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The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family

Abstract

Apoptosis is essential for normal development and maintenance of homeostasis, and disruption of apoptotic pathways is associated with multiple disease states, including cancer. Although initially identified as central regulators of apoptosis at the level of mitochondria, an important role for BCL-2 proteins at the endoplasmic reticulum is now well established. Signaling pathways emanating from the endoplasmic reticulum (ER) are involved in apoptosis initiated by stimuli as diverse as ER stress, oncogene expression, death receptor (DR) ligation and oxidative stress, and the BCL-2 family is almost invariably implicated in the regulation of these pathways. This also includes Ca2+-mediated cross talk between ER and mitochondria during apoptosis, which contributes to the mitochondrial dynamics that support the core mitochondrial apoptosis pathway. In addition to the regulation of apoptosis, BCL-2 proteins at the ER also regulate autophagy, a survival pathway that limits metabolic stress, genomic instability and tumorigenesis. In cases where apoptosis is inhibited, however, prolonged autophagy can lead to cell death. This review provides an overview of ER-associated apoptotic and autophagic signaling pathways, with particular emphasis on the BCL-2 family proteins.

Introduction

The endoplasmic reticulum (ER) has emerged as a central player in many apoptotic pathways, and evidence of an essential role for BCL-2 family members at this location is becoming increasingly apparent (Oakes et al., 2006; Hetz, 2007). For example, ER-restricted BCL-2 and BCL-xL can inhibit apoptosis initiated by diverse stimuli (Hacki et al., 2000; Germain et al., 2005; Mathai et al., 2005; Bhatt et al., 2008) and BAX/BAK (Nutt et al., 2002b; Zong et al., 2003; Chami et al., 2004), as well as a number of BH3-only proteins (Elyaman et al., 2002; Gao et al., 2005; Luo et al., 2005; Armstrong et al., 2007; Hetz et al., 2007; Puthalakath et al., 2007; Szegezdi et al., 2008; Upton et al., 2008) can promote apoptosis through their activity at the ER. BCL-2 family proteins at the ER regulate apoptosis through both direct modulation of signaling pathways leading to caspase activation and through the modulation of ER Ca2+ signaling (Oakes et al., 2006; Rong and Distelhorst, 2008). Conversely, BCL-2 family proteins themselves can be modulated by signals emanating from the ER, and therefore have an essential role in apoptotic pathways initiated by ER stress (Oakes et al., 2006; Hetz, 2007).

Accumulating evidence suggests that the ER has an essential role not only in apoptosis but also in the regulation of autophagy. Autophagy is a degradative lysosomal pathway involving the sequestration of cytoplasmic constituents (including organelles) into double-membrane-bound vesicles or autophagosomes, which eventually fuse with lysosomes for degradation. Autophagy functions as a survival pathway under starvation conditions (through nutrient recycling) and as a protective mechanism through the degradation of damaged organelles (Levine and Klionsky, 2004; Levine and Kroemer, 2008). In cells in which the apoptotic machinery cannot respond to stress stimuli, however, apoptotic stimuli can lead to prolonged autophagy resulting in cell death (Shimizu et al., 2004; Buytaert et al., 2006; Kim et al., 2006b). In addition, some chemotherapeutic agents may trigger cell death through autophagy (Shimizu et al., 2004; Buytaert et al., 2006; Kim et al., 2006b), and decreased autophagy has been associated with tumor development due to metabolic stress (Karantza-Wadsworth et al., 2007; Mathew et al., 2007a, 2007b; Jin and White, 2008). Although autophagy is effected by the evolutionarily conserved autophagy-related genes (ATG) (Yorimitsu and Klionsky, 2005), ER localized antiapoptotic and BH3-only BCL-2 family members have recently been shown to have a regulatory role in the process (Pattingre et al., 2005; Maiuri et al., 2007). This finding highlights both an additional connection between apoptotic and autophagic pathways and a novel role for BCL-2 family proteins at the ER.

Several excellent reviews are available describing the current views and controversies surrounding the core mechanisms of action of the three functional groups of proteins comprising the BCL-2 family: the multi-BH domain prosurvival members (BCL-2, BCL-xL, BCL-w, MCL-1 and A1); the multidomain proapoptotic effectors, BAX and BAK, and the proapoptotic BH3-only members (BID, BIM, BIK, BAD, NOXA and PUMA), which act to couple upstream signals to downstream regulation of the prosurvival and prodeath multi-BH domain members (Danial and Korsmeyer, 2004; Reed, 2006; Leber et al., 2007; Adams and Cory, 2007a, 2007b; Chipuk and Green, 2008; Letai, 2008; Youle and Strasser, 2008).

ER stress-induced apoptosis

The unfolded protein response (UPR): survival

The ER lumen represents both the major site of intracellular Ca2+ storage and the site at which transmembrane and secreted proteins are folded and post-translationally modified. Conditions that disrupt the luminal environment and these core ER functions, including oxidative stress, perturbation of Ca2+ or energy stores and accumulation of unfolded/misfolded proteins result in an evolutionarily conserved adaptive response termed the UPR. This protective response acts to restore homeostatic conditions through decreased global translation, increased expression of select proteins,and increased degradation of misfolded proteins. In higher eukaryotes, the UPR involves three signaling pathways, initiated by protein kinase-like ER kinase (PERK), inositol requiring kinase 1 (IRE1) and activating transcription factor 6 (ATF-6). PERK, IRE1 and ATF-6 are all transmembrane proteins, and, in response to ER stress, are activated through either disruption of an interaction with the luminal ER chaperone BiP, or through direct interaction with unfolded proteins. A number of excellent reviews have recently been written with respect to the initiation and regulation of the UPR (Schroder and Kaufman, 2005b; Malhotra and Kaufman, 2007).

Protein kinase-like ER kinase is activated through dimerization and trans-autophosphorylation, and subsequently leads to both decreased global translation and increased translation of several select proteins, including the transcription factor ATF-4. This ultimately results in a decreased overall protein load within the ER, but increased expression of proteins involved in amino-acid metabolism, oxidative stress response and chaperone function (Malhotra and Kaufman, 2007).

Inositol requiring kinase 1 is activated in the same way as PERK, but, upon activation, displays both kinase and endoribonuclease (RNase) activity. Following activation, the RNase activity of IRE1 leads to efficient translation of the transcription factor XBP1 through a nonconventional RNA splicing event. XBP1 promotes the transcription of genes involved in lipid synthesis, chaperone function and ER-associated degradation (Malhotra and Kaufman, 2007).

