A trip to the ER: coping with stress

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A trip to the ER: coping with stress D. Thomas Rutkowski and Randal J. Kaufman Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI 48109-0650, USA

The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) induces a coordinated adaptive program called the unfolded protein response (UPR). The UPR alleviates stress by upregulating protein folding and degradation pathways in the ER and inhibiting protein synthesis. With a basic conceptual framework for the UPR, including the identification of key mediators of the response, now in place, recent work has turned towards investigating how the response is regulated and how its effects radiate beyond the immediate realm of protein secretion. This review highlights advances in these areas and attempts to forecast important issues that must be addressed soon. The ability of a cell to sense, respond to and circumvent stress is essential for maintaining homeostasis. There are many ways in which stress, either endogenous or exogenous, can be manifested in a cell; these include pathogenic infection, chemical insult, genetic mutation, nutrient deprivation and even normal differentiation. The process of protein folding is particularly sensitive to such insults. For the cellular compartments in which proteins fold, there are adaptive programs that enable both the detection of misfolded proteins and the correction of protein misfolding [1]. The endoplasmic reticulum (ER) is the site of synthesis, folding and modification of secretory and cell-surface proteins, as well as the resident proteins of the secretory pathway. The quality control machinery in the ER operates in conjunction with protein folding pathways and is so selective that even relatively minor perturbations in the efficiency of protein folding can cause the rejection of nascent proteins as misfolded and, consequently, the accumulation or degradation of these proteins. Thus, the ER also contains resident molecules with a primary function of sensing protein misfolding in this organelle and initiating changes in gene expression, which impact the folding capacity of the ER. The cellular response to ER stress serves as a model for the understanding of not only the mechanisms by which stress is sensed but also the ways in which the consequences of alterations in homeostasis in one location (i.e. the ER) affect diverse areas of cellular function such as gene expression, metabolism, cell signaling and apoptosis. Although a basic unfolded protein response (UPR) is conserved throughout the eukaryotic kingdom, relative to its yeast counterpart, in higher eukaryotes the response is considerably expanded in scope and consequences. Corresponding author: Randal J. Kaufman ([email protected]).

Groundbreaking research over the past few years has led to an exhaustive description of the basic pathways involved in UPR activation, signal transduction and transcriptional activation in mammals; these advances have been thoroughly reviewed elsewhere [2,3]. By contrast, much less is known about the finer details of the control and regulation of the UPR. How does the ER distinguish between different stressors? In a multifaceted response, how are the multiple components of the UPR integrated and timed? How does the cell commit to an apoptotic fate rather than a continued effort for survival? And how is the UPR managed during persistent stress, which cannot be escaped? This review highlights these emerging areas in UPR research, with an emphasis on the coordination of the UPR with other signaling pathways in the cell. Sensing the stress: a common role for BiP The UPR is initiated whenever protein folding in the ER is compromised (Figure 1). Physiological conditions that induce the UPR by causing protein misfolding include: the differentiation and development of professional secretory cells, such as plasma or pancreatic b cells; altered metabolic conditions, such as glucose deprivation, hyperhomocysteinemia and ischemia; mutations in the genes encoding secretory or transmembrane proteins, which normally fold in the ER, such as a-1 antitrypsin and insulin; and infection by certain pathogens, such as hepatitis C. The UPR can also be induced experimentally. The inhibition of N-linked glycosylation in the ER, depletion of ER calcium stores, reductive stress and the expression of mutant, and even some wild-type, secretory or transmembrane proteins all probably saturate folding pathways and clog the ER lumen [3]. To date, three ER-resident transmembrane proteins have been identified as proximal sensors of the presence of ER stress: the kinase and endoribonuclease IRE1 (a and b), the PERK kinase and the basic leucine-zipper transcription factor ATF6 (a and b). In the cases of IRE1 and PERK, which both have cytoplasmic serine/threonine kinase domains, ER stress induces lumenal-domaindriven homodimerization, autophosphorylation and activation [4 –6]. By contrast, the accumulation of unfolded proteins in the ER lumen leads to ATF6 transit to the Golgi complex, where it is cleaved by the proteases S1P and S2P, yielding a free cytoplasmic domain that is an active transcription factor [7]. The combined effects of the activation of these molecules are an upregulation of genes encoding proteins that are involved in the secretory pathway, such as ER-resident

