Cell death induced by endoplasmic reticulum ... - Wiley Online Library

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Cell death induced by endoplasmic reticulum stress ~ oz-Pinedo Raffaella Iurlaro and Cristina Mun Cell Death Regulation Group, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Spain

Keywords apoptosis; autophagy; Bcl-2 family proteins; caspase-8; cell death; death receptors; endoplasmic reticulum stress; iDISC; mitochondrial pathway; necrosis Correspondence ~oz-Pinedo, IDIBELL - Hospital Duran C. Mun i Reynals 3a planta, Gran Via de l’Hospitalet, 199, 08908 L’Hospitalet de Llobregat, Barcelona, Spain Fax: +34 93 260 7426 Tel: +34 93 260 7130 E-mail: [email protected] (Received 6 August 2015, revised 27 October 2015, accepted 11 November 2015) doi:10.1111/febs.13598

The endoplasmic reticulum is an organelle with multiple functions. The synthesis of transmembrane proteins and proteins that are to be secreted occurs in this organelle. Many conditions that impose stress on cells, including hypoxia, starvation, infections and changes in secretory needs, challenge the folding capacity of the cell and promote endoplasmic reticulum stress. The cellular response involves the activation of sensors that transduce signaling cascades with the aim of restoring homeostasis. This is known as the unfolded protein response, which also intersects with the integrated stress response that reduces protein synthesis through inactivation of the initiation factor eIF2a. Central to the unfolded protein response are the sensors PERK, IRE1 and ATF6, as well as other signaling nodes such as c-Jun Nterminal kinase 1 (JNK) and the downstream transcription factors XBP1, ATF4 and CHOP. These proteins aim to restore homeostasis, but they can also induce cell death, which has been shown to occur by necroptosis and, more commonly, through the regulation of Bcl-2 family proteins (Bim, Noxa and Puma) that leads to mitochondrial apoptosis. In addition, endoplasmic reticulum stress and proteotoxic stress have been shown to induce TRAIL receptors and activation of caspase-8. Endoplasmic reticulum stress is a common feature in the pathology of numerous diseases because it plays a role in neurodegeneration, stroke, cancer, metabolic diseases and inflammation. Understanding how cells react to endoplasmic reticulum stress can accelerate discovery of drugs against these diseases.

The endoplasmic reticulum and the unfolded protein response sensors The endoplasmic reticulum (ER) is the organelle in which transmembrane proteins and proteins that are going to be secreted are synthesized and folded. However, this organelle is essential for multiple other cellular functions such as Ca2+ buffering and the biosynthesis of phospholipids and cholesterol. The synthesis of proteins, cholesterol and some

metabolites is an ATP-demanding process that also requires the right ionic strength. Disturbances in many homeostatic processes thus lead to a state in which protein folding slows (ER stress). The subsequent accumulation of misfolded or unfolded proteins indicates problems in cellular homeostasis that frequently end in cell death.

Abbreviations ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; CHOP, C/EBP-homologous protein; DISC, death-inducing signaling complex; DR5, death receptor 5; eIF2a, eukaryotic translation initiation factor 2a; ER, endoplasmic reticulum; iDISC, intracellular DISC; IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinase 1; MEF, mouse embryonic fibroblast; PERK, protein kinase RNA-like endoplasmic reticulum kinase; RIPK1, receptor interacting protein kinase 1; ROS, reactive oxygen species; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; UPR, unfolded protein response; XBP1, X box-binding protein 1; XIAP, X-linked inhibitor of apoptosis protein.

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FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS

~oz-Pinedo R. Iurlaro and C. Mun

Problems in protein folding can occur due to stressors as apparently disparate such as starvation or viral infection. These stimuli promote the activation of a series of signals that promote the synthesis of new proteins to cope with stress, while reducing general protein synthesis [1,2]. This is called the unfolded protein response (UPR). Another way by which the UPR aims to restore homeostasis is the stimulation of protein degradation via autophagy and the enhanced clearance of unfolded proteins by a process termed ER-associated degradation (ERAD). The UPR is orchestrated by three main sensors that reside in the ER membrane. These transmembrane proteins bind to the chaperone GRP78/BiP in the ER lumen. When most of this protein is engaged (interacting with unfolded proteins), the cytosolic sides of the sensors are autoactivated by trans-phosphorylation and they transduce signals to restore homeostasis. These three main sensors and their downstream cascades are involved at different levels in cell death induced by unresolved ER stress, with most data pointing to a role for the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/activating transcription factor 4/C/ EBP-homologous protein (PERK/ATF4/CHOP) pathway (see below), although other pathways have also been shown to regulate cell survival [3]. Inositol-requiring enzyme 1 (IRE1) is conserved from yeast to mammals. This multidomain protein is a kinase and also an endoribonuclease. The RNAse activity of IRE1 can participate in RNA degradation to reduce protein synthesis, a process known as regulated IRE1-dependent mRNA decay (RIDD) [4]. However, under low-activation conditions its RNAse activity is sequence specific, being involved in the regulation of the mRNA of a few genes. Among these, the transcription factor X box-binding protein 1 (XBP1) is particularly important for cell survival. The mRNA of XBP1 is synthesized as an unspliced, untranslated form (uXBP1). When IRE1 is activated it removes a small intron resulting in the formation of spliced XBP1 (sXBP1). This splicing is commonly used as a readout of UPR activation [5] (Fig. 1). However, sXBP1 is not only a marker, but also a key regulator of the UPR because it transcriptionally activates a number of genes responsible for restoring ER folding capacity [6]. PERK, the second sensor, participates in the UPR mainly by attenuating protein translation and regulating oxidative stress. PERK phosphorylates and activates the transcription factor NRF2, which induces antioxidant proteins. Additionally, PERK phosphorylates eukaryotic translation initiation factor 2a(eIF2a). The translation initiation factor eIF2a functions in the early steps of mRNA translation. Upon phosphorylation of the alpha FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS

