2005), the pathway of ER zonal drop-out of epithelial cells and accumulation of a large stress-induced cell death has not been fully elucidated. In the number of TUNEL-positive cells (Fig. 5B,C). By contrast, present paper, we examined the role of Apaf-1 and Apaf-1–/– mice exhibited much milder damages and no mitochondria in ER stress-induced cell death both in vitro and in vivo. Under our experimental conditions using primary MEFs, Apaf-1–/– MEFs showed no caspase- 3 activation and were resistant to ER stress-induced cell death. Furthermore, in response to ER stress, the release of cytochrome c from mitochondria accompanied by the translocation of BAX to mitochondria was observed. Bcl-XL expression protected Apaf-1+/– MEFs from ER stress-induced cell death to similar extent to Apaf-1–/– MEFs. While caspase-12 was activated downstream of Apaf-1 in response to ER stress, it played little, if any, role in the ER stress-induced cell death. Apaf-1-deficiency also protected epithelial cells from ER stress-induced cell death in the kidney in vivo. These data showed the importance of Apaf-1 and the mitochondrial pathway in ER stress-induced cell death. Contrary to this, Rao et al. have reported that Sak-2 cells, Apaf- 1-deficient immortalized MEFs, died comparably with NIH/3T3 cells and induced the processing of caspase-3, -7, -9 and -12 under their ER stress conditions (Rao et al., 2002). Although Apaf-1- deficient MEFs died at a later time point, we could not observe the processing of caspase-3 and -12 in our experimental conditions even in MEFs

Journal of Cell Science immortalized by the SV40 large T antigen. The discrepancy may be caused by the type of transformation factor, possible involvement of m-calpain activation (Nakagawa and Yuan, 2000), or the difference of the genetic background of MEFs. Phenotypes of knockout mice may occasionally depend on their genetic background. Recently, Di Sano et al. have reported the similar findings that ER stress-induced apoptosis is dependent on Apaf-1 and independent of caspase-12 (Di Sano et al., 2005). We demonstrated that caspase-12 is cleaved downstream of Apaf-1 at the site of D94 by caspase- 3 in MEFs. Although Nakagawa and Yuan previously reported that m-calpain cleaves caspase-12 under ER stress (Nakagawa and Yuan, 2000), we did not detect activity of m-calapain using fluorescent substrates Fig. 5. Loss of Apaf-1 protects the renal epithelium from tunicamycin. (A) (data not shown). These are consistent with the data +/– –/– Apaf-1 (WT) and Apaf-1 (KO) adult mice were injected intraperitoneally observed in in vitro assay using recombinant caspase- ␮ with 3 g/gram tunicamycin. After 3 days, mice were sacrificed and kidneys 12 and -3 (Hoppe and Hoppe, 2004). Furthermore, were lysed or fixed for GRP78 expression or histological analyses, knockdown or overexpression experiments showed respectively. The induction of GRP78 in the kidneys was analyzed by western blotting. (B) Photomicrographs (original magnification, ϫ50; upper panels and that caspase-12 plays little role in ER stress-induced ϫ200; lower panels) of kidney sections from Apaf-1+/– or Apaf-1–/– mice after apoptosis of MEFs. Recent papers by Obeng et al. and tunicamycin injection. Thin sections were stained with hematoxylin and eosin. by Di Sano et al. also demonstrated similar findings (C) TUNEL assay in kidneys from mice 48 hours after injection. Thin sections that ER stress-induced cell death is independent of were stained for TUNEL assay or with DAPI. Experiments were repeated caspase-12 but depends on the apoptosome (Di Sano twice with similar results. et al., 2005; Obeng and Boise, 2005). Caspase-12 is, 3964 Journal of Cell Science 119 (19)