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ER stress --Development of secretory cells --pancreatic β cells --plasma cells --Altered metabolism --glucose deprivation --hyperhomocysteinemia --Genetic mutation --DNA damage --mutated secretory proteins --Pathogenesis --polyglutamine neuropathies --viral infection --Chemical insult --inhibition of N-linked glycosylation --disruption of Ca2+ homeostasis

UPR --Translational attenuation --Transcriptional control --chaperones --degradation factors

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Figure 1. The balance of endoplasmic-reticulum (ER) stress and the unfolded protein response (UPR). Many conditions activate the UPR, all having in common the ability to induce protein misfolding in the ER. This effect could be because of the high protein load on the organelle or through the conditions that prevent the optimal operation of the ER quality-control machinery. It is thought that all of these pathways of UPR induction converge at the point of BiP association with the ER-resident stress sensors IRE1, ATF6 and PERK. Accumulated unfolded proteins titrate BiP away from associations with these sensors, freeing them for activation and the induction of the UPR. The net effect of the activation of these three molecules is to decrease the load of proteins entering the ER by attenuating translation and increasing the efflux of nascent proteins by either facilitating their folding through increased chaperone synthesis or stimulating their degradation.

chaperones and proteins involved in ER-associated protein degradation, and a downregulation of protein synthesis, reducing the influx of nascent proteins into the ER. The transcription of genes downstream of ATF6 is upregulated by the translocation of the cytoplasmic domain of ATF6 to the nucleus. IRE1-dependent transcription is upregulated when the endoribonuclease domain of the activated IRE1 molecule catalyzes the removal of a small (26-nucleotide) intron from the mRNA of the gene encoding X-box-binding protein (XBP1). This splicing event creates a translational frameshift in XBP1 mRNA to produce an active transcription factor [8– 11]. PERK activation leads to the phosphorylation of the a subunit of the translation initiation factor eIF2, which inhibits the assembly of the 80S ribosome and results in a general inhibition of protein synthesis. The activation of all three components of the UPR depends on the dissociation of the proximal signaling molecule from the abundant lumenal chaperone BiP. In nonstressed cells, ATF6 is retained in the ER lumen by an interaction with BiP [12]. BiP also associates with the lumenal domain of IRE1, possibly at multiple sites with a general hydrophobic character [13]. The same sites in IRE1 drive its dimerization in the absence of BiP. Although the exact sites of association between BiP and the lumenal domain of PERK have not yet been elucidated, the lumenal domains of PERK and IRE1 are functionally interchangeable [6], and similar to IRE1, PERK contains several http://ticb.trends.com

hydrophobic stretches in its lumenal domain that probably bind to BiP [13,14]. Importantly, the region of BiP that associates with IRE1, and presumably with PERK too, is its peptidebinding region, that is, the same region of BiP that binds to exposed hydrophobic patches in unfolded nascent proteins. Thus, a plausible model for the activation of the UPR is that at the high intralumenal concentrations of BiP (BiP is the most abundant protein in the ER lumen), the lumenal domains of IRE1, PERK and ATF6 associate more readily with BiP than with each other. However, BiP might preferentially associate with unfolded proteins instead of IRE1, PERK and ATF6, and thus the accumulation of unfolded proteins would drive the equilibrium of BiP binding away from these sensor proteins. IRE1 and PERK would then be freed for homodimerization and autophosphorylation, whereas ATF6 would be free to transit to the Golgi complex and undergo proteolytic cleavage. One prediction of this model is that there is a potential for the selective activation of only certain UPR-associated pathways, with the weakest association being titrated away more rapidly than the strongest. In general, most experimental manipulations of the UPR have involved applying high levels of stressors, which are expected to rapidly activate all three components of the UPR; thus, it is not known whether there are conditions that result in the activation of only parts of the UPR. However, in support of this model, the activation of B-cell differentiation in

