Endoplasmic Reticulum Stress Signaling in Disease

Endoplasmic Reticulum Stress Signaling in Disease

STEFAN J. MARCINIAK AND DAVID RON

Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; and

Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York

I. Introduction 1133A. Synthesis, folding, and misfolding of ER protein 1134B. ER-associated degradation 1134C. The UPR 1135

II. Endoplasmic Reticulum Homeostasis 1135A. Short-term perturbations 1135B. Long-term change 1140

III. Concluding Remarks 1143

Marciniak, Stefan J., and David Ron. Endoplasmic Reticulum Stress Signaling in Disease. Physiol Rev 86:1133–1149, 2006; doi:10.1152/physrev.00015.2006.—The extracellular space is an environment hostile to unmodifiedpolypeptides. For this reason, many eukaryotic proteins destined for exposure to this environment through secretionor display at the cell surface require maturation steps within a specialized organelle, the endoplasmic reticulum(ER). A complex homeostatic mechanism, known as the unfolded protein response (UPR), has evolved to link theload of newly synthesized proteins with the capacity of the ER to mature them. It has become apparent thatdysfunction of the UPR plays an important role in some human diseases, especially those involving tissues dedicatedto extracellular protein synthesis. Diabetes mellitus is an example of such a disease, since the demands forconstantly varying levels of insulin synthesis make pancreatic �-cells dependent on efficient UPR signaling.Furthermore, recent discoveries in this field indicate that the importance of the UPR in diabetes is not restricted tothe �-cell but is also involved in peripheral insulin resistance. This review addresses aspects of the UPR currentlyunderstood to be involved in human disease, including their role in diabetes mellitus, atherosclerosis, and neoplasia.

I. INTRODUCTION

The success of multicellular organisms owes much tothe efficiency gained from the cooperation between spe-cialized cell types. This cooperation requires communica-tion between distant components frequently achieved bythe binding of molecules from one cell to receptors onanother. Such information transfer requires proteins to besynthesized that can withstand the harsh extracellularenvironment, both as soluble ligands (hormones andtransmitters) and cell surface molecules (receptors andadhesion molecules).

The extracellular compartment differs sufficientlyfrom the cytosol that proteins destined for secretion orinsertion into the plasma membrane require modificationsinappropriate to the cytosol, such as glycosylation anddisulfide bond formation. Generation of these modifica-tions necessitates a compartment topologically distinctfrom the cytosol, which is provided by a membranousnetwork called the endoplasmic reticulum (ER). With

evolution of this compartment, eukaryotes internalized aportion of the extracellular space in which they modify,fold, and assemble secreted and membrane proteins. Theevolution of eukaryotes also created the challenge ofregulating a protein maturation machinery outside theconfines of the cytosol. Failure of this machinery to foldnewly synthesized endoplasmic reticulum “client” pro-teins presents unique dangers to the cell and is termed“ER stress.” Early in evolution, a homeostatic mechanismdeveloped to maintain the balance between the demandfor ER function and ER synthetic capacity; furthermore,as organisms became more complex, especially with theappearance of long-lived professional secretory cells, theimportance and complexity of this machinery increasedgreatly.

This review addresses the mechanisms that enablehigher metazoans to produce extracellular proteins in theface of varying demand and the consequences shouldthese mechanisms fail. In the first section, a brief over-view of the ER protein maturation machinery is pre-

Physiol Rev 86: 1133–1149, 2006;doi:10.1152/physrev.00015.2006.

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sented, along with some of the consequences of proteinmisfolding. Thereafter, the response to short-term pertur-bations in ER function is discussed, with emphasis givento human diseases caused by dysregulation of this re-sponse, followed finally by an examination of the impor-tance of ER stress signaling over the longer term tosecretory cell differentiation and lipid metabolism.

A. Synthesis, Folding, and Misfolding

of ER Protein

Translation of membrane and extracellular proteinsis performed by ribosomes on the cytosolic surface of theER (3, 94). A signal recognition particle within the cytosolcotranslationally recognizes a signal sequence within thenascent polypeptide chain and directs it to a protein-aceous pore in the ER membrane, the Sec61 complex (92,127, 149).

Proteins achieve a specific folded conformation firstby acquisition of secondary structure, e.g., helices, due tothe intrinsic properties of the amino acid sequence (39).This is followed by a further search for native structure,which involves diffusion of these preformed segmentsand assembly into larger modules (84). These processesentail the burial of amino acid side chains into a close-packed structure excluding water from the protein’s core(25). “Misfolding,” in this context, indicates that a proteinpersistently maintains the potential for nonnative interac-tions that interfere with its structure and function. Thesemay involve normally buried residues being exposed tothe solvent promoting illegitimate interactions with othercellular components. Another consequence of aberrantfolding is aggregation of proteins into insoluble higherorder structures. These may be of three varieties, all ofwhich may cause disease: disordered aggregates (e.g.,rhodopsin in autosomal retinitis pigmentosa, Ref. 163),amyloid fibrils (e.g., amyloid �-peptide/tau in Alzheimer’sdisease, Refs. 89, 148), and nonamyloid fibrils (e.g., �1-antitryspin in �1-antitrypsin deficiency, Ref. 110).

Disordered aggregates are complex insoluble accu-mulations of protein resistant to normal degradation,while both amyloid and nonamyloid fibrils are simplenoncovalent polymers, one-dimensional crystals formedfrom repeating subunits. Nonamyloid fibrils are charac-teristic of the serpinopathies, human diseases caused bymutations within the serpin family of proteins (111); thesemutations allow a specific intermolecular interaction re-sulting in polymerization. In contrast, amyloid can beformed through polymerization of a number of otherwiseunrelated proteins, each causing a distinct disease (79,148, 207). Despite intensive study of these aggregates,surprisingly little is certain about the mechanism of theirtoxicity; however, it is becoming apparent that even in thecase of amyloidoses, in which extracellular deposits are

characteristic, some of the deleterious effect comes fromtoxic gain of function due to protein oligomers within thecell (227).

The inappropriate interactions of misfolded proteinsthreaten to disrupt normal cellular function, so the bio-synthetic machinery has evolved sophisticated mecha-nisms to minimize their occurrence. Within the ER, chap-erones such as BiP bind to incompletely folded proteinsshielding them from other molecules (13, 64, 65, 126, 200).In addition, other catalysts directly promote correct fold-ing by the addition of carbohydrates (63), cis-trans

isomerization about peptide bonds (166), and the creationand rearrangement of disulfide bonds (189).