however, localized on the cytoplasmic side of the ER membrane vitro finding, in vivo administration of tunicamycin still (Nakagawa et al., 2000; Rao et al., 2001), and is actually induced epithelial cell death in Apaf-1–/– mice, albeit reduced cleaved in our ER stress condition, too. These indicate the in degree. These data thus indicate that the ER stress induces possible involvement of caspase-12 in ER stress. Caspase-12 is Apaf-1-independent, non-apoptotic cell death, as well as Apaf- phylogenetically one of the inflammatory group caspases 1/mitochondria-dependent apoptosis. The non-apoptotic cell (Martinon and Tschopp, 2004). It has been reported that murine death observed in the absence of Apaf-1 (Chautan et al., 1999; caspase-12 expression is induced by IFN-␥ (Kalai et al., 2003) Miyazaki et al., 2001) may be physiologically veiled by Apaf- and the expected NF-␬B- and AP1-binding sites are present in 1- and caspase-3-dependent canonical apoptosis, which occurs its promoter region (Oubrahim et al., 2005). In humans, a faster. The presence of non-apoptotic cell death induced by caspase-12 isozyme functions as dominant-negative regulator of genostresses and ER stresses indicates that targeting of pro-inflammatory signaling pathway (Saleh et al., 2004). In apoptotic pathway may not be enough to suppress cell death in addition, ER stress also induces NF-␬B activation mediated by diseases such as neurodegenerative disorders. TRAF2 (Mauro et al., 2006). These data indicate a role of In conclusion, in the present study we provide evidence that caspase-12 in inflammation but not apoptosis. Furthermore, Apaf-1 and mitochondria are critically involved in ER stress- Saleh et al. have recently reported that murine caspase-12 also induced cell death in MEFs as well as in renal epithelial cells. functions the regulator of inflammation in vivo (Saleh et al., Further elucidation of the molecular mechanisms linking ER 2006). It is possible that ER stress activates some of the pro- stress to mitochondrial damages will no doubt shed light to the inflammatory signal transduction pathway associated with the understandings of and therapeutic intervention of various innate immunity in situations such as viral infection. diseases in which ER stress play pathological roles. Although showing resistance to ER stress-induced cell death, Apaf-1–/– MEFs also gradually died. The cells showed Materials and Methods non-apoptotic features, such as many vacuoles. Non-apoptotic, Cell culture and reagents –/– Apaf-1+/– or Apaf-1–/– mouse embryonic fibroblasts were obtained from E14.5 necrosis-like cell death of Apaf-1 cells at later stages of embryos born to heterozygous intercrosses and cultured in DMEM supplemented apoptotic stimulation was also reported previously (Chautan et with 10% FCS, L-glutamine and ␤-mercaptoethanol. All experiments were done al., 1999; Miyazaki et al., 2001). These vacuoles might be ERs, with cells at between three and five population doublings level (PDL). Tunicamycin, because ER stress is known to induce swelling of the ER lumen thapsigargin and etoposide were purchased from Sigma. The Caspase-3 specific inhibitor Ac-DNLD-CHO was purchased from the Peptide Institute. and dissociation of ribosomes from rough ER (Hitomi et al., 2004b). In some neurodegenerative diseases where ER stress Plasmids and constructs has been allegedly involved, morphological changes including A human bcl-xl cDNA was subcloned into the retroviral expression vector, pMX- formation of cytoplasmic vacuoles have been reported while IRES-PURO (Nosaka et al., 1999). Murine casp-12 cDNA obtained from MEFs was cloned into pFLAG-CMV2 (Sigma). The substitution mutant casp-12 D94A cDNA accumulation of abnormal proteins and cell death play was generated using standard polymerase chain reaction method. Wild type and significant roles in the development of the diseases (Kobayashi mutant cDNAs tagged with FLAG-epitope were subcloned into pMX-IRES-PURO. et al., 2002; Mizuno et al., 2003). In this view, the cell death –/– Detection of cell death with vacuoles observed in Apaf-1 MEFs may have MEFs were plated in a 24-well dish at 5ϫ104 cells per well 1 day prior to treatment.