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response to the UPR [15], results in the activation of the IRE1 pathway, but does not lead to an upregulation of the CHOP transcription factor [16], which is downstream of the ATF6 and PERK pathways (Box 1) [17]. Given the importance of BiP in regulating the activation of the UPR, it seems reasonable that the threshold for UPR activation could be altered by regulating the ATPase and nucleotide exchange activities of BiP. It is thought that ATP hydrolysis is needed for BiP binding to its substrates and that nucleotide exchange stimulates substrate release. The existence of co-chaperones that stimulate ATP hydrolysis or exchange by BiP, such as the recently identified BiP-associated protein (BAP) [18], might modulate BiP release under different conditions. How these proteins are expressed in response to varying environmental conditions could influence the sensitivity of the UPR.

Box 1. B-cell differentiation and the unfolded protein response (UPR) Although the UPR has been best elucidated as a cellular response to experimental perturbations that induce protein unfolding in the endoplasmic reticulum (ER), the pathway is also important for the proper development of cells which must accommodate a high load of secretory proteins. One such cell type is the B lymphoblast, which, upon antigenic stimulation, differentiates into an antibody-secreting plasma cell, a process that is accompanied by massive ER expansion and, as has been recently discovered, the induction of the UPR [15,16,58]. The induction of plasma cell differentiation by cytokines leads to the production of XBP1, a basic leucine-zipper (bZIP) transcription factor [59]. XBP1-deficient mature B cells fail to differentiate into high-level antibody-secreting plasma cells when reconstituted into RAG-2-deficient mice, which cannot make endogenous immunoglobulins [15]. Thus, XBP1 is required for the differentiation of plasma cells. XBP1 was identified as a target of the IRE1 pathway of the UPR, independent of its role in B cell-differentiation. In the yeast, Ire1p, which is the sole sensor of ER stress – no yeast homologs of ATF6 or PERK exist – induces the UPR by catalyzing the splicing of mRNA for the bZIP transcription factor Hac1p [60]. XBP1 was identified as the mammalian counterpart of HAC1 by several independent approaches; these demonstrated that XBP1 could bind to the promoters of ER-stress-responsive genes, and that IRE1 splices XBP1 mRNA to alter the reading frame of the mRNA and produce an active transcription factor [5 –8,61]. The identification of XBP1 mRNA as a target of IRE1 led to the speculation that the UPR is activated during B-cell differentiation. In support of this notion, B-cell differentiation results in the splicing of XBP1 mRNA [58]. The process also activates the ATF6 pathway of the UPR, because ATF6 cleavage and the induction of ATF6 target genes are observed; no evidence of PERK activation has been found [16]. Is the UPR required to prepare the ER of pre-B cells for massive antibody secretion, or does the initiation of massive antibody production elicit the UPR? Proteomic analysis of developing B cells has shown that an increase in the production of ER chaperones downstream of the UPR precedes, and therefore possibly anticipates, immunoglobulin production [50]. If IRE1 and ATF6 are indeed activated before massive antibody synthesis, then what is the signal that activates the UPR? Alternatively, is it possible that immunoglobulin production and UPR activation proceed in tandem, with each providing positive feedback for the other? The position of XBP1 at the convergence of B-cell differentiation and the UPR has confirmed the importance of the UPR in the development of professional secretory cells but has raised many more questions about how XBP1 is activated and regulated in these nonpathological contexts.

