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Physiology 22: 193-201, 2007; doi:10.1152/physiol.00050.2006
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Physiology, Vol. 22, No. 3, 193-201, June 2007
© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.

REVIEW

Endoplasmic Reticulum Stress: Signaling the Unfolded Protein Response

Elida Lai*, Tracy Teodoro* and Allen Volchuk

Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network; and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada avolchuk{at}uhnres.utoronto.ca


   Abstract
 
The endoplasmic reticulum (ER) is the cellular site of newly synthesized secretory and membrane proteins. Such proteins must be properly folded and posttranslationally modified before exit from the organelle. Proper protein folding and modification requires molecular chaperone proteins as well as an ER environment conducive for these reactions. When ER lumenal conditions are altered or chaperone capacity is overwhelmed, the cell activates signaling cascades that attempt to deal with the altered conditions and restore a favorable folding environment. Such alterations are referred to as ER stress, and the response activated is the unfolded protein response (UPR). When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is initiated. This review will examine UPR signaling that results in cell protective responses, as well as the mechanisms leading to apoptosis induction, which can lead to pathological states due to chronic ER stress.


   Introduction
Top
 Introduction
UPR Signaling
 Novel ER Stress-Signaling...
 ER Stress-Associated Apoptosis
 Conclusion
 References
 
The lumen of the endoplasmic reticulum (ER) provides an environment that is specialized for the production of secretory and membrane proteins. It has been estimated that ~30% of newly synthesized proteins are rapidly degraded, possibly as a result of improper protein folding (78, 102). Thus even an acute increase in the translation of secretory proteins would impose a major problem for the cell due to a potential buildup of misfolded proteins. The situation becomes even more critical if perturbations in the ER environment occur, such as alterations in redox state, calcium levels, or failure to posttranslationally modify secretory proteins. These ER stresses will compromise the overall ability to produce properly folded proteins, and misfolded and/or unfolded proteins will accumulate. Such perturbations can be induced pharmacologically by using toxins such as tunicamycin and thapsigargin, which inhibit protein glycosylation and disrupt ER Ca2+ levels, respectively.

In the last decade or so, an intricate molecular system for monitoring and responding to alterations in the ER protein folding environment has been uncovered and termed the unfolded protein response (UPR). This molecular transduction system monitors the protein-folding capacity of the ER and signals cell responses that attempt to maintain folding capacity and prevent a buildup of unproductive and potentially toxic protein products. In mammalian cells, the basic response includes an initial transient inhibition of protein synthesis to temporarily stop production of new proteins. This is followed by transcriptional induction of chaperone genes that promote protein folding and induction and activation of the ER-associated degradation (ERAD) system that retrotranslocates terminally misfolded proteins from the ER for proteasome-dependent degradation (46, 65). If the induction of these responses fails and misfolded proteins continue to accumulate in the ER, the UPR activates cell destructive pathways.

The field of ER stress and the UPR is a rapidly expanding area of study. This is because the UPR is a fundamental process with important physiological roles in all cells, and ER stress has been linked to several diseases. ER stress signaling pathways are also essential for the normal differentiation and/or function of secretory cells such as B lymphocytes, pancreatic beta-cells, osteoblasts, and liver cells (reviewed in Ref. 99). In addition, ER stress is associated with a variety of diseases that occur as a result of the accumulation of aggregated proteins such as neurodegenerative diseases and diabetes. Several recent and excellent reviews have appeared on ER stress and its implications to disease (37, 43, 112). This review will focus on UPR signals that result in cell survival responses and the signaling systems activated during irreversible or chronic ER stress leading to cell apoptosis.


   UPR Signaling
Top
 Introduction
 UPR Signaling
Novel ER Stress-Signaling...
 ER Stress-Associated Apoptosis
 Conclusion
 References
 
The UPR consists of three main signaling systems initiated by three prototypical ER localized stress sensors: IRE1, PERK, and ATF6 (FIGURE 1Go). In addition, recent reports have also identified other sensors/pathways that may operate in distinct cell types. Despite disparate signaling mechanisms, each signaling pathway activates transcription factors that mediate the induction of a variety of ER stress response genes. These UPR signaling pathways are examined in detail below.