Cell death caused by endoplasmic reticulum stress

subunit, translation is attenuated. However, mRNAs with internal ribosomal entry sites can still be translated under these conditions. ATF4 is a transcription factor that is translated more efficiently and is induced when eIF2a is inhibited. ATF4 induces genes with roles in protein synthesis and secretion, amino acid synthesis and transport, and antioxidant stress responses [7,8]. ATF4 also induces a second transcription factor with well-known roles in the induction of cell death: C/EBPhomologous protein (CHOP/GADD153). This protein also participates in restoring homeostasis by stimulating the synthesis of GADD34, a regulatory subunit of an eIF2a-specific phosphatase complex [9]. Thus, CHOP prevents its own synthesis and that of its transcription factor ATF4, which is frequently seen to be transiently induced only after treatment with ER stressors. Importantly, eIF2a phosphorylation and the induction of ATF4 and CHOP do not only occur in response to ER stressors. Besides PERK, other kinases such as GCN2 and PKR have been shown to phosphorylate eIF2a. This eIF2a/ATF4/CHOP response is generally known as the integrated stress response, and some stimuli such as nutrient starvation can engage PERK (the UPR) and GCN2 (the integrated stress response) in parallel [10]. Activating transcription factor 6 (ATF6), the third sensor, is also a transmembrane protein, but is a transcription factor when cleaved. Although ATF6 is constitutively located in the ER, upon ER stress it translocates to the Golgi where it is cleaved to release the transcription factor [11] (Fig. 1). UPR proteins, such as chaperones, are direct targets of ATF6. Additionally, ATF6 can induce CHOP and XBP1 genes [12].

What kills downstream of IRE1, ATF6 and PERK? The IRE1 sensor is generally associated with pro-survival pathways in response to ER stress through the regulation of multiple chaperones. The mammalian genome encodes two isoforms of IRE1, IRE1a and IRE1b. IRE1a is expressed ubiquitously and has been studied more thoroughly. It has been shown that under sustained engagement, IRE1a activity can be pro-apoptotic. This protein can recruit the adapter TRAF2 activating apoptosis signal-regulating kinase 1/ MAP3K5 (ASK1) and its downstream target c-Jun Nterminal kinase 1 (JNK/MAPK8/SAPK1) [13]. JNK regulates and activates apoptotic pathways (Fig. 2) and can also participate in necrosis in response to ER stressors [14]. Thus, the JNK branch of the IRE1 pathway would promote cell death. It is currently unclear, however, whether the function of the IRE1 pathway in vivo is generally pro-death or pro-survival, and it may

2641



ER P

P

P

P

IRE1

PERK

P

ISR

Protein synthesis

ATF6

eIF2α

XBP1 mRNA

ATF4

Golgi ATF6

NRF2 XBP1s

CHOP

XBP1s

ATF4

CHOP

ATF6

NRF2

Antioxidant response, GADD34, Apoptosis

Chaperones, ERAD genes

Fig. 1. The unfolded protein response. The three main sensors of the UPR are PERK, IRE1 and ATF6. PERK phosphorylates eIF2a attenuating mRNA translation, but it specifically induces the transcription factors ATF4 and CHOP, which will regulate the expression of genes involved in the restoration of the homeostasis and GADD34, an inhibitor of eIF2a, attenuating the response. PERK phosphorylates and also activates the transcription factor NRF2, which induces antioxidant responses. This eIF2a/ATF4/CHOP response is also known as the integrated stress response and other stimuli, such as nutrient deprivation, can engage it through activation of other kinases. IRE1 is a kinase and an endonuclease that regulates the splicing of the transcription factor XBP1, resulting in the transcription of genes involved in restoring the ER folding capacity. ATF6 is translocated into the Golgi where it is cleaved to release the transcription factor that regulates chaperones expression and ER-associated degradation genes.