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Temporal control of the UPR: a distinct role for each sensor Although IRE1, PERK and ATF6 are all activated after dissociation from BiP, the signaling pathways activated by each of these sensors require unique lag times before they become fully activated; consequently, distinct parts of the UPR are regulated by each of these molecules (Figure 2). After exposure to ER stress, the pathway activated most rapidly is translational repression mediated by PERK. Because eIF2a is a direct substrate of PERK, its phosphorylation does not depend on nuclear translocation, transcription or translation. Consequently, the inhibition of protein synthesis occurs very rapidly following exposure to ER stress; for example, eIF2a phosphorylation and translational repression are complete as soon as 30 minutes after exposure to stress [19]. The immediate effect of this inhibition is to prevent further influx of nascent proteins into an already saturated ER lumen. Cleavage of ATF6 also follows fairly rapidly after exposure to stress; however, expression of the genes controlled by this sensor protein requires nuclear translocation of its cytoplasmic domain, the induction of transcription and protein synthesis. Because the genes that are specifically induced by ATF6 include most of the prominent chaperones in the ER lumen, including BiP, protein disulfide isomerase (PDI) and Grp94, the upregulation of protein folding capacity of the ER can be seen as a second stage of the UPR, following the inhibition of protein synthesis [20]. During the UPR, transcription is also upregulated by ATF4, a member of the cAMP response-element-binding (CREB) family of transcription factors. ATF4 requires eIF2a phosphorylation for its translation [21] and therefore lies downstream of PERK activation. In contrast to most proteins, ATF4 circumvents the translational block mediated by eIF2a phosphorylation because it has upstream open reading-frames (uORFs) in its 50 untranslated region (UTR) (Box 2). These uORFs, which ordinarily prevent translation of the true ATF4 ORF, are bypassed only when eIF2a is phosphorylated, allowing for ATF4 Box 2. How prevalent is translational control during endoplasmic reticulum (ER) stress? Translational regulation of ATF4 by control elements in its 50 UTR raises the possibility that other proteins are similarly upregulated at the translational level immediately after eIF2a phosphorylation. Among the possible ways through which specific proteins can escape translational attenuation are special unconventional translational mechanisms. Such mechanisms include initiation at internal ribosomal entry sites (IRESs), the reinitiation of translation and ribosome stalling [62], or in the case of the arginine –lysine transporter cat-1 mRNA, a functional coordination of an IRES and a small upstream open reading frame (uORF), both located in the 50 UTR of cat-1 [63]. By some estimates, as many as half of vertebrate mRNAs contain an IRES, an upstream initiation codon or both [64,65]. However, the presence of an IRES or other elements of interest in a 50 UTR does not guarantee that the message will escape translational inhibition by eIF2a phosphorylation. Accordingly, despite the presence of an IRES in BiP mRNA, and the evidence for translational control of BiP [66], the BiP IRES is apparently not used during ER stress [67]. In fact, ATF4 and cat-1 are the only mRNAs that are known to be translationally upregulated during ER stress [68].

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Figure 2. Distinct components of the unfolded protein response (UPR) mediated by each of the sensors resident in the endoplasmic reticulum ER. Alterations in the timing with which each pathway of the UPR can be activated, combined with positive and negative feedback loops, lead to distinct aspects of the response falling under the provenance of different sensors. The diagram shown conveys an approximate sense of timing, with events pictured farther from the ER sensing-molecules PERK, ATF6 and IRE1 taking place later than those pictured more proximal. For instance, the full activation of the IRE1 pathway requires the enhanced production of XBP1 mRNA by ATF6, resulting in an earlier induction of the transcription of genes downstream of ATF6 than those downstream of XBP1. The broken arrows represent feedback loops, both positive (green) and negative (red), known to act on the UPR. It should be emphasized that the functions of PERK, ATF6 and IRE1 are probably not as mutually exclusive as pictured here, and some genes require the action of more than one sensing pathway for their activation.

translation [21]. eIF2a phosphorylation occurs in response to not only ER stress but also other cellular stresses that lead to the activation of non-PERK eIF2a kinases such as GCN2, PKR and HRI [22]. Therefore, the genes downstream of ATF4 are generally important for the recovery from various stresses, both those originating in the ER and otherwise, whereas the targets of ATF6 are largely specific for alleviating ER stress [20,23]. IRE1 also initiates a pathway of transcriptional induction, but the full activation of this response is delayed relative to the activation of the ATF4 and ATF6 pathways [24]. This delay occurs because XBP1 mRNA, the substrate for the IRE1 nuclease domain, is expressed at fairly low levels in nonstressed cells; the synthesis of XBP1 mRNA is upregulated by ATF6 as a consequence of ER stress [11]. Thus, substantial amounts of XBP1 mRNA can only be generated after the induction of the initial transcriptional program of the UPR, ensuring that the XBP1-dependent pathway of the UPR is activated after the PERK and ATF6 pathways. Although the full complement of genes activated in response to XBP1 splicing has not been defined, these genes include the ER mannosidase http://ticb.trends.com