Activating transcription factor 6 is a transcription factor, which, like PERK and IRE1, is activated upon dissociation from BiP at the ER. ATF-6 then moves from the ER to the Golgi, where it is proteolytically processed and subsequently translocated to the nucleus. Nuclear ATF-6 leads to the transcription of genes involved in chaperone function (Malhotra and Kaufman, 2007).

Pathways leading to ER stress-induced apoptosis

The UPR is primarily a survival response, acting to resolve dysregulation of protein-folding pathways. If, however, the normal luminal environment cannot be restored, the response to ER stress is switched from survival to apoptosis. PERK, IRE1 and ATF-6 are all involved in the induction of proapoptotic as well as prosurvival pathways (Malhotra and Kaufman, 2007). Both PERK and ATF-6 induce expression of the transcription factor C/EBP homologous protein (CHOP), which in turn leads to reduced expression of BCL-2 and increased expression of a number of proapoptotic genes (McCullough et al., 2001; Ma et al., 2002; Marciniak et al., 2004). CHOP also activates the transcription of GADD34, which leads to the release of the PERK-imposed translational block (Novoa et al., 2001). IRE1, in addition to its role as an endoribonuclease, acts to initiate a signaling platform consisting of IRE1/TNF-receptor-associated factor 2 (TRAF2)/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK), resulting in the phosphorylation of JNK and subsequent induction of apoptosis through the phosphorylation of key proteins (Urano et al., 2000; Malhotra and Kaufman, 2007). IRE1 may also regulate the activation of ER-localized caspase-12 through the modulation of a TRAF2/caspase-12 complex (Malhotra and Kaufman, 2007).

Multiple pathways may be involved in ER stress-initiated apoptosis, including direct activation of initiator caspases at the ER, transcriptional activation of the apoptotic program through the modulation of BCL-2 family members or death receptors (DRs) and apoptosis through cross talk with the mitochondria. Regardless of the mechanism involved, ER stress-induced apoptosis is, in most cases, regulated by or dependent on the BCL-2 family of proteins (Hetz, 2007; Malhotra and Kaufman, 2007).

Endoplasmic reticulum stress can be experimentally induced through a variety of pharmacological methods, including the disruption of ER–Golgi trafficking (with brefeldin A; BFA), depletion of ER Ca2+ stores (with thapsigargin; TG), inhibition of N-linked glycosylation (with tunicamycin; TN), inhibition of disulfide bond formation (with dithiothreitol) or overexpression of unfolded/misfolded proteins (Malhotra and Kaufman, 2007). In most experimental systems, ER stress-induced apoptosis can be inhibited by the overexpression of either wild-type or ER-targeted BCL-2 or BCL-xL (Srivastava et al., 1999; Hacki et al., 2000; Boya et al., 2002; Murakami et al., 2007; Wlodkowic et al., 2007; Bhatt et al., 2008). In addition, BAX/BAK double-knockout (DKO) cells are resistant to ER stress-induced apoptosis, a phenotype that is only partially reversed by reintroduction of mitochondria-targeted BAX/BAK (Scorrano et al., 2003). As well, a number of BH3-only proteins, including BAD (Elyaman et al., 2002), PUMA (Luo et al., 2005), NOXA (Armstrong et al., 2007), BID (Gao et al., 2005; Upton et al., 2008) and BIM (Hetz et al., 2007; Puthalakath et al., 2007; Szegezdi et al., 2008) have been linked to ER stress-initiated cell death.

ER stress-induced apoptosis: ER-associated initiator caspases

A proximal event in apoptosis initiated by either cell surface or mitochondrial stimuli is activation of an initiator caspase (caspase-8 through DR ligation; caspase-9 through BAX/BAK-mediated mitochondrial outer membrane permeability; MOMP), which subsequently activates downstream executioner caspases (caspase-3 and/or -7), thereby leading to cell death (Danial and Korsmeyer, 2004). An analogous pathway has been proposed to exist at the ER, with murine caspase-12 and human caspase-4 acting as the initiator caspases. Once activated, caspase-12 or -4 can, in turn, activate caspase-9 or -3, thereby leading to MOMP-independent cell death (Kamada et al., 1997; Morishima et al., 2002; Rao et al., 2002a; Hitomi et al., 2004b; Lopez-Anton et al., 2006; Yukioka et al., 2008).

Caspase-12 was initially identified as an ER-localized caspase selectively activated by ER stress, and deletion of caspase-12 was shown to protect against ER stress-induced apoptosis, both in vitro and in vivo (Nakagawa et al., 2000). Murine caspase-12 has since been shown to play a role in apoptosis induced by a variety of stressors, including TN, TG and BFA (Nakagawa et al., 2000), as well as overexpression of physiologically relevant unfolded/misfolded proteins, such as poly(Q) (Huntington's disease (Kouroku et al., 2002)) and amyloid β (Alzheimer's disease (Nakagawa et al., 2000)). In a number of cases, caspase-12 activation occurred upstream of other initiator/executioner caspases, and inhibition/deletion of caspase-12 significantly decreased both cell death and activation of executioner caspases; pointing to a role for caspase-12 activation as an initiating event in ER stress-induced apoptosis (Nakagawa et al., 2000; Kouroku et al., 2002; Morishima et al., 2002; Hitomi et al., 2004b; Sanges and Marigo, 2006). In addition, overexpression of caspase-12, but not of an inactive mutant, leads to apoptotic cell death (Kalai et al., 2003).

Caspase-12 may be involved in ER stress-induced apoptosis in murine cells, but human caspase-12 has acquired a number of deleterious mutations, and is, in most cases, inactive (Fischer et al., 2002). In the small human subpopulation that does express functional caspase-12, it appears to play a role in immune response, and not in apoptosis (Saleh et al., 2004). Human caspase-4 is at least partially localized to the ER, and, like murine caspase-12, appears to be selectively activated in response to ER stress (Hitomi et al., 2004b). A functional role for caspase-4 has been shown in amyloid β-mediated cytotoxicity (Hitomi et al., 2004b) as well as in ER stress-associated death in response to a variety of stimuli, including TG and TN, as well as proteasome inhibition and kinase inhibition (Hitomi et al., 2004b; Nawrocki et al., 2005; Lopez-Anton et al., 2006; Jiang et al., 2007; Rahmani et al., 2007).