depend on the stimulus and its intensity. The pro-apoptotic or pro-survival role of the kinase/RNAse IRE1 may depend on an oligomerization threshold. mRNA decay mediated by IRE1 under high/chronic ER stress may promote cell death through degradation of the mRNAs of antiapoptotic proteins, thus tipping the balance towards apoptosis [15]. In this sense, it has been shown in a recent report that allosteric inhibition of IRE1a using a small molecule preserves survival of critical cells in mouse models of ER stress-induced diseases such as retinal degeneration or pancreatic b-cell destruction-mediated diabetes [16]. However, consistent with a general anti-apoptotic role of the IRE1 pathway, mRNA decay (regulated IRE1-dependent decay) has also been shown to promote survival through inhibition of certain pro-apoptotic proteins such as TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 [17]. 2642

The PERK/ATF4/CHOP route has been more widely explored and it plays a crucial role in cell death in vitro (Table 1) and in vivo. As a couple of examples, neuronal loss in response to ischemia is mediated by CHOP in mice and in primary hippocampal neurons upon hypoxia/reoxygenation [18], and so is pancreatic cell loss in mouse models of diabetes [19]. However, it should be mentioned that instances of cell death mediated by CHOP do not necessarily mean that PERK or ATF4 are involved, because CHOP is a transcriptional target of ATF4, but also of ATF6 and XBP1 [2]. By contrast, CHOP overexpression on its own is not necessarily sufficient to kill cells [8,20], although it represses expression of the promoter of Bcl-2 sensitizing cells to tunicamycin and thapsigargin [20]. However, adenoviral delivery of ATF4 was sufficient to induce apoptosis in mouse embryonic fibroFEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS



ER P

P

P PERK

P ATF6

P

IRE1

eIF2α ATF6

Protein synthesis

ASK1

Bcl-2

ATF4

P

CHOP

JNK

Bcl-XL Mcl-1

?

Noxa

p53

BIM

Ca2+

Puma BAX

BAK

Mitochondria Apoptosis Fig. 2. ER stress-induced cell death. Under ER stress, PERK is activated and phosphorylates and inactivates eIF2a. This results in the selective induction of ATF4 and its downstream proteins CHOP and Noxa, resulting in cell death. CHOP, which can also be induced by ATF6, induces Bim and inhibits Bcl-2. It is still not clear how p53 is induced under ER stress and induces Noxa and Puma, resulting in apoptosis. IRE1a can recruit TRAF2, which activates ASK1 and its downstream target JNK. JNK can induce apoptosis by inhibiting antiapoptotic proteins such as Bcl-2 and Bcl-xL.

blasts (MEFs), and this was enhanced by overexpression of CHOP. CHOP and ATF4 cooperate to induce cell death, although how this is achieved is still not completely understood. Genome-wide chromatin immunoprecipitation sequencing and mRNA expression analysis experiments indicated that both transcription factors cooperate and dimerize to induce the transcription of UPR genes, but not pro-apoptotic genes in MEFs in response to tunicamycin [8]. Besides Bcl-2 family members (see below), tibbles-related protein 3 (TBR3) has been described as being involved in cell death by the ATF4/CHOP axis, but its mode of action is still unclear [21]. FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS

Reactive oxygen species (ROS) may play a role in cell death, whether apoptotic or necrotic, mediated by the ATF4/CHOP axis. CHOP induces the transcription of endoplasmic reticulum oxidoreductin 1 (ERO1a), which results in a hyperoxidizing environment in the ER. This may participate in cell death in vitro and in vivo, because knocking down ERO1a in Caenorhabditis elegans protects animals from tunicamycin [9]. In addition, in human leukemia cells, tunicamycin has been shown to induce apoptosis in a p38-dependent manner. This pathway was activated transiently and very quickly after treatment. In this case, apoptosis was inhibited by N-acetylcysteine or glutathione [22]. Unfortunately, the authors did not

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Table 1. Cell death mode and key proteins under ER stress-inducing stimuli are reported for different cell lines.