Figure 1
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  		FIGURE 1. UPR signaling pathways in mammalian cells
The UPR is mediated by three ER-resident transmembrane proteins that sense ER stress and signal downstream pathways. The PERK kinase is activated by dimerization and phosphorylation. Once activated, it phosphorylates eIF2{alpha}, resulting in translation attenuation. Phosphorylated eIF2{alpha} selectively enhances translation of the ATF4 transcription factor that induces expression of UPR target genes. Activation of IRE1 by dimerization and phosphorylation causes IRE1-mediated splicing of XBP1 mRNA. Translation of spliced XBP1 mRNA produces a transcription factor that upregulates target genes via the ERSE promoter. ATF6 activation involves regulated intramembrane proteolysis. The protein translocates from the ER to the Golgi where it is proteolytically processed to release a 50-kDa transcription factor that translocates to the nucleus and binds the ERSEs of UPR target genes. All three ER-resident transmembrane proteins are thought to sense ER stress through Grp78 binding/release via their respective lumenal domains, although structural studies have also suggested that IRE1 may interact with unfolded proteins directly. The GADD34 protein, a protein phosphatase upregulated by the PERK pathway, dephosphorylates eIF2{alpha} to restore global protein synthesis.

 
The IRE1 pathway
Before the identification of ER stress signaling pathways, the presence of unfolded proteins in the ER had already been observed to induce transcription of ER chaperone genes (31, 49). Elegant genetic screens in yeast identified IRE1, a type I transmembrane protein with serine/threonine kinase activity as the ER stress sensor (11, 47) and the transcription factor HAC1 that is responsible for inducing the expression of ER stress response genes (12).

Activation of IRE1 by ER stress is typical of receptor kinase proteins, which homodimerize and transphosphorylate (80, 97). Once activated, however, a unique transduction cascade ensues. The endoribonuclease activity in the COOH-terminal domain of IRE1 catalyzes splicing of the HAC1 mRNA (12, 86). Splicing removes a 3' intron that normally inhibits translation by binding to the 5' end of the mRNA (74, 85, 86). Although HAC1 mRNA splicing can occur in the cytoplasm during ER stress (74), a recent study has demonstrated that yeast IRE1 contains nuclear localization sequences in the COOH-terminal cytoplasmic domain, which can interact with components of the yeast nuclear pore complex and target IRE1 to the inner nuclear membrane (18). This results in the COOH-terminal domain facing the interior of the nucleus where it would have access to nuclear mRNA. Once translated, HAC1 translocates into the nucleus and binds the UPR promoter element (UPRE) (31, 48, 49) to induce expression of a variety of genes required for protein folding and modification, ER-Golgi transport, and ERAD (91).

The mammalian IRE1 pathway is conserved and similar to that in yeast, although two IRE1 genes have been cloned, IRE1{alpha} and IRE1ß (90, 93). The mammalian HAC1 homolog, the X-box-binding protein 1 (XBP1) undergoes a similar IRE1-mediated mRNA splicing reaction (7, 90, 105). However, the mammalian IRE1 does not contain the nuclear localization sequences identified in yeast IRE1, and studies have shown that XBP1 mRNA splicing occurs in the cytoplasm during ER stress (1). XBP-1 once translated translocates to the nucleus and binds the ER stress-response element (ERSE) promoter in target genes (35, 104). In addition to splicing XBP1, the endoribonuclease activity of mammalian IRE1 has been shown to mediate cleavage of additional mRNAs targeted to the ER (28), as well as cleavage of the 28S ribosomal subunit (29). This suggests that IRE1 has a role in translation attenuation by degrading ER-targeted mRNA transcripts and/or the ribosomal subunits that mediate translation (28, 29).

Recently, the apoptosis-associated proteins Bax and Bak have been found to interact directly with the cytosolic domain of IRE1{alpha} , and this interaction increases in response to ER stress (26). Bax and Bak double knockout mouse embryonic fibroblasts (MEFs) have abnormal responses to ER stress and decreased XBP1 expression (26). Thus Bax and Bak are required for normal IRE1 signaling, although they are also involved in ER stress-induced apoptosis (discussed below).