Caspases -12 and -4 are likely involved in ER stress-induced apoptosis, but events upstream of their activation remain poorly defined. The activation of caspase-12 requires both removal of a prodomain and separation of the large and small subunits. Of note, although the autocatalytic activity of caspase-12 is sufficient for subunit separation, it does not allow prodomain removal (Roy et al., 2008). Without prodomain removal, caspase-12 is capable only of self-cleavage (Roy et al., 2008), and overexpression of caspase-12 under conditions in which only autoprocessing occurs therefore does not result in apoptosis, likely due to an extremely limited substrate set prior to removal of the prodomain (Nakagawa and Yuan, 2000; Fujita et al., 2002; Kalai et al., 2003; Roy et al., 2008). Removal of the caspase-12 prodomain can be achieved through a number of mechanisms, one of which is cleavage by m-calpain. A number of ER stressors lead to the release of ER Ca2+ stores, and thereby to elevation of cytosolic Ca2+ levels and calpain activation. Caspase-12 cleavage has been linked to calpain activation in response to ischemia, amyloid β accumulation, TN and TG. In addition, inhibition or genetic disruption of calpains, as well as chelation of intracellular Ca2+, has been shown to prevent caspase-12 processing in a number of systems (Nakagawa and Yuan, 2000; Schroder and Kaufman, 2005a; Sanges and Marigo, 2006; Tan et al., 2006; Imai et al., 2007). Caspase-12 may also be cleaved by caspase-7; ER stress leads to the recruitment of caspase-7 to the ER, where it forms a complex with caspase-12 and the ER chaperone GRP78. It has been suggested that this interaction results in caspase-7-mediated cleavage of caspase-12, with GRP78 acting to inhibit either caspase-7 or the active form of caspase 12 (Rao et al., 2001, 2002b; Reddy et al., 2003). How caspase-7 is recruited to the ER and activated, as well as whether activation of caspase-12 through this particular pathway represents an amplification loop (possibly downstream of MOMP), as opposed to an initiating event, remains to be determined. Finally, caspase-12 may be associated with TRAF2 under basal conditions, and ER stress induces simultaneous dissociation of this complex and homodimerization of caspase-12. TRAF2 is recruited to IRE-1 following ER stress, and overexpression of IRE-1 promotes caspase-12 homodimerization (Yoneda et al., 2001). Although an association between IRE-1 and caspase-12 has not been shown, it is possible that IRE-1 recruitment of the TRAF2/caspase-12 complex provides a scaffold for caspase-12 processing during ER stress (Yoneda et al., 2001). Less is known about caspase-4 than about caspase-12, but it requires both dimerization and interdomain processing for activation (Karki et al., 2007), and, like caspase-12, can be activated by calpain and is likely inhibited by interaction with GRP78 (Jiang et al., 2007; Oda et al., 2008).

Regulation of ER-associated initiator caspases by BCL-2 family members

Studies using BAX/BAK DKO cells have shown that caspase-12 activation is dependent on BAX/BAK, and that ER-targeted BAK can activate caspase-12 independently of caspase-7 and of the mitochondrial apoptotic pathway, in a BCL-xL inhibitable manner (Zong et al., 2003; Ruiz-Vela et al., 2005). These findings may reflect a role for BAX/BAK in the regulation of ER Ca2+ stores. BAX/BAK DKO cells have reduced levels of ER Ca2+, and are resistant to ER stress-induced apoptosis. Restoration of cell death to levels seen in wild-type cells required both mitochondrial BAX and increased ER Ca2+, pointing to a role for ER Ca2+ release in this pathway (Scorrano et al., 2003). As m-calpain can activate caspase-12, and calpain activation is Ca2+ dependent, insufficient Ca2+ release (due to low [Ca2+]ER) in BAX/BAK DKO cells may explain the absence of ER stress-induced caspase-12 cleavage in this system. The activation of caspase-12 by ER-restricted BAK may also be due to modulation of ER Ca2+ stores, as the overexpression of BAK/BAX induces an initial rise in [Ca2+]ER, followed by rapid store depletion (Nutt et al., 2002b; Chami et al., 2004).

BH3-only proteins may also be involved in caspase-12 activation, likely through BAX/BAK. TN treatment was shown to result in translocation of BIM to the ER, followed by the induction of caspase 12-dependent, mitochondria-independent apoptosis. Both BIM translocation and apoptosis were inhibited by BCL-xL, and a direct BIM/BCL-xL interaction was demonstrated. Ectopic expression of ER-targeted BIM also induced apoptosis, which was inhibited by suppression of caspase-12 (Morishima et al., 2004). Finally, PUMA and NOXA are also upregulated during ER stress, both have been found at the ER, and both play a role in ER Ca2+ release (Reimertz et al., 2003; Luo et al., 2005; Rao et al., 2006; Shibue et al., 2006; Kieran et al., 2007; Nickson et al., 2007; Hassan et al., 2008). PUMA-mediated apoptosis has been linked to activation of caspase-12 and is reduced by caspase-12 knockdown or deficiency (Shibue et al., 2006).

It should be noted that, at least in some cases, ER-localized BAX/BAK, as well as ER Ca2+ stores, have only a minor function in ER stress-initiated cell death (Scorrano et al., 2003; Hetz et al., 2006). In addition, a recent study has shown that a direct interaction between BAX/BAK and IRE1 is required for IRE1 activation as well as for downstream XBP-1 splicing and JNK phosphorylation. This BAX/IRE-1 association increased following ER stress, and loss of BAX/BAK led to impairment of adaptation to ER stress. BAK/BAX therefore appear to play a prosurvival role in the ER stress response, possibly through the stabilization of an active IRE-1 complex. In the same study, reconstitution of BAX/BAK DKO cells with mitochondria-targeted BAX was shown to restore full sensitivity to ER stress-induced cell death (Hetz et al., 2006). Induction of the UPR may, therefore, trigger an adaptive, as opposed to a prodeath, function of BAX/BAK at the ER (Hetz, 2007; Hetz and Glimcher, 2008). How this adaptive function can be reconciled with BAX/BAK-dependent activation of caspase-12 during ER stress remains to be seen. It is possible that different signaling pathways are predominant in different cell types, and/or are affected by the type, strength or duration of the ER stress signal. For example, ER localized BAX/BAK may act in an adaptive capacity under conditions of mild or transient ER stress, but as prodeath molecules following prolonged or severe ER stress. This switch in function could conceivably involve BH3-only protein-mediated dissociation of BAX/BAK from IRE-1, followed by BAX/BAK oligomerization (Hetz and Glimcher, 2008).

ER stress-induced apoptosis: mitochondrial involvement

Caspase-12/4 may provide a means for ER stress to initiate apoptosis through mitochondrial/cell surface signaling independent activation of executioner caspases. However, deletion/inhibition of caspase-12/4 generally provides only partial protection against cell death (Nakagawa et al., 2000; Hitomi et al., 2004a; Sanges and Marigo, 2006). Tissue expression of these caspases is not as ubiquitous as was initially expected, and, in a number of cases, ER stress-induced apoptosis proceeds normally in the absence of caspases-4/12, through a caspase-9-dependent pathway (Kalai et al., 2003; Obeng and Boise, 2005).