Cell death mode

Unfolded protein response/integrated stress response mediator

Key protein

Stimulus

Caspase-9, -3, and -7, but not -8 Bax, Bak Mcl-1 (anti), Noxa

Tunicamycin, thapsigargin, brefeldin A Tunicamycin, thapsigargin, brefeldin A 2-Deoxyglucose

Puma, Noxa, Mcl-1

Tunicamycin, thapsigargin

Puma Bim

Tunicamycin, thapsigargin Tunicamycin, thapsigargin

Bim Noxa Noxa, Bim Possibly Noxa Noxa

Apoptosis Apoptosis

Bim, Puma Caspase-3 Caspase-8 Caspase-8

2-Deoxyglucose Thapsigargin Thapsigargin Bortezomib Hypericin-based photodynamic therapy Thapsigargin, tunicamycin Tunicamycin Tunicamycin Tunicamycin, Thapsigargin

Apoptosis

Caspase-8

Bortezomib/MG132

Apoptosis

Caspase-8

Bortezomib/MG132

Apoptosis Apoptosis Apoptosis

Caspase-8/FADD DR5/TRAIL-R2 DR5/TRAIL-R2

Bortezomib/MG132 Thapsigargin Zerumbone, Celecoxib

CHOP ATF3/CHOP

Apoptosis

DR5/TRAIL-R2

Tunicamycin, Thapsigargin

PERK

Apoptosis

DR5/TRAIL-R2

Thapsigargin

Apoptosis

DR5/TRAIL-R2, caspase-8 DR5/TRAIL-R2, DR4/TRAIL-R1 RIPK1, caspase-8 Ca2+

Thapsigargin

PERKATF4-CHOP CHOP

Tunicamycin, Thapsigargin

CHOP

Tunicamycin Thapsigargin Glucose deprivation

IRE1

Apoptosis

Apoptosis Apoptosis Necrosis Necrosis Autophagic cell death Necroptosis

2644

Atg5 TNFR1, RIPK1

Tunicamycin, thapsigargin, brefeldin A Tunicamycin, thapsigargin, brefeldin A

Cell type

References

MEF

Masud et al. [23]

MEF

Wei et al. [74]

ATF4

Rhabdomyosarcoma

IRE1, ATF6 (protective)

Melanoma cells

Ramırez-Peinado et al. [46] Jiang et al. [47]

CHOP

HCT116, osteosarcoma MCF7, murine renal epithelial cells, thymocytes

CHOP

ATF4 PERK

ROS Atg5, Atg7

ATF4

HCT116 HeLa Neuroblastoma, melanoma T24 bladder carcinoma MEF U937 leukemia HEK293, MCF7 CASP9/ MEFs, bax/Bak/ MEFs HEK293, MDAMB231, MCF7 Atg5+/+ and Atg5/ MEFs, FADD+/+ and FADD/ MEFs, KG-1 HeLa, H460, MEFs Carcinomas HCT 116, MEFs, SW480 (colon adenocarcinoma) MCF-10ª, hTERT RPE-1, MDA-MB231 (breast), HeLa (cervix) and MM1s (multiple myeloma) MCF10A, MDA-MB231 HCT116, SK-MES-1, KSM11, RPMI-8226 A549, H1792, H460 and H157 MEFs and HEK293T Bax, Bak(/) MEFs Rhabdomyosarcoma

Reimertz et al. [51] Puthalakath et al. [57] Zarogodna et al. [75] Futami et al. [55] Zhang et al. [54] Armstrong et al. [76] Verfaillie et al. [53] Kim et al. [42] Lim et al. [22] Tomar et al. [63] Deegan et al. [64] Pan et al. [66] Young et al. [67]

Laussmann et al. [68] Yamaguchi et al. [58] Edagawa et al. [59]

rez et al. [61] Martın-Pe

rez et al. [60] Martın-Pe Lu et al. [17] Li et al. [62]

Bax, Bak(/) MEFs

Estornes et al. [65] Janssen et al. [37] n-Annicchiarico Leo et al. [36] Ullman et al. [38]

L929sA, HEK293T

Saveljeva et al. [14]



analyze whether ROS mediate activation of p38 or vice versa, or which apoptotic pathway is engaged by ROS. It should be noted that PERK deficiency, rather than preventing cell death, promotes it, possibly because the antioxidant response coordinator NRF2 is a target of the kinase activity of PERK. PERK-deficient MEF, which are oversensitive to tunicamycin, are protected by incubation with extra cysteine, b-mercaptoethanol or desferoxamine [7].