EDEM, which is involved in the degradation of nascent misfolded ER glycoproteins [24]. The stimulation of protein degradation might, therefore, represent a third step of the UPR, following translational attenuation and increased chaperone synthesis. Therefore, as an adaptive response, the UPR progresses through a series of increasingly stringent measures in an effort to alleviate the problem of unfolded proteins (Figure 2). One interesting facet of this coordination, which remains largely unexplored, is how the response is terminated if the cell escapes from the stress. It is not known if downstream pathways of the UPR are upregulated irrespective of stress persistence, or if the cell can truncate the UPR program rapidly following escape from stress. Positive and negative feedback loops As with any pathway of signal transduction, the UPR requires feedback mechanisms to ensure that the response is neither hyperactivated nor turned off prematurely. The two best-characterized negative feedback loops promoting repression of the PERK pathway of the UPR are mediated

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by GADD34, which is a member of the DNA-damage and growth-arrest-inducible gene family, and p58IPK, a co-chaperone. GADD34 requires the activation of PERK for its upregulation [19,25], and its expression is probably controlled by ATF4 [26]; GADD34 associates with protein phosphatase 1 (PP1) and contains an ER-localization motif [19,27]. PP1 dephosphorylates eIF2a, and GADD34 is required for the robust expression of ER chaperones in response to ER stress. How GADD34 can be translated when eIF2a is phosphorylated has not been established. p58IPK is also upregulated in response to ER stress, although, in contrast to GADD34, which is synthesized only a few hours after the induction of ER stress, the levels of this protein do not increase until much later (,12 hours after stress induction) [28,29]. p58IPK interacts with PERK and downregulates its phosphorylation and activity. Because p58IPK is expressed relatively late in the course of the UPR, and because its function is to downregulate signaling through the PERK pathway, the role of this protein might be to terminate the UPR when ER stress has been overcome. p58IPK lies downstream of IRE1 activation [30], suggesting that the control of the UPR is transferred from the early-acting PERK and ATF6 molecules to IRE1, which acts at later stages. However, the effects of stress withdrawal or, conversely, persistent stress on p58IPK expression and activity have not been examined. Although negative feedback is needed to attenuate the UPR, positive feedback is required to ensure that at least certain elements of the response continue if stress persists. Accordingly, although ATF6, together with the constitutively expressed transcription factor NF-Y, induces XBP1 expression, the transcription of XBP1 can be activated by the XBP1 protein, independent of NF-Y, possibly allowing for an autoregulatory feedback loop that maintains signaling through the IRE1 pathway even after the PERK and ATF6 pathways are downregulated [9]. In fact, the ability of the XBP1 protein to bind alone to the same promoter sites to which ATF6 and NF-Y bind together raises the possibility that, during chronic stress, the regulation of the expression of many UPR-responsive genes shifts from ATF6 to XBP1. To die or not to die: regulating the transition to apoptosis In addition to upregulating the genes that support adaptation to and recovery from ER stress, the UPR initiates proapoptotic pathways. However, although some of the molecules and mechanisms involved are identified, little is understood about how they are integrated and able to commit a cell to apoptosis. In general, the initiation of apoptosis is divided into intrinsic and extrinsic apoptotic pathways, which differ in the activation of the signaling pathways that lead to death [31]. The extrinsic pathway, exemplified by the ligation of tumor necrosis factor a receptor, leads to self-association of cell-surface receptors and the recruitment of caspases – notably caspase-8 – to the activated receptor. Caspase-8 then initiates a caspase-activation cascade leading to apoptosis. By contrast, intrinsic stimuli of apoptosis, such as DNA damage, act chiefly on the Bcl-2 family of proteins, http://ticb.trends.com