In line with a minor role for caspase-12/4 under some conditions, a number of studies have indicated that the induction of apoptosis by ER stress has an obligatory mitochondrial component. Cytochrome c release, loss of mitochondrial membrane potential and caspase-9 activation are seen in response to a variety of ER stressors (Hacki et al., 2000; Boya et al., 2002; Jimbo et al., 2003; Kitamura et al., 2003; Masud et al., 2007; Wlodkowic et al., 2007), and the loss of Apaf 1 (which is required, along with cytochrome c, for postmitochondrial caspase-9 activation) significantly decreases ER stress-induced apoptosis in a variety of experimental systems (Di Sano et al., 2006; Shiraishi et al., 2006; Smith and Deshmukh, 2007), as does the inhibition of mitochondrial permeability transition (involved in the release of mitochondrial cytochrome c (Kinnally and Antonsson, 2007)) (Zhang and Armstrong, 2007; Deniaud et al., 2008; Zhang et al., 2008). In addition, the activation and mitochondrial localization of BAX are seen in response to ER stress, and several studies have shown that mitochondrial BAX is required for ER stress-initiated apoptosis (Scorrano et al., 2003; Shiraishi et al., 2006; Zhang and Armstrong, 2007; Deniaud et al., 2008; Zhang et al., 2008).

Involvement of a BAX/BAK-dependent mitochondrial component implies the likely involvement of one or more BH3-only proteins at this location. As mentioned previously, both PUMA and NOXA are transcriptionally upregulated following ER stress, and both proteins can induce BAX/BAK-mediated MOMP (Luo et al., 2005; Armstrong et al., 2007; Chipuk and Green, 2008; Youle and Strasser, 2008). In support of a functional role for PUMA/NOXA, mouse embryonic fibroblasts (MEFs) deficient in either PUMA or NOXA, as well as PUMA deficient cardiomyocytes, are partially resistant to ER stress-induced cell death, and PUMA deficiency prevents ER stress-mediated loss of motoneurons in a mouse model of amyotrophic lateral sclerosis (ALS) (Li et al., 2006; Kieran et al., 2007; Nickson et al., 2007). The induction of PUMA/NOXA during ER stress can be either p53 dependent or independent (Luo et al., 2005; Li et al., 2006; Armstrong et al., 2007).

BIM is also transcriptionally upregulated during ER stress, through CHOP/C/EBPα, and, like PUMA, has been shown to play a role in motoneuron loss in a mouse model of ALS (Hetz et al., 2007; Puthalakath et al., 2007; Szegezdi et al., 2008). In addition to upregulation of BIM expression, ER stress also leads to increased stability of the BIM protein (Puthalakath et al., 2007). This increased stability is due to the inhibition of proteasomal degradation via protein phosphatase 2A (PP2A)-mediated dephosphorylation (Puthalakath et al., 2007). Of note, a recent study has shown that the catalytic subunit of PP2A is upregulated following ER stress, dependent, at least in some cases, on ER Ca2+ release-mediated activation of the transcription factor CREB (Christen et al., 2007). There is also some evidence for ER stress-mediated disruption of BIM sequestration by the dynein motor complex (Morishima et al., 2004). Disruption of this interaction is required for the translocation of BIM to the mitochondria/ER, and may, in some cases, be regulated by JNK-mediated phosphorylation (Lei and Davis, 2003; Youle and Strasser, 2008). Whether IRE-1-mediated JNK activation has a function in BIM activation during ER stress, however, remains to be determined.

In healthy cells, BAD, like BIM, is sequestered in the cytoplasm, in this case by 14-3-3 proteins. This interaction is dependent on BAD phosphorylation at specific residues, and BAD dephosphorylation is associated with dissociation from 14-3-3 and BAX/BAK-dependent apoptosis (Youle and Strasser, 2008). However, although dephosphorylation of BAD has been observed in response to several ER stress stimuli, a functional role for this protein in ER stress-mediated apoptosis has not been demonstrated (Elyaman et al., 2002; Szegezdi et al., 2008). Of potential interest, given the role of PP2A in ER stress-induced apoptosis, is the observation that survival factor withdrawal-initiated apoptosis involves PP2A-mediated BAD dephosphorylation (Chiang et al., 2001, 2003). Alternatively, in situations under which ER stress results in the release of ER Ca2+ stores, BAD may be dephosphorylated by the Ca2+-dependent phosphatase calcineurin/PP2B (Wang et al., 1999).

In addition to PUMA, NOXA, BIM and BAD, there is also convincing evidence for the involvement of BID in ER stress-induced apoptosis. A recent study has shown that BID is activated through cleavage by caspase-2 during ER stress, and that BID null MEFs display significant resistance to ER stress (Upton et al., 2008). This finding is consistent with previous studies pointing to a role for BID in caspase-2-mediated cytochrome c release and to the involvement of BID and caspase-2 in ER stress-mediated apoptosis (Dahmer, 2005; Gao et al., 2005; Cheung et al., 2006; Murakami et al., 2007). Although the mechanism of caspase-2 activation during ER stress is unclear, there is evidence that it is independent of ER-localized BAX/BAK, but potentially dependent on BCL-2/BCL-xL inhibitable activation of JNK (Murakami et al., 2007). Like other initiator caspases, procaspase-2 is activated through dimerization followed by autocatalytic cleavage (Baliga et al., 2004). On the basis of the localization of a subpopulation of procaspase-2 at the ER in healthy cells (Cheung et al., 2006), it is tempting to speculate that caspase-2 activation in response to ER stress may be mediated through induced proximity as a result of recruitment to a signaling platform, possibly dependent, either directly or indirectly, on active JNK.

Although the above described caspase-2/BID pathway appears to proceed independently of caspase-8, other studies support a role for caspase-8 in ER stress-induced apoptosis (He et al., 2002; Jimbo et al., 2003; Yamaguchi et al., 2003; Yamaguchi and Wang, 2004). Taken together, these studies indicate that CHOP and/or ER Ca2+ depletion-dependent upregulation of both DR5 and the DR5 ligand TRAIL (TNF-related apoptosis-inducing ligand) induce caspase-8 activation and subsequent BID cleavage during ER stress (He et al., 2002; Yamaguchi et al., 2003; Yamaguchi and Wang, 2004).