Apoptosis or necrosis? Conditions that activate the UPR have been shown to induce mitochondrial apoptosis in numerous cell types. Apoptosis proceeds via two main pathways. In the extrinsic or death receptor pathway, caspase-8 activated by homo-oligomerization cleaves and activates caspase-3. In the mitochondrial pathway, an outer mitochondrial membrane pore regulated by Bcl-2 family proteins allows the release of cytochrome c. This activates caspase-9 in the apoptosome. Caspase-9 activates downstream, executioner caspases, caspase-3 and caspase-7. Several inducers of ER stress have been shown to require executioner caspases in multiple cell lines. For example, caspase-9 or caspase-3-deficient MEFs are considerably resistant to tunicamycin (an antibiotic that inhibits N-glycosylation), brefeldin A (inhibitor of ER–Golgi transport), thapsigargin (that inhibits the SERCA pump) and the Ca2+ ionophore A23187. In this report, the toxicity of the two latter stimuli was additionally reduced by the absence of caspase-7 [23]. The initiator caspase downstream of death receptors, caspase-8, was shown not to play a role in cell death in these cells. However, depending on the stimulus or the dose, this caspase may be required or cell death may proceed in a nonapoptotic manner (see below). X-linked inhibitor of apoptosis protein (XIAP) is an inhibitor of apoptosis protein that can inactivate caspase-9 and caspase-3. It has been shown that XIAP belongs to that select pool of mRNAs that can still be translated when eIF2a is phosphorylated and it contributes to the survival to ER stress induced by glucose deprivation [24]. However, it has also been reported that tunicamycin and thapsigargin reduce XIAP levels in a number of mammalian cell lines [25]. XIAP translation was reduced in a PERK-mediated manner, and in addition, ATF4 promoted its degradation, which may contribute to caspase activation. Thus, the role of XIAP in ER stress-induced death or survival requires clarification. Caspase-12 has been reported by many laboratories to be a mediator of ER stress-induced apoptosis. It has FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS


been shown to be cleaved during apoptosis induced by ER stressors. It should be noted, however, that cleavage of a caspase can occur downstream of other caspases and that many experiments were performed by using ‘specific caspase inhibitors’ that are not specific [26]. More recently, many articles have demonstrated that this caspase does not participate in UPR-mediated apoptosis: caspase-12 is activated downstream of Bax and Bak in the mitochondrial apoptotic pathway (see below) and MEF deficient in caspase-12 do not display different sensitivity to ER stressors [27–29]. However, caspase-12 is not present or functional in the majority of humans [30] and it has been suggested that its relative the inflammatory caspase-4 is the apical caspase in ER stress-induced apoptosis of human cells [31,32]. It remains to be determined how this inflammatory caspase functions in promoting the apoptotic pathway. Even more controversial is the role of caspase-2. It is still unclear whether this protein functions as an initiator caspase to induce apoptosis. It has been reported that caspase-2 participates in cell death by ER stressors [33]. However, in other reports, no role for this caspase in ER stress-induced apoptosis could be found [34]. Recently, a report [35] indicated that ER stress inducers, brefeldin A, thapsigargin and tunicamycin do not upregulate caspase-2 levels, or induce caspase-2-dependent apoptosis in different cell types, in contrast to a previous publication [33]. Besides a well-documented role for apoptosis, a recent study found that tunicamycin can kill in a necrotic (necroptotic) manner. This form of regulated necrosis is mediated by the enzymatic activity of RIPK1, RIPK3 and the pseudokinase MLKL. In L929, a murine fibrosarcoma cell line, tunicamycin can induce caspase-independent, death ligand-independent, tumor necrosis factor receptor 1 (TNFR1)-mediated necroptosis [14]. Intriguingly, in this system, silencing TNFR1, RIPK1 or MLKL did not prevent cell death, but induced a switch to apoptosis that was reverted by caspase inhibitors. In a recent report, our group has described that ATF4 mediates necrosis of rhabdomyosarcoma in response to glucose deprivation, a potent ER stressor [36]. In these cells, however, the mode of cell death was not necroptotic or ferroptotic. The pore on the mitochondrial outer membrane through which cytochrome c is released is controlled by Bcl-2 family proteins, many of which are regulated by the UPR (see below). In one study, Bax/Bak-deficient cells were found to be equally sensitive to thapsigargin as their wild-type counterparts [37]. Cell death was mediated by cytosolic Ca2+, but not by JNK. The involvement of Ca2+ and mitochondrial depolarization suggests that cell death in this context is necrosis medi-

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ated by the mitochondrial permeability transition pore. In a similar system, however, another group characterized this form of necrosis as autophagic cell death because it was prevented by reduction of the autophagy protein Atg5 [38].

