ER stress: Can the liver cope?

www.elsevier.com/locate/jhep

Journal of Hepatology 45 (2006) 321–333

Review

ER stress: Can the liver cope?

Cheng Ji*, Neil Kaplowitz

Gastroenterology/Liver Division, Keck School of Medicine and the Research Center for Liver Disease, University of Southern California

and the USC-UCLA Research Center for Alcoholic Liver and Pancreatic Disease, Los Angeles, CA 90033, USA

Hepatocytes contain abundant endoplasmic reticulum (ER) which is essential for protein metabolism and stress signal-

ing. Hepatic viral infections, metabolic disorders, mutations of genes encoding ER-resident proteins, and abuse of alcohol

or drugs can induce ER stress. Liver cells cope with ER stress by an adaptive protective response termed unfolded proteinresponse (UPR), which includes enhancing protein folding and degradation in the ER and down-regulating overall protein

synthesis. When the UPR adaptation to ER stress is insufficient, the ER stress response unleashes pathological conse-

quences including hepatic fat accumulation, inflammation and cell death which can lead to liver disease or worsen under-

lying causes of liver injury, such as viral or diabetes-obesity-related liver disease.

� 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: HCV; HBV; Ischemia; a1- AT deficiency; Alcoholic liver disease; Hyperhomocysteinemia; Steatosis; Insulin

resistance; NASH

1. Introduction

Proteins are continuously synthesized and turnedover inside cells. When subjected to stress from dena-tured or malfunctioning proteins, eukaryotic cellsapparently use two mechanisms to maintain protein sta-bility. One is heat-shock response which occurs in the

0168-8278/$32.00 � 2006 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2006.06.004

Available online 15 June 2006* Corresponding author. Tel.: +323 442 3452; fax: +323 442 5425.

E-mail address: [email protected] (C. Ji).Abbreviations: ASK1, apoptosis signaling kinase 1; BHMT, beta-

ine-homocysteine methyltransferase; CBS, cystathionine-b-synthase;CHOP, C/EBP homologous protein; eIF2a, eukaryotic initiation fac-tor-2a; EOR, ER overloading response; ERAD, ER-associated deg-radation; ERO1a, ER oxidase 1a; ERSE, ER stress response elements;GADD34, growth arrest and DNA damage-inducible protein 34;GRP78, glucose-regulated protein 78 also known as BiP; Herp, hom-ocysteine-induced protein; Hcy, homocysteine; HHcy, hyperhomocys-teinemia; HSP, heat-shock proteins; IRE1a, type-I ER transmembraneprotein kinase; JIK, c-Jun-N-terminal inhibitory kinase; MS,methionine synthase; nrf-2, NF-E2-related factor-2; PARP, poly(ADP-ribose) polymerase; PERK, PKR like ER kinase; PKR,RNA-dependent protein kinase; RIP, regulated intramembrane pro-teolysis; SAH,S-adenosylhomocysteine; SAM, S-adenosylmethionine;SREBP, sterol regulatory element-binding protein; UPR, unfoldedprotein response; UPRE, UPR response elements; XBP-1, X-box DNA-binding protein 1; sXBP-1, alternative spliced XBP-1.

cytosol featured by the synthesis of heat-shock proteins(HSPs) [1] and the other is the unfolded protein response(UPR) [2] which occurs in the largest organelle of mosteukaryotic cells, the endoplasmic reticulum (ER), and ischaracterized by upregulation of molecular chaperoneswhich are a class of proteins that are highly conservedin all life forms and help other polypeptides reach aproper conformation or cellular location without them-selves becoming part of the final structure. Unlike theheat-shock-response which is exclusively a stressresponse, the UPR is a normal physiological processrequired for organelle expansion to promote more pro-tein folding and secretion during differentiation of spe-cialized secretory cells such as mature B cells andthose in the liver or pancreas [2–5] and also for safe-guarding protein synthesis, post-translational modifica-tions, folding and secretion, calcium storage andsignaling, and lipid biosynthesis [6–11]. Under normalconditions the ER maintains high concentrations of res-ident calcium-dependent chaperone proteins, such asglucose-regulated protein-78 (GRP78 also known asBiP) and GRP94 [12,13], a high level of calcium andan oxidized environment. Only properly folded proteinsare allowed to reach their final destination, whereasunfolded and misfolded proteins are exported or

Published by Elsevier B.V. All rights reserved.

mailto:[email protected]

322 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) 321–333

dislocated from the ER and degraded by cytoplasmicproteasomes. Perturbations, such as elevated secretoryprotein synthesis, over-expression and/or accumulationof mutant proteins, glucose deprivation, altered glyco-sylation, ER calcium depletion, shifting of redox statusto a more reduced state, and overloading of cholesterol,create stress in the ER leading to the UPR.