How IRE1 is activated by ER stress is currently one of the most interesting and unresolved questions in the field. Early biochemical studies identified the ER resident chaperone glucose-regulated protein 78 (Grp78) as a potential indirect regulator of IRE1 activation (3). Under homeostatic conditions, Grp78 binds to the IRE1 lumenal domain rendering IRE1 monomeric and inactive (3). During ER stress, Grp78 dissociates from IRE1 to assist in protein folding, which allows IRE1 to dimerize and become activated (3, 39). Recently, crystal structures of the yeast (13) and mammalian (113) IRE1 lumenal domains have been obtained. Despite a high degree of sequence and structural similarity, different conclusions have been drawn on the mechanism of ER stress sensing and IRE1 activation. Credle et al. (13) suggest that IRE1 is directly activated by unfolded proteins, which interact with a major histocompatibility complex (MHC)-like groove in the lumenal domain that stimulates the formation of oligomers and subsequent activation of the cytoplasmic portion of the molecule. Zhou et al. (113), however, contend that the MHC-like groove is too narrow for unfolded protein binding and suggest that dimerization is the main mechanism for full IRE1 activation, which is regulated by Grp78 binding as originally proposed. Additional structural studies, particularly with interacting proteins, are needed to resolve the true molecular mechanism of IRE1 activation. Structural studies of the PERK kinase lumenal domain, which undergoes a mechanistically similar activation process (see below), will also be highly informative.

Recent studies have also identified additional physiological roles for IRE1 in mammals in addition to its role in mediating classical UPR responses. IRE17{alpha} can be activated by glucose under normal physiological conditions in pancreatic ß-cells and may contribute to proinsulin biosynthesis (38). Interestingly, this activity does not involve XBP-1 splicing, although how IRE1 activates proinsulin biosynthesis is not established. In addition, studies have shown that the IRE1 pathway is essential for B cell lymphopoiesis (71, 109). It is possible that the IRE1 pathway may have an important role in the development of other tissues since XBP-1 is essential for development of tissues such as the liver (70).

The PERK pathway
In mammalian cells, the first response to ER stress is transient global translation attenuation. This is mediated by the PERK signaling pathway. PERK is a type I ER-resident transmembrane protein that senses ER stress through its lumenal domain (3, 22, 83). Like IRE1, PERK is believed to bind the chaperone protein Grp78 under normal conditions. As unfolded proteins accumulate during ER stress, Grp78 dissociates, allowing PERK to autophosphorylate and dimerize, which induces transphosphorylation (3, 39). Once activated, PERK phosphorylates serine-51 of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), its only identified target (22, 83).

When phosphorylated, eIF2{alpha} is unable to efficiently initiate translation, leading to inhibition of global protein synthesis (66, 72). However, phosphorylated eIF2{alpha} also preferentially initiates translation of the ATF4 mRNA, which contains multiple upstream open reading frames (20, 40). The transcription factor ATF4 upregulates ER stress target genes including amino acid transporters and cellular redox control genes, among others (23). Translational recovery is mediated by the stress-induced phosphatase growth arrest and DNA damage-inducible gene 34 (GADD34), which is upregulated by ATF4 and dephosphorylates eIF2{alpha} (59, 72).

Translation attenuation is thought to be an adaptive response that helps cells survive ER stress. Evidence for this includes studies of PERK knockout and eIF2{alpha} (S51A) knock-in mice (21, 77, 110). Cells in these animals cannot regulate translational control via PERK/eIF2{alpha} . Consequently, cell types with high secretory activity, such as endocrine and exocrine pancreatic cells, are highly susceptible to ER stress and appear to undergo apoptosis. Thus the PERK pathway is required for normal physiological control of ER protein synthesis. The situation, however, is likely to be more complex. A recent report has shown that PERK may also be required for normal proliferation and differentiation of certain cell types, such as pancreatic ß-cells (111). Furthermore, eIF2{alpha} is highly phosphorylated in pancreas tissue in fasted mice and becomes dephosporylated on physiological glucose stimulation (110), suggesting that the PERK pathway may regulate ß-cell translation independent of ER stress per se. The PERK gene is also important for normal endocrine and other cell function in humans as individuals with Wolcott-Rallison syndrome, who have mutations in the Perk gene, have early onset insulin-dependent diabetes, dwarfism, and altered bone development, indicating that PERK is required for normal development of several tissues (15).