The mitochondrial pathway Whether ATF4/CHOP- or IRE1-mediated, cell death induced by ER stressors has been widely shown to occur through the mitochondrial pathway of apoptosis [15,39]. This pathway is mediated by the opening of the mitochondrial outer membrane pore involving the Bcl-2 family members Bax and Bak [40]. Whereas antiapoptotic Bcl-2 family proteins like Mcl-1, Bcl-2 and Bcl-xL inhibit pore formation, pro-apoptotic BH3-only proteins activate Bax and Bak directly or indirectly and induce formation of the mitochondrial pore and the release of cytochrome c. It is intriguing that pro- and anti-apoptotic Bcl-2 family members have been shown to localize to the ER, an effect that may mediate Ca2+ signals [41]. In general, anti-apoptotic members reduce the free ER Ca2+ concentration, whereas Bax and Bak increase it. However, the role of ER-located Bcl-2 proteins is still not fully elucidated. Some reports indicate that Bak, Bim or Puma targeted to the ER are able to transduce apoptotic signals in response to ER stressors, whereas other reports indicate the opposite [42,43]. An additional layer of complexity was added by a report indicating that Bax and Bak can directly interact with and regulate IRE1 [44]. Mcl-1 is a short-lived protein and is tightly regulated by translation efficiency. Mcl-1 has been shown to be downregulated due to the inhibitory effect on protein synthesis of the PERK pathway in response to thapsigargin [45]. In addition, Mcl-1 levels were reduced post transcriptionally in an ATF4-dependent manner in response to the inhibitor of N-glycosylation 2-deoxyglucose [46]. Conversely, it has been shown that Mcl-1 is upregulated in response to ER stressors in cells that do not die and it is responsible for resistance of melanoma to tunicamycin and thapsigargin [47]. Bcl-2, as mentioned above, is downregulated at the transcriptional level by CHOP [20]. In addition, JNK activation through the IRE1 pathway may lead to Bcl-2 and Bcl-xL phosphorylation and their subsequent inactivation [48,49]. Taken together, these results indicate that ER stress-induced apoptosis inactivate Bcl-2 anti-apoptotic proteins in different manners to lead to cell death. Puma, Noxa and Bim are the three major BH3-only proteins involved in ER stress-induced cell death in multiple systems. It should be noted that several BH32646


only proteins may participate in apoptosis in the same cells simultaneously. For instance, MEFs knocked out for Bim or Puma show reduced cell death in response to thapsigargin or tunicamycin, whereas double-knockout MEFs are more resistant than cells deficient in each one of them [42]. Puma and Noxa are induced in a p53-dependent manner in several systems, i.e. in MEFs subjected to treatment with tunicamycin, thapsigargin or brefeldin A [50]. The mechanism of p53 activation in response to ER stress, however, requires further characterization. Puma is induced through transcriptional upregulation in a variety of human cell lines in response to tunicamycin. Moreover, HCT116 cells deficient in Puma were remarkably protected from thapsigargin [51]. In this report, induction of Puma was shown to be independent of p53, because other p53 target genes were not induced in these cells, and Puma upregulation was observed in p53-deficient osteosarcoma [51]. Noxa can be directly upregulated by ATF4 in complex with ATF3 [52]. This makes Noxa a good candidate to mediate apoptosis in many systems in which the PERK/ATF4 pathway participates in apoptosis, including cell death by starvation or treatment with the glycosylation inhibitor 2-deoxyglucose [46]. Indeed, Noxa has been shown to mediate apoptosis induced by directed production of ROS at the ER [53] and by thapsigargin in HeLa [54] and HCT116 [55]. In this study, Puma, Bid, death receptor 5 (DR5) and caspase-8 were shown to participate together with Noxa, suggesting either pathway redundancy or that caspase-8 is upstream of the mitochondrial pathway in this setting (see below). In addition, PERK-deficient MEFs, which are hypersensitive to ER stressors, also die in a Noxa-dependent manner when subjected to these stimuli [56]. Bim has been shown to be transcriptionally induced by CHOP. Importantly, reduction of Bim using siRNA protects several human and murine cell types from ER stress [57]. In this study, it was also reported that bim/ thymocytes treated with tunicamycin or thapsigargin survived significantly better than their wild-type counterparts. In another report, it was shown that silencing Bim and Noxa, but not other eight BH3-only proteins prevented cell death in HeLa cells treated with thapsigargin [54]. All these data suggest that the involvement of specific BH3-only proteins is cell type and stimulus specific.

ER stress induces death receptor expression to mediate cell death ER stress-induced cell death can also occur through the induction of the extrinsic pathway of apoptosis. In FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS