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downregulating the antiapoptotic and activating the proapoptotic members of this family. Central to this pathway is mitochondrial release of cytochrome-c, which is induced by the action of Bak, Bax and similar other proteins on the mitochondrial membrane. Cytochrome-c released into the cytosol mediates the formation of a complex between Apaf-1 and procaspase-9, leading to caspase-9 activation. In turn, the active caspase-9 activates the ‘executioner’ caspase-3 and other caspases. Although the cytochrome-c-mediated pathway is not the only proapoptotic pathway in cells, and not even the sole death pathway emanating from mitochondria, it seems to play at least a strong amplifying role, if not an absolutely essential one, in the initiation of apoptosis by most pathways. In fact, the relatively simple dichotomy of intrinsic and extrinsic apoptotic pathways obscures the considerable overlap between them. Indeed, the relative importance of cytochrome-c, Bcl-2-family proteins and caspases is currently poorly understood. It is against this backdrop that the initiation of apoptosis by ER stress further clouds a comprehensive understanding of the initiation of apoptosis. Although ER stress is better described as an intrinsic, rather than extrinsic, apoptotic signal, apoptosis induced by ER stress appears to rely on elements of both pathways (Figure 3), and thus the relative importance of either of these pathways is unclear. Surprisingly, Apaf-12/2 cells, which cannot activate caspase-9 through the release of cytochrome-c, nonetheless activate caspase-9 and caspase-3 in response to ER stress [32]. The ER-stress-specific caspase-12 can activate caspase-9 independent of cytochrome-c and Apaf1 in vitro [32]. Caspase-12 is localized at the ER membrane through an interaction with IRE1 and the adaptor protein TRAF2 [33]. ER stress dissociates TRAF2 from caspase-12, with the subsequent dimerization and activation of this caspase. Supporting a role for caspase-12 in UPRmediated apoptosis, caspase-122/2 cells are mildly resistant to apoptosis stimulated by ER stress [34]. However, it should be emphasized that these cells are not completely resistant to ER-stress-mediated apoptosis, suggesting that other pathways also contribute to apoptosis and can compensate for the absence of caspase-12. Furthermore, as well as its role in caspase-12 activation, the IRE1 – TRAF2 interaction, which is regulated by ER stress [35], also allows for the recruitment and activation of ASK1, a stress-activated MAP3K [36], and the c-jun N-terminal kinase (JNK) [36], which might interact to initiate proapoptotic phosphorylation cascades. It is with respect to signaling through the IRE1– TRAF2 complex that ERstress-induced apoptosis most closely resembles the extrinsic pathway of apoptosis, except that the receptor that initiates apoptosis recognizes a signal not outside the cell but instead in the lumen of the ER. The regulation of calcium concentrations in the ER, mitochondria and cytoplasm might represent an additional trigger of apoptosis (Figure 3). Indeed, some data suggest that calcium regulation is required even for the activation of the caspase-12 pathway, because caspase12 activation might occur through its cleavage by the calcium-dependent protease calpain [37]. The exact mechanism of calcium release from the ER during the

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Figure 3. Multiple proapoptotic pathways emanate from the endoplasmic reticulum (ER). Caspase-12 is an ER-localized caspase, which is activated by ER stress and can possibly lead to the cleavage of caspase-3, independent of mitochondrial participation. However, caspase-12 activation seems to be insufficient for ER-stress-induced apoptosis, and it is fairly clear that mitochondria participate intimately in the process. Proapoptotic signals are also sent through the induction of the CHOP protein, which is downstream of both the PERK and ATF6 pathways of the unfolded protein response (UPR). The current level of understanding in the field of ER-induced apoptosis is largely restricted to the identification of the proteins involved in the process; a consensus on the relative importance of any of them or the degree to which the various pathways overlap has not been achieved. An important question to address is how the proapoptotic signals from the UPR are balanced with the prosurvival signals to regulate the commitment to death.