Inhibiting translational recovery by pharmacologically inhibiting eIF2{alpha} dephosphorylation protects some cells from ER stress-induced apoptosis (5), suggesting that translation attenuation can be beneficial. Transient inhibition of translation has also been suggested to contribute to the cell survival role of PERK by repressing cyclin D and p53 expression, which leads to cell cycle arrest (6, 107). In addition, PERK-mediated eIF2{alpha} phosphorylation and subsequent translation attenuation has been found to activate NF-{kappa}B as a result of inhibited I{kappa}B translation, potentially leading to cell protective gene expression changes (16, 30). However, not all cell types are protected from ER stress by preventing eIF27{alpha} dephosphorylation (10), indicating that PERK/eIF2{alpha} may have subtle cell type-specific roles in translational control.

The ATF6 pathway
Two isoforms of ATF6 exist is mammalian cells (ATF6{alpha} and ATF6ß), which have fairly ubiquitous tissue distributions (24). ATF6 pathway activation involves an unusual mechanism termed regulated intramembrane proteolysis (RIP), whereby the protein translocates from the ER to the Golgi for proteolytic processing. The ER stress-sensing mechanism of ATF6 involves dissociation of Grp78 from its lumenal domain during ER stress, similar to the mechanism proposed for PERK and IRE1 (8, 81). In the case of ATF6, Grp78 dissociation reveals two Golgi localization signals, allowing ATF6 to enter COPII vesicles and translocate to the Golgi compartment (53, 81). In addition, the redox state of the ATF6 lumenal domain has recently been shown to be important for ATF6 activation (52). Disulfide bonds in the ATF6 lumenal domain are thought to keep ATF6 inactive. On ER stress, these bonds are reduced, resulting in an increased ability of ATF6 to exit the ER. Although disulfide bond reduction is required for ATF6 activation, it is not sufficient, suggesting that both the ATF6 redox state and Grp78 binding are involved in sensing ER stress and activating ATF6 (52).

After translocating from the ER to the Golgi, the full-length 90-kDa ATF6 is proteolytically processed by two Golgi resident enzymes: site-1 protease (S1P) and site-2 protease (S2P) (82, 101). This releases a 50-kDa cytosolic basic leucine zipper (bZIP) transcription factor, which translocates into the nucleus (25, 104) and binds to several different promoter elements in ER stress response genes (32, 95, 104). ATF6 binding to some promoters requires the presence of an additional transcription factor, NF-Y, which binds at a different site than ATF6 within the ERSE (106). In addition, ATF6 transcriptional activity can be enhanced by phosphorylation (41).

Some target genes of the ATF6{alpha} pathway have been identified by microarray profiling and include genes encoding ER-resident chaperones (e.g., Grp78, Grp94, PDI, among others) (60). In addition, ATF6{alpha} has recently been shown to synergistically activate (together with the transcription factor CREBH) acute inflammatory response genes during ER stress in liver cells (108). Knockout mouse models of the ATF6 pathway have not been reported; thus the exact role of ATF6 in the UPR is not completely established. However, CHO cells defective in S2P, which are unable to proteolytically process ATF6, have constitutive stress in the ER and are unable to upregulate Grp78 in response to ER stress (54), highlighting the importance of this pathway in regulating ER homeostasis in both stressed and unstressed cells.


   Novel ER Stress-Signaling Transducers
Top
 Introduction
 UPR Signaling
 Novel ER Stress-Signaling...
ER Stress-Associated Apoptosis
 Conclusion
 References
 
Recently, other potential ER stress sensors have been identified in certain cell types. These include CREB4, CREB-H, Luman, and OASIS, which belong to the bZIP transcription factor family of proteins related to ATF6 (33, 61, 62, 68, 88, 108). They all have putative trans-membrane domains and reside in the ER of unstressed cells. Like ATF6, on ER stress they undergo RIP. These proteins, however, have significantly shorter lumenal domains than ATF6, and consequently ER stress sensing may not be regulated by Grp78 interaction.

Recent studies have examined the function of some of these putative ER stress sensors in different tissues. The OASIS protein has been implicated in mediating UPR signaling in astrocytes (33). OASIS is localized to the ER membrane and in response to ER stress translocates to the Golgi, where it undergoes proteolytic processing and translocation to the nucleus. OASIS can bind ERSE, and CRE promoter elements and can activate Grp78 chaperone transcription, but the full complement of transcriptional targets remains to be identified (51). At the mRNA level, OASIS is expressed in several tissues, suggesting a role in mediating the UPR in cells other than astrocytes (61).

CREBH is expressed exclusively in liver and has been shown to undergo translocation and proteolysis during ER stress (108). However, it appears that the main target genes of CREBH include acute inflammatory response genes (108). Future studies are required to elucidate the roles of CREB4, Luman, and OASIS in the different cell types that they are expressed in and the response genes these factors activate.