this pathway, interaction of death ligands (TRAIL, TNF or Fas ligand) with their receptors (TRAIL-R1/ DR4, TRAIL-R2/DR5, TNFR1, Fas) recruits the adapter protein FADD and activates caspase-8 leading to apoptosis. It was shown that ER stress induced by thapsigargin results in cell death due to the upregulation of TRAIL receptor 2 (TRAIL-R2/DR5) in human carcinoma cell lines [58]. In this setting, silencing DR5 blocks the conformational change of Bax, activation of caspase-3 and the resulting cell death. A CHOP-binding site was found on the 50 -flanking region of the DR5 gene. In addition to CHOP, the stress response gene ATF3 has been shown to be required for ER stress-mediated DR5 induction upon treatment with zerumbone (an anti-proliferative sesquiterpene) or celecoxib (a selective inhibitor of cyclooxygenase 2) in human p53-deficient colorectal cancer cells [59]. Both agents activated PERK and induced the expression of ATF4 and CHOP, which was remarkably suppressed by reactive oxygen species scavengers. Cotreatment with any of these drugs sensitized cells to apoptosis induced by TRAIL, showing that combining TRAIL treatment with ER stress inducers can be used as a suitable tool to kill tumor cells. Along the same lines, it was recently described that constitutively active ERBB2 (HER2/Neu) sensitizes human breast epithelial cells to thapsigargin treatment resulting in cell death due to an altered unfolded protein response [60]. Mutant ERBB2 triggers a deregulation of ERK, AKT and the mTOR pathway, which results in UPRdependent apoptosis in those cell lines. It was shown that PERK–ATF4–CHOP pathway induces the upregulation of DR5 receptor and TRAIL-independent activation of caspase-8. The same group previously showed that thapsigargin and tunicamycin treatment sensitizes both nontransformed and transformed human cell lines to TRAIL-dependent apoptosis [61]. Also in these models, the ER stressors were shown to promote the upregulation of DR5 that was dependent on PERK, but independent of CHOP and IRE1a. More recently, it was shown that thapsigargin induces apoptosis through UPR-dependent activation of DR5 in different cell lines [17]. In this study, ER stress induced CHOP-dependent DR5 upregulation, whereas the sensor IRE1a induced DR5 mRNA decay allowing cells to adapt to the stress. Under reversible ER stress, CHOP induces, whereas regulated IRE1-dependent decay suppresses DR5 transcription. If the ER stress is resolved, then the DR5 transcripts return to basal levels. Intriguingly, authors described that under persistent ER stress conditions, DR5 accumulated in the ER or at the Golgi apparatus depending on the cell type, resulting in DR5 activation independently of TRAIL. FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS


Activation of the death receptor TRAIL-R1/DR4 has also been associated with ER stress [62]. It was shown that CHOP interacts with the phosphorylated form of the transcription factor JUN in a complex that binds to the AP-1 binding site within the DR4 promoter region. It was also observed that GCN5, a histone acetyltransferase, physically interacts with the N-terminal region of CHOP and together with phospho-JUN forms a complex regulating DR4 and DR5 promoters. In addition, the authors confirmed that DR4 mediated apoptosis triggered by thapsigargin and tunicamycin in human lung cancer cells. In summary, several reports indicate that ER stress promotes the induction of TRAIL receptors that may result in cell death.

Persistent ER stress induces apical caspase-8 activation on an autophagome-associated platform: the iDISC/stressosome Recently, a new platform inducing cell death upon treatment with ER stressors has been described (Fig. 3). Different groups have shown that persistent ER stress or the related proteotoxic stress can activate caspase-8 independently of death ligands on an intracellular autophagosomal platform. Although autophagy is a homeostatic process that helps degrade misfolded proteins and alleviates ER stress, it can also participate in cell death. Under an ER stress scenario, K63 polyubiquitinated caspase-8 molecules were shown to be recruited by sequestosome 1/p62 and LC3, two major regulators of autophagy, leading to recruitment of caspase-8, FADD and Atg5 on autophagosomal membranes. This intracellular death-inducing signaling complex (iDISC) or stressosome has been described upon tunicamycin treatment in human colon tumor cells and in breast cancer cell lines and it results in caspase-8-dependent cell death [63]. In this model, TRIM13, a RING-E3 ubiquitin ligase, polyubiquitinates caspase-8 on K63, which stabilizes it and is responsible for its activation during ER stress. TRIM13 was also shown to be necessary for the translocation of caspase-8 to autophagosomal membranes upon ER stress stimuli, where it interacts with p62 and the lipidated form of LC3. Moreover, it was recently shown that ER stressors such as tunicamycin and thapsigargin induce, in cells lacking caspase-9 or Bax and Bak, apoptotic cell death depending on caspase-8 as the apical caspase [64]. Knockdown of autophagic genes such as Atg5 and Atg7 protected from caspase-8-dependent cell death. They identified a protein complex composed of Atg5, FADD, and pro-cas-

2647



ER

P

P

?

ER stress

PERK eIF2α

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Autophagosomal membranes

Autophagy ATF4

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LC3 FADD

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RIPK3

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Ub Ub Ub Necroptosis ATF3

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Nucleus Apoptosis Fig. 3. Persistent ER stress can result in cell death through caspase-8 recruitment and activation at iDISC. PERK can induce, through phosphorylation of eIF2a, ATF4 expression, which results in CHOP and ATF3 expression. These two proteins have been shown to bind the promoter of DR5 and DR4 genes and upregulate their expression thus promoting caspase-8 activation and cell death. Autophagy can be induced upon ER stress condition and a death platform on autophagosomal membranes can be formed upon ubiquitination (Ub) of caspase-8 and its recruitment by p62 on this platform. A complex of LC3, Atg5, FADD, p62 and caspase-8 has been described under ER stress on autophagosomal membranes, resulting in oligomerization and full activation of caspase-8. ER stress can also induce necroptosis through activation of the RIPK1–RIPK3–MLKL pathway.