B. ER-Associated Degradation

Some proteins never attain their correct conforma-tion, perhaps due to a mutation impeding correct foldingor because the cell lacks the energy to drive sufficientcycles of chaperone interaction. After a lag period of30–90 min, misfolded ER client proteins are disposed ofby ER-associated degradation (ERAD) (107). The timerthat dictates this delay, at least for misfolded glycopro-teins, is likely to be ER mannosidase I, which operates bytrimming mannose residues from N-linked glycans (38,76). Evidence that this regulates degradation comes fromits inhibition, which retards ERAD (115, 186), and from itsoverexpression, which enhances ERAD (72). Recently,EDEM, a stress-inducible catalytically inactive mannosi-dase homolog, has been shown to interact with misfoldedglycoproteins and effect their extraction from the foldingcycle (43, 73, 122).

The path by which unfolded proteins are retrotrans-located into the cytosol is not clear. Some evidence sup-ports a role for the original Sec61 translocon complex(150, 228), although a new complex containing derlin-1and p97, a cytosolic ATPase, has been shown to be in-volved in retrotranslocation of major histocompatibilitycomplex (MHC) class I molecules (106, 219). Once withinthe cytosol, ERAD substrates are degraded by the ubiq-uitin/proteosome pathway (49, 51, 151).

For these synthetic and quality assurance processesto proceed efficiently, there needs to be coordinationbetween the input load of unfolded client proteins and thematuration machinery of the ER. If the client protein loadis excessive compared with the reserve of ER chaperones,the cell is said to be experiencing “ER stress.” If un-checked, ER stress threatens to overwhelm the process-ing capacity of the ER, leading to the accumulation ofunfolded proteins and collapse of the secretory pathway.Consequently, signaling pathways have evolved that re-spond to ER stress by regulating processes on both sidesof the ER membrane through an adaptive mechanismtermed the unfolded protein response (UPR).

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C. The UPR

The nature of ER stress encountered by a cell dic-tates the nature of its UPR. During normal function asecretory cell will experience dramatic variations in theflux of new proteins through its ER in response tochanges in demand. During a transition from low proteinsynthesis to high, there is a need to increase ER protein-folding capacity to avoid overloading of chaperones. Con-sequently, the UPR regulates transcription factors whosetargets include genes for components of the ER proteinmaturation machinery. However, inherent in this re-sponse is a temporal delay, since new chaperones cannotbe made instantly. To avoid the accumulation of mis-folded client protein during this hiatus, the rate of secre-tory protein synthesis must also be under UPR-directedmodulation through a more rapid transcription-indepen-dent mechanism. Furthermore, when protein-folding effi-ciency falls, for instance, in ischemic tissue when lack ofenergy impedes the proper function of ER chaperonesand enzymes, the primary adaptation may be throughreduced client synthesis rather than by increasing thelevels of this machinery, which itself would be costly inenergy. Therefore, both transcriptional and translationalsignals emanate from the stressed ER to allow a coordi-nated response.

Not all changes in ER protein flux are transitory norcan all perturbations be survived. In some cases thechange in ER protein synthesis accompanies a change incellular phenotype, such as during differentiation into asecretory cell. Here the prolonged activation of UPR sig-naling itself appears to play a role in the differentiationprocess, leading to dramatic alterations in ER structure.When the level of ER stress is too great to allow adapta-tion, the cell may die. It remains unclear to what extentthe UPR plays a role directly in cell death, but studies ofER stress lethality have revealed novel potential thera-peutic targets for intervention in human disease.

These concepts will be elaborated upon in more de-tail below with emphasis on the human diseases to whichthey are most relevant.

II. ENDOPLASMIC RETICULUM HOMEOSTASIS

A. Short-Term Perturbations

Tissues whose primary function is the secretion ofprotein should depend most strongly on the UPR, espe-cially those liable to make large changes in ER clientprotein load. A good example is the pancreatic �-cell,which produces insulin in response to changes in circu-lating glucose. It constantly executes changes in ER syn-thetic capacity to track current glucose levels. Althoughthe precise mechanisms remain unclear, it has been es-

tablished that upswings in glucose, at least in the shortterm, promote proinsulin synthesis through enhancedprotein translation (75, 203). This stimulation of proinsu-lin translation by glucose is specific to the �-cell anddependent on cis-acting elements in untranslated regionsof proinsulin mRNA (206). The resultant large fluctuationsin ER client protein load are compensated for by the UPR,but render the �-cell highly vulnerable to defects in ERstress signaling in animal models (54) and in human dis-ease (34).

1. Regulating client protein load

Clues to the homeostatic importance of regulatingnew protein synthesis in response to changes in ER loadhave come from a rare human disease. Wolcott-Rallisonsyndrome is an autosomal recessive condition character-ized by early development of diabetes mellitus with asso-ciated bone, liver, renal, and neuronal defects (209). It hasbeen mapped to 2p12 where candidate gene analysisyielded mutations in EIF2AK3 (34), better known as PKR-like eukaryotic initiation factor 2 (eIF2�) kinase (PERK)(56) or pancreatic eIF2� kinase (PEK) (171).

PERK is an ER-resident transmembrane protein ubiq-uitously expressed, but highly enriched in professionalsecretory cells (56, 170, 171, 175). Its cytosolic portion ishighly homologous to a yeast stress-responsive kinase,Gcn2p (56, 170). Unlike PERK, Gcn2p is a soluble proteinthat enables yeast to adapt their rate of new proteinsynthesis to the levels of available amino acids (125, 190,191, 208). When amino acids are limiting, Gcn2p phos-phorylates the �-subunit of eIF2�.

The eIF2 complex is essential in all eukaryotes fornew protein synthesis, since it recruits the initiator me-thionyl tRNA to ribosomes about to begin translation (69).Phosphorylation of eIF2� inhibits this activity and thusglobally reduces protein translation. In metazoans, theGCN2 gene has expanded into a family of related eIF2�kinases, all of which inhibit protein translation in re-sponse to stress. These kinases all possess homologouscatalytic domains but have different stress-sensitive reg-ulatory domains. GCN2 persists to respond to amino acidstarvation, while new members sense disparate stressesincluding viral infection (PKR and PKZ) and iron defi-ciency (HRI) (9, 10, 23, 44, 159). The existence of PERK,an ER transmembrane eIF2� kinase, led to the apprecia-tion that cells are able to adjust their level of new proteinsynthesis to match their ER folding capacity and preventoverload of the secretory pathway (55, 56).