   ER Stress-Associated Apoptosis
Top
 Introduction
 UPR Signaling
 Novel ER Stress-Signaling...
 ER Stress-Associated Apoptosis
Conclusion
 References
 
All three UPR pathways contribute to inducing cell apoptosis when the cell protective changes mediated by the UPR fail to restore folding capacity. As mentioned in the introduction, ER stress-induced apoptosis may contribute to diseases associated with chronic ER stress, such as diabetes. The C/EBP homologous protein transcription factor (CHOP), c-Jun NH2-terminal kinase (JNK), and caspases have been implicated in mediating apoptotic signals in response to ER stress (FIGURE 2Go), although the mechanisms are only beginning to be delineated.


Figure 2
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  		FIGURE 2. ER stress pathways implicated in mediating cell apoptosis
I:activation of the PERK and ATF6 (not shown) pathways leads to the induction of CHOP, which downregulates the expression of the anti-apoptotic protein Bcl-2 and induces GADD34 and ERO1{alpha}. The latter promote ER stress by increasing ER protein load (via translational recovery by eIF2{alpha} dephosphorylation) and altering ER redox conditions. II:activated IRE1 binds JIK and recruits TRAF2, which leads to the activation of ASK1 and JNK. JNK phosphorylates Bcl-2 and BH3-only protein (Bim), initiating mitochondria-mediated apoptosis (not shown). III:the recruitment of TRAF2 to IRE1 also permits TRAF2 to dissociate from procaspase-12 (pCP12) residing on the cytoplasmic side of ER membrane, allowing pCP12 activation. During ER stress, Bax and Bak in the ER membrane oligomerize and allow the release of Ca2+ from the ER to the cytosol, which activates m-Calpain, which subsequently cleaves and activates pCP12. Active caspase-12 (CP12) cleaves and activates procaspase-9, which in turn activates downstream caspases, including caspase-3. In addition, Ca2+ released from the ER is taken up by the mitochondria, causing mitochondrial inner membrane depolarization and cytochrome c release into the cytoplasm. This allows the formation of the apoptosome (consisting of Apaf-1, cytochrome c, ATP, and procaspase-9), activation of procaspase-9, and subsequent downstream caspases leading to cell apoptosis.

 
CHOP/GADD153
CHOP is a bZIP-containing transcription factor that was identified as a member of the CCAAT/enhancer binding protein (C/EBP) family (73). CHOP is also known as growth-arrest and DNA-damage-inducible gene 153 (GADD153), although it is induced by ER stress more than growth arrest or DNA damage (94). During prolonged ER stress, CHOP is one of the most highly upregulated genes (20, 60). All three UPR signaling pathways are involved in inducing CHOP transcription, although the PERK pathway is essential (20, 42, 60).

Several studies have implicated CHOP in ER stress-induced apoptosis. CHOP–/– MEFs are partially resistant to ER stress and have reduced ER stress-induced apoptosis (63, 64, 114). Conversely, overexpression of CHOP promotes apoptosis in response to ER stress caused by thapsigargin and tunicamycin (45).

CHOP, being a transcription factor, is unlikely to induce apoptosis directly. However, upregulation of certain CHOP target genes, such as GADD34 and ERO1{alpha}, promote ER stress conditions. As mentioned previously, GADD34 dephosphorylates eIF2{alpha} on serine 51, leading to translational recovery. Recovery of protein synthesis will increase protein load and promote ER stress. Indeed, ER stress-induced apoptosis is reduced in CHOP–/– and GADD34 mutant cells compared with controls (44), whereas pharmacologically inhibiting eIF2{alpha} dephosphorylation protects cells from tunicamycin-induced apoptosis (5). CHOP also upregulates ERO1{alpha} expression, an ER oxidase that provides oxidizing equivalents to protein disulfide isomerase enzymes (PDIs) (44). Thus CHOP-mediated ERO1{alpha} activation causes hyperoxidizing conditions in the ER, which will increase the levels of misfolded proteins. Future studies are required to determine precisely how these CHOP-induced changes lead to apoptosis. In addition, CHOP downregulates the expression of the anti-apoptotic protein Bcl-2 (discussed below) and increases cellular reactive oxygen species, which likely contributes to ER stress-associated cell death (23, 45).