pase-8 whose assembly coincides with caspase activation and cell death induction. In addition, a death receptor-independent, caspase8-mediated form of apoptosis has been reported under persistent ER stress [65]. It was described that receptor interacting protein kinase 1 (RIPK1) acts as a regulator of tunicamycin-induced cell death because RIPK1deficient MEFs were protected from death. RIPK1 was found to function upstream of caspase-8 activation, but this effect was independent of its kinase activity. Caspase-8 was not found in complex with RIPK1 like in the canonical DISC formed upon TNFR1 stimulation. The authors hypothesized that RIPK1 regulates caspase-8 activity indirectly through its interaction with IRE1, although in a JNK-independent manner. 2648

Different groups have shown that not only ER stress, but also proteotoxicity due to autophagy or proteasome inhibition can result in iDISC/stressosome formation and subsequent cell death. An iDISC on an autophagosomal membrane platform has been described after proteasome inhibition with MG132 in breast cancer cells and in human and mouse kidney cells, formed independently of death ligands and death receptors [66]. In this study, it has been shown that this treatment induced oligomerization and activation of caspase-8 in the cytosolic side of intracellular membranes. LC3 and p62 were found to interact with endogenous caspase-8 in these membranes and the ubiquitination of caspase-8 was essential for its full activation in this platform. Proteasome inhibition using bortezomib has also been shown to FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS


induce apoptosis upon the formation of a complex of caspase-8 with FADD, p62, LC3 and Atg5, independently of death ligands, [67] and of caspase-8 with FADD and Atg5 [68]. This last report described how cell death in Bax- and Bak-deficient MEFs and HeLa cells was not dependent on death ligands, but was dependent on FADD. Thus, proteasome inhibition, by leading to impairment in the degradation of misfolded or unwanted proteins, can trigger ER stress and cell death. Both proteasome inhibition and ER stress inducers can promote the formation of an autophagosomal membrane platform acting as a scaffold for recruitment and activation of caspase-8 to induce apoptosis.

Conclusions and perspectives ER stress and its downstream cell death pathways can be engaged by many stimuli that alter homeostatic cellular function. Indeed, there is probably no single stimulus that may be regarded as a ‘pure’ ER stressor. For this reason, reports indicating that ‘ER stress’ induces necrosis or apoptosis should be read with caution. Perhaps the challenges ahead lie in understanding which signaling pathways are engaged upon pathological ER stress and are more susceptible to pharmacological treatment. One caveat of in vitro versus in vivo studies is that acute stressors employed in vitro usually damage the ER irreversibly, which may not represent the closest scenario mimicking chronically stressed cells in vivo. However, they may indeed be close to certain pathological situations such as ischemia or viral infections. One subject that requires extensive work is the determination of pro-survival versus pro-death roles of kinases involved in sensing ER stress. Currently, numerous compounds are being developed against the three sensors and JNK kinase. However, we do not fully understand what determines that a particular sensor behaves as a pro- or anti-apoptotic signal [1]. Mild ER stress usually triggers adaptation rather than cell death, possibly because mRNA decay (regulated IRE1-dependent decay) preferentially targets proapoptotic proteins with a shorter half-life [69]. Many studies have also suggested that a switch between the generally ‘pro-survival’ IRE1 pathway and the generally ‘pro-death’ PERK/ATF4 pathway determines ‘life versus death’ responses. However, a recent study suggests that it is the relative timing of these responses that determines the fate of individual cells [70]. It is also intriguing that downstream of individual sensors such as PERK or IRE1, many different survival/adaptation or death responses are engaged in the same FEBS Journal 283 (2016) 2640–2652 ª 2015 FEBS


population. Particularly interesting is the role of the eIF2a/ATF4 pathway, which has been shown to be pro- or anti-apoptotic in studies of cells undergoing starvation [36,46,71–73], possibly reflecting the fact that this pathway can be engaged not only by PERK and ER stress, but also upon activation of other kinases. Many more studies are required to understand what determines that a cell adapts to ER stress or it is lost.

Acknowledgements We apologize for not citing many articles that have contributed to elucidate the mechanisms of ER stressinduced cell death. Clara Le on-Annicchiarico and Dıdac Domınguez are acknowledged for discussions and help with figures. Work in CMP’s laboratory related to this topic is funded by grant PI13/00139 from Instituto de Salud Carlos III, adscribed to Ministerio de Economıa y Competitividad of Spain and supported by Fondo Europeo de Desarrollo Regional (FEDER). RI is supported by a fellowship of SUR of the ECO of the Government of Catalonia.

Author contributions RI and CMP conceived and wrote the article.

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