To respond to ER stress, PERK must transduce aluminal signal across the ER membrane to its cytosolickinase domain. The nature of this stimulus is intriguing.The ER is a site of secretory protein maturation and sowill, by necessity, harbor incompletely folded and activelyfolding protein intermediates. The level of these is a reg-

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ister of client protein throughput, rather than an indicatorof the ER’s capacity to fold them. A homeostatic mecha-nism that responded solely to unfolded protein could not,therefore, measure the efficiency of ER protein biosyn-thesis, but, instead, only respond to the absolute clientload. Evolution appears to have found a more subtlesolution: PERK responds not to the level of misfoldedprotein within the ER, but instead to changes in the ERchaperone reserve, that is, to the unfolded/misfolded pro-tein-to-chaperone ratio. BiP is an abundant chaperonewithin the ER that binds to folding proteins through in-teraction with exposed hydrophobic residues. BiP over-expression has long been known to suppress the UPR (41,197). Two potentially complementary models have beenproposed for the mechanism(s) involved. During un-stressed conditions, BiP also binds to the luminal domainof PERK (12, 108). This interaction correlates with theinactive, monomeric state of PERK. When an increase inER client load is experienced, BiP dissociates from PERK,perhaps through sequestration to unfolded clients or bymore direct mechanisms. The dissociation of the PERK-BiP complex is hypothesized to allow PERK to cluster inthe plane of the membrane, leading to activation of thecytosolic kinase domain through a process of trans-auto-phosphorylation and a dramatic increase in affinity to-wards eIF2� (12, 116).

Recently, crystal structure data on IRE1, whose lu-minal domain is homologous to that of PERK, raise thepossibility that direct binding of sensing molecules tounfolded proteins might also take place (32). Dimeriza-tion of the luminal portion of IRE1 appears to generate agroove similar to that found in the peptide-binding pocketof MHC molecules. If the groove formed by dimerizationof IRE1 can be shown to bind unfolded proteins, thiswould suggest an alternative model whereby unfoldeddomains that are unbuffered by chaperones signal di-rectly in the unfolded protein response, perhaps combin-ing direct elements (unfolded protein) and indirect ele-ments (chaperone reserve).

Humans with this Wolcott-Rallison syndrome sharesome clinical features with PERK�/� mice, which areborn with essentially normal islets that are capable ofnormal insulin synthesis and secretion (54). Indeed, whenisolated islets from prediabetic PERK�/� mice are chal-lenged with glucose, their secretion of insulin is greaterthan wild-type controls. During the first few weeks of lifethese pups develop overt diabetes due to progressive�-cell destruction. These findings indicate that PERK isessential for �-cells to cope with normal physiological ERstress arising from day-to-day insulin synthesis. This in-volves restraining the insulin synthetic response to glu-cose by attenuating new protein translation. In otherwords, protein synthesis is limited in normal animals byeIF2� phosphorylation to a level with which the existingER machinery can cope; in the PERK�/� animals and

Wolcott-Rallison patients, new proteins continue to enterthe ER regardless of its ability to fold them. This mani-fests as the accumulation of misfolded products (109) andexcessive stress signaling and culminates in cell death(54). Interestingly, individual mutations of the otherknown eIF2� kinases (PKR, HRI, and GCN2) do not resultin mice with diabetes, indicating that only eIF2� phos-phorylation in response to ER stress is involved in thisphenotype (52, 217, 226).

The importance of translational regulation in re-sponse to ER stress may not be restricted to rare geneticdiseases, as a recent study, using transgenic mice subtlyimpaired in their ability to phosphorylate eIF2� has re-vealed a phenotype remarkably similar to that of humantype II diabetes. PERK and all eIF2� kinases phosphory-late a single residue on their substrate: serine-51. Muta-tion of this serine to an alanine (eIF2�S51A) prevents itsphosphorylation in response to any stress. HeterozygouseIF2�S51A mice are capable of significant translationalregulation by phosphorylation of the remaining popula-tion of eIF2� molecules and are born with normal pan-creatic islets and under normal conditions do not developdiabetes (164). In contrast, eIF2�S51A homozygotes areborn with severe �-cell deficiency by late embryonic stage(165). However, when heterozygous mice are fed a high-fat diet, a new and interesting phenotype is revealed(164). These mice are more prone to obesity because of afailure to increase energy expenditure in response to theexcess calories. Remarkably, they more rapidly exhibithyperleptinemia, mild hyperinsulinemia, and raised fast-ing glucose, features of the “metabolic syndrome” cur-rently dominating the epidemic of type 2 diabetes in de-veloped countries. When islets from high-fat diet-fedeIF2�S51A mice were analyzed in vitro, they demonstratedincreased basal insulin secretion but reduced stimulatedinsulin secretion. Prolonged high-fat feeding led to disten-sion of the ER by unfolded proteins, including proinsulin,and to profound glucose intolerance. This surprising find-ing highlights the exquisite sensitivity of pancreatic�-cells even to subtle dysregulation of eIF2a phosphory-lation and raises the intriguing possibility that minor vari-ations in efficiency of eIF2� phosphorylation could, inprinciple, contribute to significant morbidity. As yet, nostudies have addressed the possible existence of suchvariations in eIF2� signaling between patient groups, forexample, obese subjects with or without associated fea-tures of metabolic syndrome.

2. Integrated stress response

The effects of eIF2� phosphorylation are not re-stricted to attenuation of protein translation, but alsoinclude activation of a transcriptional program. This isillustrated in a family of white matter hypomyelinationdisorders, termed CACH/VWM leukodystrophies. These




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are autosomal recessive conditions with a spectrum ofseverity from congenital to adult-onset disease, involvingthe progressive loss of mental and motor faculties due todeterioration of brain white matter. In all cases the caus-ative mutations have been found within subunits of aneIF2�-interacting protein, eIF2B (46, 47, 100, 158). As partof its normal active cycle, eIF2 undergoes rounds of GTPbinding, hydrolysis, and guanine nucleotide exchange.Only in the GTP-bound state is eIF2 capable of bringingmethionyl tRNA to the ribosome. The guanine nucleotideexchange factor (GEF) for eIF2 is, in fact, eIF2B; how-ever, once phosphorylated, eIF2 binds avidly to its GEFinhibiting further exchange (27). In this way, phosphory-lation of a small pool of eIF2 can inhibit most GEF activity(Fig. 1). The mutations that cause the CACH/VWM disor-ders appear to interfere with eIF2B GEF activity, butrecent studies with cells from patients with CACH/VWM

have shown that, rather than affecting global translationrates during stress, these mutations increase signaling viaa transcription factor known as ATF4 (82, 193).