BAX/BAK activation, at both the ER and the mitochondria, can be regulated by antiapoptotic, as well as BH3-only, BCL-2 family members. ER stress decreases BCL-2 expression through CHOP (McCullough et al., 2001), and may also affect BCL-2 stability/activity through phosphorylation (Bassik et al., 2004; Lin et al., 2006). BCL-2 may be in a complex with PP2A at the ER, and either knockdown of PP2A or knock-in of a nonphosphorylatable BCL-2 mutant protects against ER stress-induced apoptosis (Lin et al., 2006). The phosphorylation of BCL-2, in most cases, leads to decreased antiapoptotic activity and/or increased degradation (Dimmeler et al., 1999; Bassik et al., 2004; Simizu et al., 2004; Tamura et al., 2004; Lin et al., 2006). As JNK is activated during ER stress, and JNK-mediated BCL-2 phosphorylation/inactivation has been shown at the G2/M phase of the cell cycle (Yamamoto et al., 1999) and in response to vincristine (Muscarella and Bloom, 2008), JNK may downregulate BCL-2 activity during ER stress.

Protein phosphatase 2A not only preserves BCL-2 antiapoptotic activity, but also activates several proapoptotic BCL-2 family members (Chiang et al., 2001, 2003; Puthalakath et al., 2007), and, in at least one system, has an inhibitory, as opposed to an activating, effect on BCL-2 (Ruvolo et al., 1999, 2002). These apparently contradictory results may be due to the involvement of substrate/organelle-specific PP2A regulatory subunits, stimulus-dependent phosphorylation site specificity or availability, or additional post-translational modifications.

Endoplasmic reticulum stress has also been implicated in the regulation of BCL-2 family members through several additional intermediates. Glycogen synthase kinase 3β (GSK3β), for example, has been causally linked to ER stress-induced apoptosis, upstream of caspase-9 activation, in a number of systems (Song et al., 2002; Srinivasan et al., 2005; Brewster et al., 2006; Takadera et al., 2007). GSK3β is activated by dephosphorylation at Ser9; an event which may be mediated either directly or indirectly by PP2A (Song et al., 2002; Chen et al., 2004a; Srinivasan et al., 2005; Lin et al., 2007). How GSK3β is involved in ER stress-induced apoptosis is unclear. However, in response to other apoptotic stimuli, active GSK3β has been shown to phosphorylate MCL-1 and/or BAX; leading to MCL-1 degradation and BAX activation/mitochondrial translocation (Linseman et al., 2004; Maurer et al., 2006; Ding et al., 2007; Zhao et al., 2007), and it is possible that similar pathways may operate during ER stress. It should be noted, however, that GSK3β also has a prosurvival function during ER stress, through the increased degradation of p53 (Qu et al., 2004; Baltzis et al., 2007).

Like GSK3β, both JNK and p38 mitogen-activated protein kinase are activated during ER stress, and are capable of phosphorylating multiple substrates. BAX activation/mitochondrial translocation has been linked to JNK-mediated phosphorylation following exposure to H2O2, ultraviolet irradiation, staurosporine or etoposide (Kim et al., 2006a). Alternatively, JNK may lead to the disruption of an inhibitory BAX/14-3-3 interaction through the phosphorylation of 14-3-3 (Tsuruta et al., 2004). Although phosphorylation of BAX may be an activating event, dephosphorylation (presumably at different sites) can also lead to activation, and there is evidence of a role for PP2A-mediated dephosphorylation in both activation of BAX and disruption of a BAX/BCL-2 interaction (Xin and Deng, 2006). Again, as with GSK3β, whether JNK and/or PP2A activate BAX during ER stress remains to be seen.

In summary, ER stress-induced apoptosis is a complex and heterogeneous process, involving activation of ER-associated initiator caspases, mitochondrial permeabilization and multiple BH3-only proteins. The specific pathways involved seem to be highly dependent on the type, strength and duration of the ER stress signal, as well as on the cell type under study. It is also likely that multiple pathways operate within the same cell, with initiating as opposed to amplifying components being determined by the genetic/proteomic makeup of the system under study. In support of coordinate activation of multiple signaling pathways, there is no one component, with the possible exception of BAX/BAK, whose inhibition or deletion completely abrogates ER stress-induced apoptosis.

Apoptosis and ER Ca2+ release

Release of ER Ca2+ stores is an important component of many apoptotic pathways, including, among others, those initiated by ER stress, oncogenic stress, staurosporine, oxidative stress and lipid second messengers (Nutt et al., 2002a; Demaurex and Distelhorst, 2003; Hajnoczky et al., 2003; Scorrano et al., 2003; Dong et al., 2006). In some cases, increased cytosolic Ca2+ levels during apoptosis can lead to the activation of calpains and/or calcineurin (Dong et al., 2006), thereby promoting apoptosis through calpain-mediated cleavage of BAX/BID/BCL-2/BCL-xL/caspase-12 (Wood et al., 1998; Gao and Dou, 2000; Nakagawa and Yuan, 2000; Gil-Parrado et al., 2002; Oh et al., 2004; Tan et al., 2006) and/or calcineurin-mediated dephosphorylation of BAD (Wang et al., 1999; Roderick and Cook, 2008). Alternatively, or in parallel, privileged ER-mitochondria Ca2+ transmission can induce cytochrome c mobilization within the mitochondrial intermembrane space, thereby allowing cytosolic translocation following BAX/BAK-mediated MOMP (Petrosillo et al., 2004; Germain et al., 2005; Walter and Hajnoczky, 2005; Pizzo and Pozzan, 2007). In healthy cells, cytochrome c is both sequestered within mitochondrial cristae and held in place through interaction with cardiolipin; BAX/BAK-induced MOMP alone cannot support efficient release (Ott et al., 2002; Scorrano et al., 2002). A ‘sensitizing’ cytochrome c mobilization signal, acting to functionally ‘open’ mitochondrial cristae and disrupt the cytochrome c-cardiolipin interaction, may therefore be a general requirement of mitochondrial-dependent apoptotic pathways.