Journal of Cell Science physiological relevance. The cells were left untreated or treated with tunicamycin or thapsigargin at indicated Bcl-XL conferred resistance to wild type cells against ER concentration. At various time points after treatment, cells were trypsinized and stress-induced cell death just like Apaf-1-deficient cells, collected with the supernatants, and cell viability was determined by propidium iodide (PI, Sigma) and Annexin V (Sigma) staining using flow cytometry. confirming the involvement of Apaf-1/mitochondrial pathways of apoptosis in ER stress-induced cell death. Interestingly, Bcl- Western blotting analysis –/– ϫ 5 XL bestowed additional resistance to Apaf-1 MEFs against MEFs were plated at 5 10 cells per dish in 100 mm dish one day prior to treatment. ER stress-induced cell death. Lines of evidence have shown After treatment, cells were scraped and collected with the supernatants, and lysed with NP40 lysis buffer. Proteins were separated by SDS-PAGE (10% for GRP78, that various pro-apoptotic factors are released from 12% for caspase-12, 15% for caspase-3 and Bcl-XL), transferred onto nitrocellulose mitochondria including Smac/DIABLO (Du et al., 2000), membranes, and incubated with antibodies reactive to GRP78 (Santa Cruz), Bcl-XL Omi/HtrA2 (Suzuki et al., 2001), AIF (Joza et al., 2001; Susin (BD Transduction Laboratories), Caspase-3 mixed with Cleaved caspase-3 or caspase-12 (Cell Signaling). Optimal antibody concentrations were determined in et al., 1999), and Endo G (Li et al., 2001) as well as cytochrome pilot assays. c. Smac/DIABLO and Omi/HtrA2 block IAP-family proteins, which inhibit caspases at post-mitochondrial phases. AIF and Electron microscopy Endo G cause large fragmentation of chromosomal DNA Before or after apoptotic treatment, MEFs were fixed on ice with 1ϫ fixing buffer (2.5% glutaraldehyde, 0.1 M sucrose, and 3 mM CaCl2 in 0.1 M cacodylate buffer leading cell death either dependent on or independent of [pH 7.4]). The cells were processed through 1% OsO4 for 1 hour at 4°C, dehydrated caspases (Arnoult et al., 2003). Considering the lack of in graded ethanol and in propylene oxide, and embedded in Epon 812 resin. Thin caspase-3 activation in Apaf-1–/– MEFs, the additional effects sections were stained with 5-uranyl acetate for 30 minutes and lead acetate for 25 minutes, then examined under a JEM-2000-EX (JEOL, Tokyo) electron microscope. of Bcl-XL may be due to blocking the release of AIF and Endo G from mitochondria by mitochondrial membrane Immunocytochemistry for cytochrome c stabilization. Another possibility is that Bcl-XL may function MEFs were grown on 1mm glass coverslips for 24 hours and stimulated with tunicamycin (1 ␮g/ml) or thapsigargin (0.1 ␮M). Twenty-four hours after at the ER membrane. Bcl-XL blocks the release of pro- stimulation, cells were fixed with 4% paraformaldehyde, and then permeabilized apoptotic factors by blocking functions of Bax and Bak with 0.1% Triton X-100 in phosphate buffered saline at room temperature for 5 channels on mitochondria. Therefore, Bcl-XL may regulate the minutes. MEFs were immunostained with the antibody reactive to cytochrome c function of Bax and Bak at the ER (Zong et al., 2003). (BD PharMingen) and BAX (Upstate). However, neither Bcl-X overexpression nor Apaf-1- L Retrovirus-mediated gene transduction deficiency protected cells from ER stress-induced cell death Human bcl-xl, murine casp-12, FLAG-casp-12, or FLAG-casp-12(D94A) observed at later phases of stimulation. Consistent with the in subcloned into pMX-IRES-PURO were transfected into the PLAT-E packaging cell Apaf-1 and ER stress-induced apoptosis 3965

line using the calcium phosphate method to obtain viruses. MEFs were infected with Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki, T., the virus, treated with polybrene (Sigma) a day after infection, and then culture Elia, A. J., Cheng, H. Y., Ravagnan, L. et al. (2001). Essential role of the medium was replaced with the selection medium containing puromycin. Twenty- mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410, 549- four hours after selection, surviving cells were used for experiments. 554. Kalai, M., Lamkanfi, M., Denecker, G., Boogmans, M., Lippens, S., Meeus, A., Knockdown of casp-12 Declercq, W. and Vandenabeele, P. (2003). Regulation of the expression and processing of caspase-12. J. Cell Biol. 162, 457-467. The mammalian expression vector, pSUPER.retro.puro (Oligoengine) was used for Kaufman, R. J. (1999). 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