CHOP induction, however, is not the only pathway involved in mediating ER stress-induced apoptosis. CHOP is not upregulated in Perk–/– and eIF2{alpha} (S51A) mutant cells in response to ER stress, yet these cells still undergo ER stress-associated apoptosis (23, 77). In addition, disruption of the CHOP gene delays, but does not prevent, pancreatic ß-cell apoptosis in heterozygous Akita mice that express a folding-deficient insulin gene (63).

JNK
In response to ER stress, IRE1{alpha} has been found to recruit the adaptor protein TNF receptor-associated factor 2 (TRAF2) to the ER membrane (92). This recruitment is regulated by c-Jun NH2-terminal inhibitory kinase (JIK), which has been reported to interact with both IRE1{alpha} and TRAF2 (103). The IRE1{alpha}/TRAF2 complex then recruits apoptosis signal-regulating kinase 1 (ASK1), causing activation of ASK1 and the downstream JNK pathway leading to cell death (FIGURE 2Go) (57, 58). Overexpression of JIK promotes interaction between IRE1{alpha} and TRAF2 and JNK activation in response to tunicamycin, whereas overexpression of an inactive JIK mutant inhibits JNK activation (103). The importance of ASK1 in mediating ER stress-induced apoptosis has been demonstrated in ASK–/– primary neurons and MEFs, which are resistant to ER stress inducers and are defective in JNK activation and apoptosis (57). The downstream mechanism by which ASK1 and JNK lead to apoptosis is not completely clear but may involve the regulation of Bcl-2 family of proteins (discussed below).

Caspases
Caspases also participate in ER stress-induced apoptosis. In mice, procaspase-12 is localized on the cytoplasmic side of the ER and is cleaved and activated specifically by ER stress, but not by death receptor-or mitochondria-mediated apoptotic signals (55, 56). Calpains, a family of Ca2+-dependent cysteine proteases, have been shown to play a role in caspase-12 activation (55, 69), and calpain-deficient MEFs have reduced ER stress-induced caspase-12 activation and are resistant to ER stress-associated apoptosis (89). In addition, elevation of cytoplasmic Ca2+ level caused by tunicamycin and thapsigargin in primary MEFs leads to the accumulation and activation of m-calpain at the ER membrane, where it can activate caspase-12 (89).

Caspase-7, which translocates from the cytosol to the cytoplasmic side of the ER membrane in response to ER stress, has been reported to interact with and cleave caspase-12, leading to its activation (69). In this study, a dominant negative catalytic mutant of caspase-7 inhibited caspase-12 activation and cell death (69).

In addition to the role of calpains and caspase-7 in the activation of caspase-12, TRAF2 has been shown to promote the clustering of procaspase-12 at the ER membrane (103). The interaction between TRAF2 and procaspase-12 is inhibited by ER stress conditions or by overexpressing IRE1. Therefore, it has been proposed that, during ER stress, caspase-12 activation requires the dissociation of procaspase-12 from TRAF2, which may subsequently be recruited to IRE1 (103).

Although caspase-12 is activated during ER stress, the involvement of caspase-12 in ER stress-induced apoptosis is still controversial. Initial reports on caspase-12–/– mice and MEFs showed resistance to apoptosis in response to ER stress (56). In contrast, a recent study by Saleh et al. (75) reports that caspase-12–/– mice are not protected from cell death induced by ER stress. Instead, caspase-12 has been suggested to play a role in inflammation. Furthermore, Sanges et al. (76) have proposed that ER stress-induced apoptosis is mediated by calpain, but not by caspases, based on the observation that calpain inhibitors, but not a pan-caspase inhibitor, block tunicamycin and thapsigargin-induced apoptosis. Finally, humans lack functional caspase-12 due to the presence of a frameshift mutation that results in premature stop codons in the gene (17). However, Hitomi et al. (27) have proposed that caspase-4, which is homologous to mouse caspase-12, performs the function of caspase-12 in humans. Caspase-4 cleavage is specifically induced by ER stress but not by other apoptotic signals, and knockdown of caspase-4 decreases ER stress-induced apoptosis. Although studies have shown that caspase-12 can activate caspase-9 (50), the precise function and downstream targets of caspase-12 require further study.