Both mitochondrial Ca2+ uptake and tBID can trigger intermembrane space cytochrome c mobilization, and this likely involves transient opening of the permeability transition pore, reactive oxygen species, cardiolipin peroxidation and components of the mitochondria fission/fusion machinery (Scorrano et al., 2002; Petrosillo et al., 2004; Germain et al., 2005; Cipolat et al., 2006; Frezza et al., 2006; Zhang et al., 2008). A potentially interesting observation is that dynamin-related protein 1 (DRP1), a regulator of mitochondrial fission, may be required for cristae remodeling (Germain et al., 2005). DRP1-mediated cristae remodeling requires enzymatic activity (Germain et al., 2005), and DRP1 activity can be positively regulated by calcineurin (Cribbs and Strack, 2007). Given that calcineurin is activated by Ca2+, ER Ca2+ release may contribute to cristae remodeling through both mitochondrial Ca2+ transmission and DRP1 phosphorylation. However, as calcineurin activity is not restricted to proapoptotic conditions (Groenendyk et al., 2004; Rong and Distelhorst, 2008), DRP1 activation during apoptosis likely involves additional regulatory factors. Potential candidates for apoptosis-specific DRP1 activation include BAX and/or BAK. Stable association of DRP1 with mitochondrial membranes during apoptosis has recently been shown to be dependent on BAX/BAK, and has been correlated with BAX/BAK-dependent sumoylation of DRP1 (Wasiak et al., 2007). This stable association occurs downstream of organelle fission, but, as in the case of cristae remodeling, before the loss of membrane potential or cytochrome c release. DRP1-dependent cristae remodeling, but not mitochondrial fission, may, therefore, be dependent on BAX/BAK (Wasiak et al., 2007), and DRP1-initiated cytochrome c mobilization during apoptosis could require the coordinate activation of calcineurin and BAX/BAK.

Although DRP1, linked to ER Ca2+ signaling, is likely an important factor in mobilization of cytochrome c during apoptosis, numerous other factors are almost certainly also involved. Chief among these is Opa1. A detailed discussion of this and other contributors to cristae remodeling and/or disruption of the cytochrome c/cardiolipin interaction during apoptosis is beyond the scope of this review, but can be found in the following papers: (Bayir et al., 2006; Garrido et al., 2006; Heath-Engel and Shore, 2006; Gonzalvez and Gottlieb, 2007; Orrenius, 2007; Ott et al., 2007; Pellegrini and Scorrano, 2007)

Apoptosis and ER Ca2+ release: BAX, BAK and antiapoptotic BCL-2 family members

Regulation of apoptosis by BCL-2 family members at the ER is largely dependent on their ability to modulate ER Ca2+ signaling. Ca2+ signaling during apoptosis is enhanced by the proapoptotic family members BAX/BAK and dampened by the antiapoptotic BCL-2 and BCL-xL proteins. Overexpression of BAX/BAK at the ER induces a transient ER Ca2+ accumulation, followed by store depletion due to the induction of apoptosis (Nutt et al., 2002b; Zong et al., 2003; Chami et al., 2004), whereas the loss of BAX/BAK leads to a decrease in ER Ca2+ stores, and resistance to Ca2+-dependent apoptotic stimuli (Nutt et al., 2002a; Shi et al., 2005), even following reconstitution with mitochondria-targeted BAX/BAK (Scorrano et al., 2003). Overexpression of wild-type or ER-restricted BCL-2/BCL-xL, on the other hand, provides resistance to Ca2+-dependent apoptotic stimuli, due to decreased release of ER Ca2+ and a resultant decrease in mitochondrial Ca2+ uptake (Annis et al., 2001; Pinton et al., 2001; Rudner et al., 2001). Ca2+ release from the ER occurs primarily through the inositol 1,4,5-triphosphate receptor (IP3R) (Foskett et al., 2007) and the ryanodine receptor (Zalk et al., 2007) protein families, whereas Ca2+ reuptake is dependent on the sarco/ER Ca2+-ATPase (SERCA) (Wuytack et al., 2002). BCL-2/BCL-xL may regulate ER Ca2+ signaling through interaction with the IP3R (Chen et al., 2004b; White et al., 2005; Hanson et al., 2008), and/or alteration of the IP3R phosphorylation state (Oakes et al., 2005; Xu et al., 2007). In addition, decreased ER Ca2+ levels in BAX/BAK-deficient cells may be due to changes in IP3R permeability due to the unopposed activity of BCL-2/BCL-xL (Oakes et al., 2005; White et al., 2005). Finally, a recent study has indicated that the ER transmembrane protein Bax inhibitor 1 (BI-1) is required for BCL-xL-mediated lowering of ER Ca2+ stores (Xu et al., 2008). Notably, BI-1 is required for resistance to ER stress-initiated apoptosis, pointing to a physiologically relevant role for this effect (Chae et al., 2004).

The mechanistic aspect of BCL-2/BCL-xL function at the ER is the subject of significant debate, with some studies pointing to a link with decreased ER Ca2+ stores (Foyouzi-Youssefi et al., 2000; Pinton et al., 2000; Palmer et al., 2004), some indicating decreased stimulus-dependent ER Ca2+ release, without a change in basal [Ca2+]ER (Chen et al., 2004b; Zhong et al., 2006; Hanson et al., 2008), and others supporting a protective role through adaptation to increased spontaneous [Ca2+]i spiking (White et al., 2005; Li et al., 2007). Several studies have also shown evidence for BCL-2/BCL-xL modulation of ER Ca2+ uptake through the inactivation of SERCA (Dremina et al., 2004, 2006), as well as for changes in IP3R/SERCA expression levels (Kuo et al., 1998; Li et al., 2002; Vanden Abeele et al., 2002). These possibilities, however, are not mutually exclusive, and, in any case, would all have a protective effect with respect to apoptosis. The role of BCL-2/BCL-xL in ER Ca2+ signaling has recently been reviewed elsewhere (Distelhorst and Shore, 2004; Pinton and Rizzuto, 2006; Rong and Distelhorst, 2008).

Apoptosis and ER Ca2+ release: BH3 only proteins

In addition to multi-BH domain members, a number of BH3-only proteins localize to the ER, and, as with other members of the BCL-2 family, modulate ER Ca2+ stores. The most extensively studied of these proteins, with respect to mechanism of action at the ER, is the p53-inducible protein BIK (Mathai et al., 2002). Once expressed, BIK is localized at the ER where it initiates ER Ca2+ release, leading to mitochondrial Ca2+ uptake and mobilization of cytochrome c stores (Mathai et al., 2005). BIK-induced ER Ca2+ release is dependent on the presence of BAX/BAK, can be inhibited by ER-restricted BCL-2, and is accompanied by BCL-2 inhibitable oligomerization of BAK at the ER (Mathai et al., 2005). It therefore seems likely that BIK leads to ER Ca2+ release via activation of BAX/BAK at the ER, possibly through disruption of an inhibitory interaction between BCL-2/BCL-xL and BAX/BAK. A ‘two-hit’ model was proposed, where ER BIK-initiated Ca2+ signaling resulted in cristae remodeling (to mobilize cytochrome c stores), a mitochondrial-directed signal resulted in the activation of BAX/BAK (to permeabilize the outer membrane) and the two in combination resulted in the complete release of cytochrome c (Germain et al, 2005). Although prolonged BIK expression alone can result in cell death, the physiological relevance is questionable as BIK is typically induced in response to physiological stress as part of a larger cohort of proapoptotic proteins.