Whilst the majority of RNA transcripts experiencedecreased translation during periods of increased eIF2�phosphorylation, a small and still poorly defined subset istranslated more efficiently (53, 68, 121, 124). This para-doxical effect is due to the presence of multiple upstreamopen reading frames (uORFs) 5� to the coding sequenceinitiation codon (1, 68, 112, 194). The best-characterizedexample in mammals is the stress-inducible transcriptionfactor ATF4 (112, 194). This transcript has two uORFs,the second overlapping out of frame with the true ATF

coding sequence. During resting unstressed conditions,ribosomes scan along the mRNA translating uORF1 andthen recapacitate by rebinding eIF2/GTP/Met-tRNA ter-nary complex in time to translate inhibitory uORF2. Dur-ing stress, limiting levels of eIF2 ternary complex lead toa delay in recapacitation of these scanning ribosomes,such that they fail to reinitiate at uORF2 but instead scanto the ATF4 initiation codon. By then, a proportion havereacquired ternary complex allowing translation of theactive transcription factor.

In yeast, the single eIF2� kinase Gcn2p regulates atranscription program governing the response to aminoacid deprivation through induction of the transcriptionfactor GCN4, by a mechanism analogous to that of ATF4

(37, 68, 125). As the family of eIF2� kinases has diversi-fied to respond to numerous stresses, so too has thetranscriptional program it regulates in metazoans. It con-tinues to regulate genes essential for amino acid suffi-ciency, but now ATF4 also induces antioxidant genes andgenes of the ER protein maturation machinery (57). Be-cause eIF2� phosphorylation triggers a final commonpathway in response to many stresses, this transcriptionalprogram in combination with protein translation modula-tion has been termed the integrated stress response (ISR)(57) (Fig. 1).

Oxidative protein folding is inextricably linked to thegeneration of reactive oxygen species (ROS), in large partthrough the activity of ER oxidase 1 (ERO1), which gen-erates much of the oxidizing potential of the organelle(48, 152, 187, 188). Without ER stress-regulated activationof ATF4, PERK�/� �-cells, and those from Wolcott-Ral-lison patients, lack the prosurvival effects of the ISR withits antioxidant expression program (54, 57). It is worthremarking, therefore, that recent data indicate the oxida-tion of cysteines to form disulfide bonds leads directly tothe generation of ROS and cell death (58) and that ERstress-dependent ERO1� induction promotes ER oxida-tion (117). Insulin, with its three disulfide bonds per mol-ecule, might therefore be expected to impose a consider-able ROS load on the cell. The finding that targeted dele-tion of a widely expressed ER-associated reductase,

FIG. 1. The integrated stress response (ISR). A: basally PERK ismaintained in an inactive state, perhaps through interactions with BiP,while GTP-bound eIF2 complex efficiently supports new protein trans-lation initiation. The guanine nucleotide exchange factor (GEF) eIF2Bmaintains the GTP-bound population of eIF2 complexes. B: during en-doplasmic reticulum (ER) stress BiP dissociates from PERK, whichbecomes active. Unfolded protein might interact directly with the lumi-nal domain of PERK to promote activation. Phosphorylation of eIF2 onits �-subunit leads directly to inhibition of eIF2B activity. When GTP-bound eIF2 levels are limiting, most protein translation is inhibited,while translation of ATF4 is enhanced. ATF4 migrates to the nucleus totransactive genes of the ISR.




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Ncb5or, leads to a selective loss of �-cells, once moreunderlines their sensitivity to redox stress (212).

3. Recovery of protein translation

A) EIF2� PHOSPHATASES. The execution of an adaptivegene expression program like the ISR can only be per-formed if the attenuation of protein translation accompa-nying eIF2� phosphorylation is reversed (29, 114, 131).Indeed, persisting loss of protein synthetic activity wouldcompromise cell survival, regardless of a need for adap-tation to stress. For these reasons, phosphatase activityexists to relieve eIF2� phosphorylation, restoring normaltranslation initiation activity.

Viral infection leads to eIF2� phosphorylationthrough activation of PKR (10, 90). This is part of aninnate antiviral response antagonizing viral protein trans-lation. Frequently, the relationships between infectiveagents and host are characterized by the adaptations ofone party being countered by adaptations within theother. For example, viruses have evolved proteins thatdirectly inhibit the activity of PKR (21, 153); however, inthe case of herpes simplex virus (HSV), viral proteintranslation can be maintained despite continued PKR ac-tivity. This is achieved through enhanced dephosphoryla-tion of eIF2� by a virally encoded protein, ICP34.5, whichbinds to a host serine/threonine phosphatase, proteinphosphatase 1 (PP1), and directs its activity towardseIF2� (61, 62).

It is now well appreciated that protein phosphatases,including PP1, are represented within the genome byrelatively few genes and achieve their specificity throughthe formation of complexes with regulatory subunits (28).It was therefore gratifying when suppressor screens of ERsignaling revealed two mammalian genes, CReP andGADD34, with striking homology to ICP34.5 (80, 131).These proved to be responsible for reversing PERK-in-duced translational attenuation, as they too are regulatorysubunits of PP1 that specifically promote eIF2� dephos-phorylation (29, 80, 131). CReP is constitutively ex-pressed, while GADD34 is strongly induced during ERstress. The induction of GADD34 in particular appearscentral to the reversal of stress-induced translational at-tenuation (18, 87, 114, 131, 132).

B) ROLE OF CHOP. Activation of ER stress signaling hasbeen correlated with the induction of cell death in manymodels of ER stress. The mechanisms by which this mightpromote cell death remain unclear; however, CHOP, atranscription factor downstream of the PERK-ATF4 axis,was thought to be important to the process, since itsdeletion ameliorates tissue damage during ER stress (20,136, 137, 174, 229). The downstream mediators of thiseffect remained unknown until recent work demonstratedthat some of the toxic effects of CHOP are the result ofGADD34 induction (117).

The CHOP dependence of ER stress toxicity is wellillustrated in a murine model of diabetes, the Akitamouse. Mice, unlike humans, have two genes encodinginsulin (Ins1 and Ins2). These appear functionally redun-dant, as deleting both alleles of either gene has no effecton glucose homeostasis (101). It is therefore remarkablethat a spontaneous mutation in the Ins2 gene (Akita)leads to a severe diabetic phenotype inherited as a semi-dominant trait (196, 223).