UPR is not clear, although several important proteins that participate in the response have been identified. One such group of potential mediators is the BH3-only class of proteins including Bax, Bad and Bak. In response to ER stress, Bax and Bak undergo conformational changes and/ or oligomerization at the ER membrane, and ER-localized Bak leads to calcium depletion and the activation of caspase-12 in the ER [38]. Calcium release from the ER rapidly results in calcium uptake in mitochondria, through those regions of mitochondria that are closely and stably associated with the ER membrane [39]. Calcium influx into mitochondria might lead to the collapse of the mitochondrial inner-membrane potential commonly associated with apoptosis. Blocking this collapse suppresses, but does not completely abrogate, ER-stress-induced apoptosis [40]. Thus, the participation of Bcl-2-family members in ER-stress-induced apoptosis recalls the prototypical intrinsic pathway of apoptosis in these respects. Other proteins recently implicated in ER-stress-induced apoptosis include c-Abl [41], Bbc3 – PUMA [42] and BAP31 [43], although the roles of these proteins and their effects, if any, on calcium homeostasis are not clear. Bcl-2-family members counteract the effects of BH3only proteins, and their localization appears to be regulated in response to ER stress. Bcl-2 itself can localize to either mitochondria or the ER, and its permanent localization in the ER membrane can block apoptosis in http://ticb.trends.com

certain cases [44,45]. CHOP, a proapoptotic transcription factor upregulated by ATF4 and ATF6 [17], suppresses the transcription of Bcl-2 [46], suggesting a role for CHOP in regulating the transition of the UPR from prosurvival to proapoptotic signaling. CHOP2/2 cells are modestly resistant to ER-stress-induced apoptosis [46,47], although the relative importance of CHOP, or any other protein implicated in the ER-stress-mediated apoptosis, remains unclear. Despite the description of several proapoptotic pathways involved in the transition from ER stress to apoptosis, the control of ER-stress-induced apoptosis remains poorly understood. What is the required threshold for a cell to undergo apoptosis? At what point does the initiation of proapoptotic pathways become irreversible? How essential are caspase-12 cleavage, cytochrome-c release and calcium flux to this process? The fact that different forms of ER stress seem to promote different pathways of apoptosis [48] suggests that the nature of the stressor itself might be an important contributing factor in how and when apoptosis is induced. ER-stress radiates beyond the immediate realm of protein secretion The initiation of apoptosis in response to ER stress clearly involves cellular processes, in addition to those strictly involved in protein secretion, and requires interorganellar crosstalk. Signaling pathways radiate from the ER to the

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nucleus and mitochondria, and probably to other organelles as well. It is becoming apparent that, even during the prosurvival phase of the UPR, the response is far more complex than a simple program of transcriptional upregulation. A question that is now being actively pursued is how cellular function, apart from the secretory pathway, is altered during ER stress. ER stress desensitizes cells to further stresses. The induction of UPR-responsive genes persists long after the original ER stressor is withdrawn [49]. Thus, the UPR not only assists cells in coping with the immediate presence of unfolded proteins in the ER but also prepares them for further insult through long-term changes in genetic programming. This preconditioning is most vividly seen during B-cell differentiation, which requires IRE1 activation and XBP1 splicing. This differentiation is accompanied by a dramatic expansion in the ER content, which might anticipate, rather than occur as a consequence of, the massive immunoglobulin synthesis carried out in the antibody-secreting plasma cell (Box 1) [50]. Tolerance to secondary stress appears to involve the induction of chaperone expression [51,52], alteration in calcium homeostasis and the activation of phosphorylation cascades [53]. Desensitization might also be augmented by alteration of the metabolic status of the cell. The activation of the PERK pathway of the UPR upregulates the expression of the mitochondrial Lon protease, facilitating the assembly of the cytochrome-c-oxidase complex of the electron-transport chain [54]; the functional consequences of this alteration are not clear. UPR activation also upregulates genes that help with maintaining the redox balance in the cell to both offset the generation of reactive oxygen species owing to ER stress and promote cell survival [23]. Finally, PERK activation leads to cellcycle arrest [55], which might allow the cell to concentrate resources on mitigating stress rather than on growth and division. The importance of adapting to persistent ER stress is underscored by the apparent connection between ER stress in the pancreas – a tissue that experiences a high burden of protein secretion – and diabetes. Both PERK2/2 mice and mice homozygous for phosphorylation-incompetent eIF2a display impaired physiological glucose and insulin regulation, as well as overt diabetes reminiscent of Type I diabetes in humans [56,57]. Moreover, mice with a single eIF2a allele incompetent for phosphorylation develop b-cell dysfunction similar to Type II diabetes when maintained on a high-fat diet (D. Scheuner and R. J. Kaufman, unpublished). Because, in humans, Type II diabetes develops later in life, it is tempting to speculate that it is at least partially a consequence of chronic ER stress on the b cells of the pancreas, which must increase insulin secretion to compensate for the elevated glucose levels brought on by a high-fat diet and sedentary lifestyle. Concluding remarks – the future of the UPR Emphasizing the regulation of the UPR is not meant to imply that understanding the basic pathway lacks only the dotting of ‘i’s and the crossing of ‘t’s. Perhaps there are other sensors of ER stress, as well as new pathways of communication between the ER and other organelles. The http://ticb.trends.com