In addition to BIK, both PUMA and NOXA are capable of initiating ER Ca2+ release (Shibue et al., 2006). PUMA was shown to induce both caspase-dependent and caspase-independent MOMP, with the caspase-dependent pathway proceeding through calpain-associated activation of caspase-12 (Shibue et al., 2006), whereas NOXA led to both cytochrome c release and activation of JNK/p38 mitogen-activated protein kinase (Hassan et al., 2008).

Apoptosis and ER Ca2+ release: death receptor pathway

The apoptotic arm of DR signaling results in the activation of caspase-8. If the level of caspase-8 enzyme activity is sufficient to lead to processing of downstream procaspase-3 or -7, a direct pathway to cell death is achieved. Alternatively or in addition, a limited number of noncaspase targets of caspase-8 have been identified, including BID and BAP31, which amplify the caspase-8 signal through the mitochondria and ER, respectively (Breckenridge et al., 2003a). BAP31 is an integral membrane protein of the ER and has a function in ER protein trafficking, likely by escorting newly synthesized integral membrane proteins from their site of biogenesis to those complexes in the ER that determine the fate of the cargo protein in question (Wang et al., 2008). Caspase-8 cleavage of BAP31 within its cytosolic domain (Ng et al., 1997) is required for Fas-mediated apoptosis in certain contexts (Nguyen et al., 2000) and results in the release of ER Ca2+ stores, activation of DRP1 and sensitization of mitochondria to tBID (Breckenridge et al., 2003b). Although the mechanism of Ca2+ release by caspase-cleaved BAP31 remains to be elucidated, it appears to recapitulate a contribution of ER signaling in the DR pathway in supporting the mitochondrial propagation of the caspase-8 signal. Cleavage of BAP31 has also been observed in a number of ER stress-associated apoptotic systems, possibly due to caspase-8 activation following CHOP-mediated upregulation of DR5 (Hidvegi et al., 2005; Li et al., 2005; Liu et al., 2006; Iizaka et al., 2007). However, whether caspase-cleaved BAP31 has a functional role in Ca2+ signaling during ER stress remains to be determined.

ER regulation of autophagy: role of the BCL-2 family proteins

The mammalian ortholog of yeast Atg6, Beclin 1, was originally identified as a BCL-2-binding protein in a yeast two-hybrid screen (Liang et al., 1998). Beclin 1 is a haploinsufficient tumor suppressor and induces autophagy by promoting autophagosome formation when in complex with hVps34/class III phosphatidylinositol 3-kinase (PI3K) (Kihara et al., 2001; Yue et al., 2003). The interaction between BCL-2 antiapoptotic proteins and Beclin 1 represents a possible convergence point for apoptosis and autophagy pathways and has important functional consequences. Through direct interaction, BCL-2 antiapoptotic proteins negatively regulate Beclin 1-dependent autophagy and Beclin 1-dependent autophagic cell death (Pattingre et al., 2005). Interestingly, the role of BCL-2 in antagonizing Beclin 1-dependent autophagy is restricted to BCL-2 located at the ER. Beclin 1 binding with BCL-2 has been suggested to interfere with the ability of Beclin 1 to form a complex with hVps34/PI3K, thus resulting in a loss of Beclin 1-associated PI3K autophagy-inducing activity. Moreover, the interaction between Beclin 1 and BCL-2 was found to be dependent on the nutrient status of the cell. During autophagy-inducing conditions, such as starvation, the interaction between Beclin 1 and BCL-2 is minimal, suggesting that the release of Beclin 1 from BCL-2 during starvation allows the induction of autophagy. The exact mechanism by which nutrient status regulates Beclin 1 and BCL-2 binding, however, has yet to be determined (Pattingre et al., 2005).

Through binding competition experiments and crystal structure studies, Beclin 1 has recently been identified as a novel BH3-only protein (Maiuri et al., 2007; Oberstein et al., 2007). This observation provides a structural basis for the interaction between Beclin 1 and antiapoptotic proteins BCL-2 and BCL-xL. The interaction between Beclin 1 and BCL-2 or BCL-xL is abolished when key residues within the BH3 domain of Beclin 1 or the BH3-binding groove of BCL-2/BCL-xL are mutated. These Beclin 1 mutants are more efficient at stimulating autophagy than wild-type Beclin 1, whereas the BCL-2 and BCL-xL mutants are unable to inhibit autophagy induced by Beclin 1 (Pattingre et al., 2005; Maiuri et al., 2007). It is important to note, however, that Beclin 1 does not behave like other BH3-only members of the BCL-2 family, in that it does not induce apoptosis.

The BH3 dependency of the interaction between Beclin 1 and BCL-2 suggests that another possible mechanism for its regulation is through competitive binding to the BH3 acceptor groove of BCL-2. The small molecule BH3 mimetic ABT-737, which binds and inhibits BCL-2 and BCL-xL (Oltersdorf et al., 2005), competitively inhibits the interaction between Beclin 1 and BCL-2/BCL-XL (Maiuri et al., 2007). The ability of ABT-737 to bind BCL-2 and liberate Beclin 1 results in increased Beclin 1-dependent autophagy. Moreover, ABT-737 was only able to diminish Beclin 1 binding with wild-type and ER-restricted BCL-2 and had no effect on Beclin 1 binding with mitochondrial BCL-2, further supporting the unique function of ER BCL-2 in the inhibition of autophagy (Maiuri et al., 2007).

In a physiological context, BH3-only proteins such as BAD, which is known to be activated upon growth factor withdrawal (Danial et al., 2003), may have the potential to regulate Beclin 1-dependent autophagy. A correlation was found during starvation-induced autophagy between the decreased binding of Beclin 1 and BCL-2 and the increased association of BCL-2 and BAD. Ectopic overexpression of BAD was sufficient to induce autophagy, whereas the knockdown of BAD by siRNA only partially reduced the amount of starvation-induced autophagy (Maiuri et al., 2007). This suggests that BAD is not the only factor involved in the induction of starvation-induced autophagy, and that there is a contribution from other BH3-only proteins or other BH3-independent factors as well. Further studies are needed to determine if certain signals dictate whether BH3-only proteins function exclusively as autophagy or apoptosis inducers, or whether there is cross talk between the two pathways.