Insulin is translated as a single proinsulin polypep-tide that undergoes oxidation within the ER to form threeintramolecular disulfide bonds. After excision of a shortpeptide from the prohormone, these three bonds hold theremaining two insulin chains together. The Akita muta-tion converts a conserved cysteine to tyrosine preventingformation of one of these bonds (196). This mutant insulinis not secreted, but instead degraded (8, 196). Mice har-boring the Akita mutation have apparently normal pan-creatic islets at birth, but go on to develop diabetes in theensuing weeks due to selective �-cell loss in the absenceof inflammation. This was initially attributed to nonspe-cific effects (196); however, when this mutation was in-troduced into mice with a targeted deletion of the CHOP

gene, onset of diabetes was significantly delayed due tothe preservation of �-cell numbers (136).

As indicated above, CHOP is a transcription factorhighly induced during ER stress (197, 229). The protectiveeffect of CHOP deletion appears not restricted to theAkita mouse, since it also affords protection against cy-tokine-induced �-cell death, in which nitric oxide triggersER stress by the depletion of ER calcium stores (20, 137),nor is it restricted to the pancreas, as CHOP�/� animalsare resistant to renal damage caused by the ER stress-inducing toxin tunicamycin (229). CHOP deletion alsoprotects mice from dopaminergic neuron loss following6-hydroxydopamine injection (174). This drug, which iscommonly used as a model for Parkinson’s disease sinceit selectively kills dopaminergic neurons in vivo, causesER stress in dopaminergic neurons in tissue culture (71,162).

One popular hypothesis held that CHOP, a metazoanspecific gene, evolved to promote the death of individualcells in response to insurmountable levels of ER stress,affording particular benefit to multicellular organisms(135). In accordance with this, CHOP overexpression hasbeen shown to sensitize cells to the toxicity of ER stress(118). Analysis, however, of the CHOP transcriptionalprogram failed to reveal an obvious connection to path-ways that promote cell death. Instead, CHOP appears todefend ongoing protein secretion, in part through induc-tion of GADD34 (117). In this light, it appears that theER-stressed cell is challenged with balancing the need todefend its chaperone reserve, by limiting secretory pro-tein synthesis, against the need of both cell and organismfor ongoing ER synthetic function. According to this




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model, the protection from death afforded by deletion ofthe CHOP gene in some models of ER stress reflects,therefore, an enforced shift of this balance from syntheticfunction towards the defense of chaperone reserve (Fig.2). In accordance with this, GADD34 null animals havebeen shown to be protected from ER stress-induced neph-rotoxicity equally well as CHOP�/� animals (117).

Of course, there exists the formal possibility thatCHOP-dependent GADD34 induction evolved to promotecell death rather than ongoing protein synthesis; however,two pieces of evidence argue against this. First of all, intissue culture GADD34 mutant cells are more prone tocell death in response to ER stress induced by thapsigar-gin (132). This agent causes ER stress through depletionof ER calcium stores and is particularly effective at shut-ting down protein synthesis, indicating that recovery ofprotein translation is necessary for the adaptive prosur-vival effects of UPR signaling. Second, in murine modelsof Pelizaeus-Merzbacher leukodystrophy (due to a prote-olipid protein mutation), CHOP expression appears tohave an antiapoptotic effect, a strange phenotype for aprodeath signal (176).

It remains unclear whether the toxic effects of CHOPin models of ER stress are primarily mediated throughGADD34. Although this may be true for tunicamycin-induced nephrotoxicity, it has yet to be proven for othermodels. For example, it is possible that other CHOP targetgenes, such as ERO1�, may mediate toxicity in a tissue-

specific fashion. In cell culture, CHOP overexpressionleads to ROS production (118), and CHOP-dependentERO1� induction appears responsible for increased oxi-dation in the stressed ER (117). This may be relevant totissues known to be sensitive to redox perturbation, suchas the pancreatic �-cell.

Perhaps there are circumstances experienced bymulticellular organisms during which the cost of worsen-ing ER stress is compensated for by enhanced proteinsecretion, at least in a subset of cells. Because osteoblastsare replenishable secretory cells they may behave in thisfashion and, consistent with this, recent data suggest thatthe bones of CHOP�/� animals demonstrate defects dueto impaired osteoblast function (147). If the importance ofCHOP in the defense of protein secretion is a generalfeature of replenishable secretory cells, it is tempting tospeculate that terminally differentiated effector cells ofthe immune system might also behave thus. ChallengingCHOP�/� and GADD34 mutant animals with infectiousagents could test this hypothesis.

An exciting prediction arising from these findings isthat careful titration of GADD34 inhibitors might be oftherapeutic benefit in diseases involving ER stress,through the preservation of ER chaperone reserve. It isencouraging, therefore, that a recent screen for smallmolecules that protect cells from ER stress yielded acompound that enhances eIF2� phosphorylation and, inHSV-infected cells, appears to block eIF2� dephosphory-lation by ICP34.5 (15). If compounds can be synthesizedthat have specificity for GADD34 over CReP, there existsthe potential for selective effects on ER-stressed tissue,perhaps limiting systemic toxicity.

4. NF-�B and ER activity

A distinct antiviral response initiated by the accumu-lation of viral proteins in the ER has previously beenpostulated (140, 141, 143). This followed from observa-tions that virally encoded proteins could activate NF-�Bsignaling in a cell autonomous fashion (119, 144, 145).This is in contrast to most canonical NF-�B stimuli, e.g.,tumor necrosis factor (TNF)-�, which originate from out-side the cell. This “ER overload response” (EOR), as itwas termed, appeared not to be specific for viral proteins,since other membrane proteins, e.g., MHC class I, whenoverexpressed could also induce NF-�B signaling (145). Itwas subsequently extended to include NF-�B inductionfrom ER accumulation of other nontransmembrane pro-teins, including �1-antitrypsin polymers (67, 95). Thetransduction mechanism for this pathway remained ob-scure, although release of ER calcium and ROS were bothsuggested (140, 143).

Recent discoveries have implicated PERK signalingin the activation of NF-�B in response to ER perturba-tions, challenging the notion that the EOR is distinct from

FIG. 2. Failure of ER homeostasis. The ratio of client protein load(gray spheres) to the availability of chaperones (red caps) varies de-pending on new protein translation rates. When levels of new proteinexceed the capacity of ER chaperones to bind them, aggregation ensues.PERK promotes eIF2� phosphorylation and thus inhibits protein trans-lation, which is represented by a rightward shift in this figure. GADD34induction, through eIF2� dephosphorylation and disinhibition of eIF2BGEF activity, promotes protein translation and a leftward shift with anincreased risk of client protein aggregation. CHOP�/� and GADD34

mutant cells experience lower levels of new protein aggregation becausereduced new protein synthesis defends the reserve of ER chaperones.