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emerging interplay between ER stress and the functioning of cellular pathways only tangentially related to protein secretion should also come into sharper focus over the next few years. For example, the mechanistic link between ER stress and oxidative stress is not understood. How does protein misfolding in the ER affect the formation of reactive oxygen species, thought to be generated in the mitochondria? Similarly, the connection between ER stress and diabetes interjects yet another component into the already complex dynamics of proteins involved in glucose homeostasis. The position of the UPR within that framework must still be elucidated. Finally, the pathways leading from ER stress to apoptosis, which undoubtedly underlie numerous UPR-related pathologies, must move from a phenomenological to a mechanistic understanding. Complex questions such as these will guide the field for the foreseeable future. Acknowledgements We thank X. Shen and K. Zhang of the Kaufman laboratory for critically evaluating the manuscript. We apologize to those whose work could only be cited by reference to review articles.

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Have you seen our series on ’Interdisciplinary Biology’? Articles published to date: Building bridges through collaboration – a pathway for interdisciplinary research (Editorial) Alan R. Horwitz Trends Cell Biol. (2003) 13, 2–3 Cell-biological applications of transfected-cell microarrays Randy Z. Wu, Steve N. Bailey and David M. Sabatini Trends Cell Biol. (2002) 12, 485–488 Computational modeling of the EGF-receptor system: a paradigm for systems biology H. Steven Wiley, Stanislav Y. Shvartsman and Douglas A. Lauffenburger Trends Cell Biol. 13, (2003) 43–50 Fluorescence imaging of signaling networks Tobias Meyer and Mary N. Teruel Trends Cell Biol. (2003) 13, 101–106 An array of insights: application of DNA chip technology in the study of cell biology Satchidananda Panda, Trey K. Sato, Garret M. Hampton and John B. Hogenesch Trends Cell Biol. (2003) 13, 151–156 Chemical genetics: tailoring tools for cell biology Thomas U. Mayer Trends Cell Biol. (2003) 13, 270–277 Functional genomics and proteomics: charting a multidimensional map of the yeast cell Gary D. Bader, Adrian Heilbut, Brenda Andrews, Mike Tyers, Timothy Hughes and Charles Boone Trends Cell Biol. (2003) 13, 344–356 Quantitative cell biology with the virtual cell Boris M. Slepchenko, James C. Schaff, Ian Macara and Leslie M. Loew Trends Cell Biol. (2003) 13, 570–576 Enzyme activity – it’s all about image Amos Baruch, Douglas A. Jeffery and Matthew Bogyo Trends Cell Biol. (January 2004, in press) Other reviews planned for the series: Structural genomics of biological complexes Steve Almo et al. Mathematical models applied to cell biology Martin Nowak et al. High-throughput screens to study cell morphology Norbert Perrimon et al. Computational analysis of regulatory networks in mammalian cells Ravi Iyengar et al. http://ticb.trends.com