The ability of BCL-2 to regulate Beclin 1-dependent autophagy through direct interaction remains a controversial topic within the field. This controversy is mainly due to the fact that the subcellular localization of endogenous Beclin 1 is unclear. In the literature, Beclin 1 has been published to localize primarily to either the trans-Golgi network (Kihara et al., 2001), the ER and mitochondria (Pattingre et al., 2005), or to uncharacterized cytoplasmic granular structures (Hoyer-Hansen et al., 2007). It was also noted that varying expression levels of BCL-2 in cells had no effect on the localization of Beclin 1 (Pattingre et al., 2005). This suggests that not all of Beclin 1 is found bound to BCL-2, which may account for the inconsistency to detect their interaction. It is possible that the interaction between ER BCL-2 and a subpopulation of Beclin 1 is sufficient to regulate and inhibit autophagy-inducing signals.

In addition, there have been other reports in the literature that propose alternative mechanisms for autophagy regulation by BCL-2 at the ER. Rises in cytosolic Ca2+ by various Ca2+ mobilizing agents were found to be sufficient in inducing Beclin 1-dependent autophagy (Hoyer-Hansen et al., 2007). ER-localized BCL-2 was able to inhibit this process by lowering ER Ca2+ levels and inhibiting Ca2+ flux from the ER. Beclin 1, on the other hand, had no effect on ER Ca2+ levels or on the levels of induced rises in cytosolic Ca2+, suggesting that ER BCL-2 inhibits Ca2+-dependent autophagy in a Beclin 1-independent manner (Hoyer-Hansen et al., 2007). As well, inhibition of the IP3R has been found to stimulate autophagy, which is also inhibitable by ER BCL-2 and BCL-xL. In contrast, autophagy regulation by IP3R is not attributed to the changes in the levels of ER or cytosolic Ca2+. BCL-2 and BCL-xL have previously been demonstrated to regulate and interact with the IP3R at the ER (Chen et al., 2004a; White et al., 2005), however, the exact mechanism of how this contributes to the regulation of IP3R-mediated autophagy has not been elucidated.

Although it is evident that the ER has a prominent function in autophagy, we still do not have a clear understanding of how the antiapoptotic proteins at the ER negatively regulate autophagy. The mechanisms of autophagy regulation may not necessarily be exclusive and may depend on cell-type specificity as well as on the type of autophagy-inducing stimulus. We also have yet to determine why the role of autophagy regulation is unique to BCL-2 at the ER membrane. The BCL-2 family is emerging as a family of proteins that have multidimensional functions within the cell. Perhaps proteins specific to the ER interact with and sequester BCL-2 to perform its anti-autophagy-specific function. These ER-specific proteins may also potentially serve as cofactors that enhance the interaction between BCL-2 and Beclin 1.

Conclusions

The ER has by now been established as an essential site for the regulation of apoptotic pathways, and has recently been recognized as an important component of autophagic signaling. With respect to apoptosis, the ER acts both as a scaffolding platform for the activation of initiator caspases and proapoptotic signaling complexes such as IRE-1/TRAF/JNK, and as an initiating point for proapoptotic Ca2+ signaling. Apoptotic stimuli as diverse as ER stress, oncogenic stress, lipid second messengers and kinase inhibitors activate many of the same ER-associated signaling molecules/pathways, including ER-localized BH3-only proteins, ER-associated initiator caspases and Ca2+ signaling. Apoptotic pathways initially thought to be specific to ER stress may therefore, in fact, be triggered by multiple stimuli. In addition, ER-derived Ca2+ signaling may frequently provide an obligatory sensitizing signal for mitochondrial cytochrome c release (Figure 1). An intriguing hypothesis, therefore, is that most apoptosis initiating signals involve an ER-related execution pathway, with the relative importance of this pathway in ultimate cell death being dependent on the cellular context.

Figure 1
figure1

ER mitochondrial cross talk: Ca2+ as a sensitizing signal for cytochrome c release. See text for details.

The BCL-2 family proteins are important regulators of apoptosis at both the ER and the mitochondria, and, in some cases, may have a similar function at the two organelles. Analogous to mitochondrial release of cytochrome c, BH3-only proteins can induce ER Ca2+ release, dependent on BAX/BAK, and associated with their activation/oligomerization (Mathai et al., 2005; Nieto-Miguel et al., 2007; Liao et al., 2008). This process can be inhibited by the overexpression of BCL-2/BCL-xL at the ER, likely due to sequestration of either BH3-only proteins or BAX/BAK (Mathai et al., 2005). How this is coupled to Ca2+, however, remains to be determined.

Although the above mechanism may function under some circumstances, BCL-2 and BCL-xL also act to inhibit apoptosis through a reduction in the strength of ER Ca2+ signaling (Rong and Distelhorst, 2008). In addition, the loss of BAX/BAK leads to reduced ER Ca2+ stores, and sensitivity to a number of apoptotic stimuli can be restored by the correction of ER Ca2+ levels, without the reintroduction of BAX/BAK (Scorrano et al., 2003). This finding indicates that it is the [Ca2+]ER level, and not the presence of BAX/BAK, that determines sensitivity to certain apoptotic stimuli, and also that the release of ER Ca2+ is, in these cases, independent of BAX/BAK. It will be interesting to determine the relative dependence of various apoptotic stimuli on BAX/BAK at the ER, as well as whether all BH3 only proteins are dependent on BAX/BAK for ER Ca2+ release. The mechanism by which BCL-2/BCL-xL regulates ER Ca2+ stores, under basal conditions as well as during apoptosis, also remains to be fully defined.

Finally, the emerging role of BCL-2 family proteins at the ER in the regulation of autophagy has raised a number of additional questions with respect to the function and regulation of these proteins. How, given the fact that autophagy (usually) promotes survival, while apoptosis leads to cell death, do BCL-2 proteins coordinately regulate the two processes? And what impact does this connection between the two pathways ultimately have on cell survival and cancer development/treatment? From a mechanistic viewpoint, both autophagy and apoptosis can be negatively regulated by BCL-2/BCL-xL at the ER, and positively regulated by BH3-only family members, but BAX/BAK do not seem to have a function in autophagy. Both processes can also be regulated by ER Ca2+ release and IP3R-dependent pathways. BCL-2 family members at the ER evidently regulate apoptosis and autophagy through divergent mechanisms, which are as yet poorly understood. Further delineation of the functions of these proteins in the two processes will therefore be extremely interesting.

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Acknowledgements

NCC is a recipient of the Canadian Institutes of Health Research Canada Graduate Scholarships Doctoral award.

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Correspondence to G C Shore.

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Heath-Engel, H., Chang, N. & Shore, G. The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene 27, 6419–6433 (2008). https://doi.org/10.1038/onc.2008.309

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Keywords

  • BH3
  • ER stress
  • calcium
  • mitochondria
  • Beclin-1

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