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the UPR (36, 78). There exists a protein family of NF-�Binhibitors termed I-�B, which, in unstimulated cells, isexpressed constitutively and sequesters NF-�B inactivewithin the cytoplasm (160). Classically, NF-�B is acti-vated through the relief of this inhibition, for example,TNF receptor activation triggers I-�B phosphorylation,causing its ubiquitination and degradation by the proteo-some (146). In the case of ER stress, at least when sig-naled by PERK, disinhibition of NF-�B is achieved onceagain by reducing I�B levels, but through a novel mech-anism. I-�B has a far shorter half-life than NF-�B. Conse-quently, translational attenuation preferentially lowersI�B levels, releasing NF-�B to execute its transcriptionprogram. A similar effect can be obtained by treatment ofcells in culture with other agents that inhibit proteinsynthesis, e.g., cycloheximide (36, 78). This novel mech-anism extends to NF-�B activation in response to otherstresses, including ultraviolet (UV) irradiation and aminoacid deprivation, where GCN2 appears to be the relevantkinase (77, 78, 211). Some controversy persists within thisfield, since the existence of a PERK-dependent NF-�Bcascade does not preclude activation of NF-�B by otherER-originating signals. This will be clarified once EORstressors have been more extensively studied in PERKmutant and eIF2�S51A cell lines.

The physiological significance of ER stress-inducedNF-�B signaling likely reflects the importance of NF-�Bsignaling in other settings, namely, the coordination of in-flammatory response and promotion of cellular survival. Inbacterial infection, the induction of NF-�B by microbialcomponents sensed by Toll-like receptors leads to upregu-lation of early effectors of the innate immune response,including chemokines, proinflammatory cytokines, and im-mune receptors (7, 91, 215). These early effectors then gen-erate a further cycle of NF-�B induction and generation ofan antimicrobial response (104). The induction of such acascade by detection of viral components within the ER, andsubsequent PERK-dependent NF-�B activation, might repre-sent an analogous antiviral response.

Developmentally, NF-�B is a prosurvival signal insome cell types, for example, in the maturation of B andT lymphocytes and in the development and regenerationof the liver (6, 103, 183, 210). While many stimuli thatinduce NF-�B also promote cell death, paradoxicallyNF-�B tends to oppose signal-induced cell death by in-duction of antiapoptotic genes including c-IAP1, c-IAP2,

and FLIP (120, 195). Indeed, NF-�B can have proliferativeeffects through its targets c-myc and cylcinD1 (142, 155).It is therefore possible that NF-�B induction may also linksecretory activity to trophic signals.

B. Long-Term Change

In contrast to the continuous modulation of ER syn-thetic activity, differentiation of a cell into a highly secre-

tory phenotype requires a dramatic expansion of ER. Thisis exemplified in the development of B-lymphocytes intoplasma cells. In this regard, defects in plasma cell differ-entiation have helped reveal an additional role for theUPR in the regulation of absolute ER mass within the cell.

1. Linking ER mass to demand

Multiple myeloma is a hematological neoplasm char-acterized by presence in the blood of a monoclonal im-munoglobin or Bence Jones protein (free monoclonal �-or �-light chains). These are produced by myeloma cellswithin the bone marrow, which over time expand todisplace normal marrow leading to anemia and immuno-logical deficiency. The plasma concentration of the mono-clonal paraprotein can reach exceeding high levels (�100g/l), even leading to complications through increasedblood viscosity. The causative myeloma cells are mono-clonal expansions of plasma cells, immune cells normallycharged with large-scale immunoglobin manufacture.

To understand the genesis of plasma cells and my-elomas, the transcription factors necessary for their dif-ferentiation have been determined. Early work demon-strated that myeloma cell lines frequently express a basicleucine zipper transcription factor, XBP-1, to extremelyhigh levels (26, 204). XBP-1 was also shown to be essen-tial for normal plasma cell differentiation (156, 157, 204).Myeloma cells and mature plasma cells share the ability tosecrete huge quantities of immunoglobin, thanks to theirhighly developed secretory pathway. Histologically thesecells have characteristically basophilic cytoplasm due tothe large numbers of ribosomes associated with theirextensive ER (Fig. 3). It seems likely that XBP-1’s role inplasma cell differentiation is related to regulation of ERexpansion in response to increased demand (96). Further-more, new therapeutic agents being developed to treatmyeloma may exploit the dependence of this neoplasm onUPR activation (2, 66). Proteosome inhibitors haveproven to be highly effective in the killing of myelomacells and in early trials have achieved remarkable clinicalremissions in otherwise drug-resistant disease (81). Theirselectivity toward myelomas might reflect the reliance ofthese cells on efficient ERAD, which itself is dependenton XBP-1 signaling (96, 221). The first drug of this class,bortezomib (Velcade), has now been approved by theFDA for use in otherwise refractory disease (17).

XBP-1 regulates many genes of the unfolded proteinresponse in metazoans, including genes involved in gen-eration of the ER membrane (177). It is upregulated at theprotein level during ER stress by a remarkable mecha-nism. The XBP-1 primary transcript is translated, albeitinefficiently, to produce a protein without significanttransactivation activity (99). ER stress-dependent splicingof XBP-1 mRNA involves excision of a short intron (26ntin mammals) causing a frame shift in its open reading




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frame, which introduces a new COOH-terminal transacti-vation domain to generate the active transcription factor(19, 99, 169, 222). The endonuclease responsible for re-moving the inhibitory intron is an ER transmembraneprotein, IRE1, whose luminal domain shares significanthomology with that of PERK, allowing it a similar regu-latory interaction with BiP (11, 12, 86, 108, 134). As withPERK, ER stress-induced dissociation of BiP allows clus-tering of IRE1 and its activation through transautophos-phorylation. In mammals there are, in fact, two IRE1isoforms. IRE1� is detectable in all tissues (185), while

IRE1� appears limited to the gut epithelium (198). Thesignificance of this tissue-dependent isoform expressionis unclear but may point toward cell type specific differ-ences in UPR signaling. IRE1��/� animals are not viable,whereas IRE1� mutants appear well, although are moreprone to chemical-induced colitis (11, 185, 192).