ER stress and mitochondria-mediated apoptosis
Recent studies have linked ER stress with apoptosis mediated by mitochondrial mechanisms involving the Bcl-2 family of proteins. Bcl-2, Bax, and Bak associate not only with mitochondria membranes but also with the ER (34, 115). Several studies have shown that the Bcl-2 family of proteins have a role in mediating ER stress-induced apoptosis. MEFs lacking both Bax and Bak (Bax–/– Bak–/–) are resistant to apoptosis induced by thapsigargin, tunicamycin, and brefeldin A (96), and to tunicamycin-induced JNK phosphorylation (26). Two pathways have been proposed by which Bax and Bak may promote apoptosis in response to ER stress. During ER stress, Bax and Bak undergo conformational changes and oligomerization in the ER membrane (115). This causes the release of Ca2+ from the ER to the cytoplasm, depleting ER Ca2+ stores (79, 115). The increase in Ca2+ concentration in the cytosol activates m-calpain, which cleaves and activates procaspase-12 as mentioned previously (55). Activated caspase-12 then cleaves and activates procaspase-9, which in turn activates the downstream caspase cascade including caspase-3 (50, 89). The second pathway involves cytosolic Ca2+ being taken up by mitochondria, causing depolarization of mitochondrial inner membrane and cytochrome c release (14). Cytochrome c in the cytosol stimulates the assembly of the apoptosome, comprised of Apaf-1, procaspase-9, cytochrome c, and ATP, leading to activation of caspase-9, which in turn activates caspase-3, DNA fragmentation, and cell death (14, 84).

Conversely, overexpression of Bcl-2 and Bcl-xL reduces thapsigargin-induced JNK activation and apoptosis (87). Furthermore, overexpressing wild-type or ER-targeted Bcl-2 protects against tunicamycin effects on mitochondrial transmembrane potential and the release of cytochrome c from the mitochondria (4, 19).

How does ER stress affect Bcl-2 proteins to promote apoptosis? Several studies have provided evidence for the involvement of CHOP and JNK. Overexpression of CHOP has been shown to cause a decrease in Bcl-2 expression, which may allow for the activation of proapoptotic Bcl-2 proteins (45). Under normal conditions, Bcl-2 can bind and sequester BH3-only proteins, preventing these proteins from triggering oligomerization and activation of Bax and Bak (9, 96).

In addition, JNK, which is activated via the IRE1{alpha}/TRAF2/ASK1 pathway as described previously, can phosphorylate Bcl-2 (100). Phosphorylated Bcl-2 is inhibited from binding to and sequestering the proapoptotic proteins Bax and Bid (2). Phosphorylated Bcl-2 also enhances Ca2+ efflux from the ER and increases Ca2+ uptake by the mitochondria (2). JNK can also phosphorylate the BH3-only protein Bim (36). The proapoptotic activity of Bim is normally inhibited by binding to dynein motor complexes (67). Phosphorylation by JNK releases Bim from the complex and allows the initiation of Bax-dependent apoptotic cascade (36). Together, these results suggest that the ER stress signals to the mitochondria via the regulation of Bcl-2 proteins by both CHOP and JNK.

Most of the studies on ER-associated apoptosis have been done using toxins that cause severe ER stress. Whether these pathways also contribute to ER stress-induced cell death in more physiological systems such as in pancreatic beta-cells of the Akita mouse or other pathological states associated with chronic ER stress requires future study.


   Conclusion
Top
 Introduction
 UPR Signaling
 Novel ER Stress-Signaling...
 ER Stress-Associated Apoptosis
 Conclusion
References
 
ER stress is a potentially deleterious condition that must be counteracted by activating the UPR. The UPR system deals with ER stress conditions by sensing the ER environment and eliciting appropriate responses to various perturbations that restore ER homeostasis, as well as providing other physiological functions in various cell types (99). Although the general signaling pathways are well established, much is still not well understood, such as the complete mechanism of ER stress-induced apoptosis and the signal that sends the cell on the apoptotic route when the cell protective responses of the UPR fail to adequately restore ER homeostasis. A more complete understanding of the ER stress response is certain to provide new approaches to treating diseases that occur as a consequence of ER stress and defective protein folding (98).


   Footnotes
 
* E. Lai and T. Teodoro contributed equally to this review. Back


   References
Top
 Introduction
 UPR Signaling
 Novel ER Stress-Signaling...
 ER Stress-Associated Apoptosis
 Conclusion
 References
 

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