The IRE1-dependent arm of the UPR is the mostancient in evolutionary terms, since the entire UPR oflower eukaryotes, including that of yeast, is regulated bythis molecule (30, 31, 40, 184, 185). Although in yeast thetarget transcription factor HAC1 bares little homology toXBP1, the activation of Ire1p is identical. It was, in fact,historically the first UPR pathway to be described inmolecular terms (14, 22, 50, 85, 123, 167, 173, 202). Inyeast, after Ire1p-mediated intron excision from theHAC1 mRNA, religation by the tRNA ligase Rlg1p gener-ates an efficiently translated message (123, 161, 172). Theanalogous ligase in higher organisms has yet to be iden-tified and remains an important unanswered question.

2. Insulin resistance and ER stress

In addition to linking secretory capacity to demandthrough regulation of ER expansion, IRE1 also plays animportant role in integrating ER stress signaling withother stress and growth factor sensitive pathways.

An important feature of type 2 diabetes is peripheralinsulin resistance. Normally, activated insulin receptorsphosphorylate proximal signaling molecules, such as in-sulin receptor substrate 1 (IRS-1), on tyrosine residues,which transduce the effects of insulin through interactionwith cytosolic targets (180, 182). In obesity (genetic ordietary), this tyrosine phosphorylation is inhibited byJNK-dependent serine phosphorylation of IRS-1 (4, 5).Surprisingly, the mechanism of obesity-related JNK acti-vation appears to involve ER stress.

For unclear reasons, liver and adipose cells fromobese mice show biochemical evidence of ER stress withPERK activation, eIF2� phosphorylation, and BiP induc-tion (139). It has previously been shown that ER stress,through IRE1 activation, can directly trigger the JNK cas-cade (192). Activated IRE1 recruits the scaffolding pro-tein TRAF2 to the ER membrane (192), which triggers amitogen-activated protein (MAP) kinase cascade leadingto JNK activation (129, 130). Thus IRE1 offers a plausiblemechanistic link between obesity and peripheral insulinresistance. In this model, obesity-associated ER stresswould contribute to insulin resistance by causing JNKactivation through IRE1/TRAF2 with subsequent IRS-1serine phosphorylation (Fig. 4). Consistent with this hy-pothesis, IRE1�/� cells fail to active JNK or phosphory-late IRS-1 during ER stress unlike wild-type controls.Supporting a causal link between peripheral ER stressand insulin resistance, a recent study has demonstratedprotection against obesity-induced type 2 diabetes in mice

FIG. 3. The unfolded protein response (UPR). A: photomicrograph(�1,000) of neoplastic plasma cells stained with eosin and hematoxylinfrom a bone marrow aspirate in a case of multiple myeloma. Note thelateral displacement of the nucleus by the extensive basophilic ER.Scale bar is 10 �m. (Image kindly provided by Dr. Wendy Erber, Adden-brookes Hospital, UK.) B: ER stress causes signaling through threedistinct mediator proteins in the ER membrane offering redundancyand, perhaps, a means to adjust the UPR to fit the circumstances of thestress. Activated PERK phosphorylates eIF2� and inhibits new proteintranslation. Additionally, ATF4 induction activates genes of the ISR,including GADD34, which relieves translational attenuation, andERO1�, which promotes oxidataive protein folding. Antioxidant targetsof ATF4 act to buffer increased reactive oxygen species (ROS) producedby ERO1�. ATF6 cleavage releases soluble ATF6c allowing transactiva-tion of UPR target genes that increase the ER protein maturation ma-chinery. IRE1-mediated activation of XBP1, which is more sustained dueto XBP1 autoinduction, leads to induction of genes involved both in thematuration of ER client proteins and also genes that promote ER asso-ciated protein degradation.




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by overexpression of an ER chaperone, while knockdownof the same chaperone was diabetogenic (128). Further-more, in the Akita mouse, systemic rather than �-celloverexpression of an ER chaperone appeared to improveperipheral insulin sensitivity (138).

In light of this, it is possible that the peripheralinsulin resistance seen in the eIF2�S51A heterozygousmice (above) might have resulted from increased signal-ing through the IRE1-JNK arm of the UPR attempting tocompensate for impaired PERK-dependent signaling.Consistent with this speculation, PERK�/� cells, whichare impaired in eIF2� phosphorylation, experience ba-sally higher IRE1 activation (55).

It can be seen that the role of UPR signaling indiabetes is far from straightforward. At the level of thepancreatic �-cell, the UPR protects against developingtype I diabetes by ensuring that this highly secretorytissue maintains efficient function despite wide swings inER protein flux. Conversely, chronic ER stress signaling(perhaps related to obesity) appears involved in the eti-ology of peripheral insulin resistance. We can at presentonly speculate as to how the UPR progresses from regu-lation to dysregulation over the passing years. It is knownthat chronic exposure to elevated saturated free fattyacids induces ER stress (83). Consequently, obesity-re-lated ER stress may reflect the evolutionarily recent phe-nomenon of chronic nutrient excess. The dysregulation ofER stress signaling seen in this disease may reflect thelack of selection pressure to adapt this homeostatic mech-anism to chronic inappropriate activation.

3. ER stress and lipid metabolism

A) HOMOCYSTEINE AND ATHEROSCLEROSIS. Among theknown risk factors for atherosclerosis, ER stress wouldcurrently be ranked low, if at all. This may begin tochange as evidence accumulates implicating the UPR indisordered lipid metabolism. The relationship betweenthe UPR and lipid accumulation is a complex one. On theone hand, many target genes of the UPR involve lipidsynthesis, in part to allow expansion of the ER itself; onthe other hand, there is increasing evidence that pertur-bation of the lipid environment within the ER can activateUPR signaling.

Homocysteinemia is associated with the develop-ment of atherosclerosis, certainly in patients with rareinherited disorders of amino acid metabolism, but also inthe wider population, as indicated by large epidemiolog-ical studies (42, 70, 201). Although homocysteine is asulfur-containing amino acid, some of its toxic effectsappear to involve dysregulation of cholesterol and triglyc-eride biosynthesis (205). Patients with inherited hyperho-mocysteinemia or laboratory animals fed a homocysteine-rich diet develop hepatic steatosis in the absence ofmarked rises in plasma lipids. The likely explanation islocal lipid synthesis, and consistent with this, homocys-teine has been shown to activate lipogenic signaling viathe sterol regulated element-binding proteins (SREBPs)(205). Surprisingly, ER stress contributes to the activationof SREBP by homocysteine. Livers of homocysteine-fedmice contain raised levels of ER chaperones, as do cul-tured cells exposed to high levels of homocysteine invitro. The importance of ER stress in this lipogenic sig-naling was demonstrated by BiP overexpression, whichameliorated SREBP induction in response to homocys-teine.

The relationship between the UPR and SREBP, whileclearly evident, is still not entirely explained in mechanis-

FIG. 4. Insulin resistance in obesity mediated by ER stress. A: innonobese subjects, glucose homeostasis is maintained by effectors ofinsulin signaling. Insulin binding to its receptor causes tyrosine phos-phorylation of IRS1, which in turn promotes activation of effectorproteins. B: in obesity, ER stress causes insulin resistance. IRE1 activa-tion may then recruit TRAF2 to the ER membrane causing activation ofJNK, which in turn would promote phosphorylation of serine residues inIRS1. Serine phosphorylation of IRS1 inhibits its tyrosine phosphoryla-tion by activated insulin receptors, thus impairing insulin signaling.




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tic terms. The SREBPs are transmembrane proteins that,under sterol-sufficient conditions, are retained within theER through interaction with other ER membrane pro-teins, Insig-1 and Insig-2 (213, 216). When cholesterollevels are low, this interaction is weakened, whereuponSREBP progresses to the Golgi to be cleaved sequentiallyby two serine proteases, S1P and S2P (33). This cleavageliberates the cytosolic portion of SREBP as a solubleprotein, which enters the nucleus to bind specific DNAelements, thus transactivating target genes involved inlipid synthesis. One possible mechanism of ER stress-induced SREBP activation, for which there is evidence,involves stress-dependent loss of Insig-1 from the ER (98).

B) SREBP AND ATF6. Interestingly, the third known classof ER stress sensor, exemplified by ATF6, is unrelated toPERK or IRE1. ATF6 is another ER transmembrane pro-tein, that undergoes processing similar to SREBP (24, 60,102). It is normally retained within the ER through inter-action, not with Insig proteins, but with BiP (168). DuringER stress, ATF6 is released from the ER and moves on tothe Golgi. There it is processed by the very same pro-teases that act on SREBP, to liberate a soluble transcrip-tion factor (218). Unlike SREBP, the targets of ATF6 areclassical UPR genes (133, 199, 220). Indeed, ATF6 wasisolated in a search for ligands of an ER stress responsiveDNA element (ESRE). Despite their separate transcrip-tional programs, there may still be some cross-talk be-tween ATF6 and SREBP. A recent report suggests thatwithin the nucleus, cleaved ATF6 can bind SREBP2 di-rectly to inhibit its transactivation potential and opposelipogenesis (224).

Following from the identification of ATF6, there havebeen several related transcription factors identified,which share similar domain structures and localization tothe ER [Luman (35, 113, 154), OASIS (88), CREBH (225),ATF6b (59), CREB4 (178)]. The existence of such a vari-ety of UPR signaling molecules at first seems puzzling, buta clue to their roles may come from tissue distribution. Inthe cases of OASIS and CREBH, at least, there is clearevidence for tissue specificity, OASIS being found in as-trocytes of the central nervous system, while CREBH isrestricted to the liver. This raises the possibility that ERstress may elicit tissue-specific transcriptional responses.In the case of CREBH for example, it has already beenshown that its activation by ER stress leads to inductionof genes of the acute phase response, providing a directlink between hepatic ER stress and systemic inflamma-tion (225).

C) CHOLESTEROL’S IMPACT ON THE ER. The dependence ofcholesterol metabolism on ER stress signaling may reflectthe impact of sterols on ER function. This is well illus-trated by the macrophage in development of atheroscle-rosis. During the initial phase of atherosclerotic lesionformation, foam cells are a characteristic histologicalfinding within the vessel intima. These are macrophages

that have taken up oxidized lipoprotein particles andbecome laden with cholesterol. This cholesterol is storedas esters within large lipid vesicles, which give foam cellstheir foamy appearance. Over time, death of these cells,likely due to the toxic effects of unesterified cholesterol,results in deposition of extracellular cholesterol withinthe plaque. Unlike its esters, free cholesterol efficientlyinserts into lipid bilayers and can alter membrane physi-cal properties. The ER membrane, being poor in freecholesterol (16), may be especially sensitive to choles-terol loading. Indeed, recent findings indicate that for freecholesterol to have its toxic effect it must be trafficked tothe ER (45). This trafficking results in activation of UPRsignaling and caspase activation, and eventually in mac-rophage apoptosis. In addition, ER stress caused by freecholesterol loading of macrophages promotes chemokinesecretion, and this may contribute to the formation of“vulnerable” atherosclerotic lesion, which are prone torupture as a function of their high inflammatory cell infil-trate (105).

III. CONCLUDING REMARKS

The complexity of the mammalian UPR, mediated asit is by three distinct classes of ER sensors and numeroustranscription factors, may provide a degree of redun-dancy; however, perhaps a more interesting interpreta-tion of this complexity may be to allow higher organismsa more subtle response to ER stress. For example, thedivision of UPR genes between multiple transcriptionfactors likely enables portions of the gene expressionprogram to be induced as is appropriate either to theduration or intensity of ER stress. While ATF6 and XBP-1transactivate many of the same genes, their expressionprograms show important differences (97). Binding ofATF6 to the BiP promoter is sufficient for maximal trans-activation, while XBP-I binding is sufficient for maximalinduction of EDEM (97, 221). In contrast, many genesrequire both arms to be active in order for best induction(214). It has been suggested that an early, ATF6-domi-nated, response may attempt to compensate for ER stressthrough chaperone induction alone. While XBP-1 activa-tion, which is more sustained, may, in addition, promoteprotein degradation through the induction of ERAD com-ponents. Accordingly, IRE1��/� cells have been showndefective in glycoprotein ERAD (221).

Repeatedly, models of ER dysfunction have gener-ated models of diabetes, and investigations into rare ge-netic diabetic syndromes have revealed unexpected linksto ER stress. The field remains fertile for further study,since mutations in ER stress-inducible genes such asWFS1 (Wolfram syndrome) (74, 179, 181) and P58IPK

(93), which lead to diabetic syndromes, have yet to befully understood. Furthermore, the study of apparentlyunrelated disease states including atherosclerosis, viral




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infection, and multiple myeloma may also benefit fromour increasing understanding of ER stress in their patho-physiology.

ACKNOWLEDGMENTS

Address for reprint requests and other correspondence:S. J. Marciniak, Cambridge Institute for Medical Research, Univ.of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cam-bridge CB2 2XY, UK (e-mail: [email protected]); and D. Ron,Skirball Institute of Biomolecular Medicine, New York Univer-sity School of Medicine, New York, NY 10016 (e-mail:[email protected]).

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