2. The unfolded protein response

The UPR initially activates intracellular signalingpathways mediated by three ER-resident sensors inmammalian cells: the type-I ER transmembrane proteinkinase (IRE1) [14,15], the activating transcription factor6 (ATF6) and the PKR like ER kinase (PERK) (Fig. 1)[16,17]. The ER lumenal domains of these sensorproteins are usually associated with or bound to

Fig. 1. The protective responses of the unfolded protein response (UPR). During

such as Bip/GRP78 from ER-resident kinases-IRE1a (type-I ER transmembr

factor-ATF-6. Activated PERK phosphorylates eIF2a (eukaryotic initiation fac

up-regulate genes encoding ER chaperones leading to increased capacity for

response elements; GRP78, glucose-regulated protein 78 also known as BiP;

autophosphorylation; eIF-2a-P, phosphorylated eIF-2a. nrf-2, NF-E2-related fa

elements; XBP-1, X-box DNA-binding protein 1; sXBP-1, alternative spliced X

intralumenal GRP78 in the absence of ER stress. Asunfolded proteins accumulate, engaging more GRP78in the ER, GRP78 dissociates from IRE1, ATF6, andPERK liberating these signal transducers to promote acompensatory protective response [18–21]. IRE1 dimer-izes and is autophosphorylated, which allows it to actas an endoribonuclease in the alternative splicing of themRNA of the X-box DNA-binding protein 1 (XBP-1)[22], which removes a 26 base pair intron and results ina translation frameshift that permits XBP-1 to act as atranscriptional activator (sXBP-1). Bcl-2 family mem-bers BAX and BAK may modulate the expression ofXBP-1 by a direct interaction with IRE1a [23]. sXBP-1upregulates genes such as GRP78, GRP94, and calreticu-lin, all of which contain a common motif of upstream ERstress response elements (ERSE) within their promoterregions [24]. Concomitantly, ATF6 is transported tothe Golgi where its cytosolic transactivation domain is

early UPR, unfolded proteins cause dissociation of molecular chaperones

ane protein kinase) and PERK (PKR like ER kinase) and transcription

tor-2a) resulting in translational attenuation. Activated IRE1a and ATF6

protein-folding. ERAD, ER-associated degradation; ERSE, ER stress

Herp, homocysteine-induced protein; nATF-6, activated ATF6; Auto-P,

ctor-2; RIP, regulated intramembrane proteolysis; UPRE, UPR response

BP-1.

C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) 321–333 323

cleaved from the membrane by specific proteases (S1Pand S2P); a process termed regulated intramembraneproteolysis (RIP) [25,26]. The cleaved ATF6 (nATF6)localizes to the nucleus where it interacts with the consti-tutively expressed transcription factor NF-Y and withERSE, thereby activating transcription of UPR-respon-sive genes, including GRP78, XBP-1, ERp72, andHcy-induced ER protein (Herp) [27–32]. Therefore, acti-vation of ATF6 and IRE1 as well as downstream XBP-1(IRE1–XBP-1) increases the expression of ER-residentchaperones. Comparing ATF6 and IRE1, ATF6 regu-lates genes encoding ER-resident chaperones and foldingenzymes via ERSE, whereas the IRE1–XBP-1 pathwayalso regulates the expression of ER-resident chaperonesthat are essential for protein folding and maturationvia ERSE as well as the expression of genes involved inER-associated degradation (ERAD) via a distinctunfolded protein response element (UPRE) [33].

In addition to transcriptional regulation of a group ofgenes encoding ER-resident proteins and enzymes, theUPR also induces a rapid attenuation in protein synthe-sis which is mediated by PERK. Once dissociatedfrom GRP78, PERK phosphorylates eukaryotic initia-tion factor-2a (eIF-2a), which blocks global mRNA

Fig. 2. Major mechanisms of injury mediated by ER stress response. Prolonge

factor receptor associated factor 2) which activates ASK1 (apoptosis signal

Upregulation of CHOP (C/EBP homologous protein) by ATF4 represses me

(oxidative stress) and GADD34 (reversal of translation inhibition leading to mor

cytosolic Ca2+ and activation of NF-jB lead to inflammation. Activation

accumulation. ERO1a, ER oxidase 1a; PP1, phosphatase 1; eIF ptase, eIF p

GADD34, growth arrest and DNA damage-inducible protein 34.

translation initiation and helps reduce the proteinburden on the ER [17]. At the same time the phospho-eIF-2a increases expression of a subset of genes byselectively enhancing the translation of the transcriptionfactor ATF4 and also leads to increased translation ofGADD34 which associates with protein phosphatase 1to act as an eIF-2a phosphatase to reverse the suppres-sion of translation [34]. Under physiological conditions,the latter is designed to turn-off the UPR so that proteinsynthesis can resume. PERK has recently been shown toenhance nrf-2 phosphorylation and translocation to thenucleus where this transcription factor up-regulates theexpression of antioxidant genes.

3. ER stress response: pathological consequences of

prolonged UPR

The UPR deals with adverse effects of ER stress in atimely and efficient manner at the early stage and thusenhances cell survival. However, prolonged ER stresshas severe consequences, including apoptosis, andis referred to as the ER stress response (Fig. 2). Forexample, to resolve ER stress, sustained UPR consumes

d ER stress leads to interactions of IRE1a with TRAF2 (tumor necrosis

ing kinase 1), caspases, JNK and p38MAPK, modulating cell death.

mbers of Bcl2 family which promotes cell death and increases ERO1ae ER stress). Reactive oxygen species (ROS) resulting from interaction of

of SREBP (sterol regulatory element-binding protein) increases fat

hosphatase; EOR, ER overload response; nSREBP, activated SREBP;


energy in retrotranslocating unfolded or misfolded pro-teins retained in the ER to the cytoplasm for ubiquitina-tion and the ERAD [35]. Energy depletion cancontribute to programmed cell death [36]. During ERstress, the activated IRE1 interacts with the c-Jun-N-ter-minal inhibitory kinase (JIK) which recruits cytosolicadapter TRAF2 to the ER membrane [37,38]. TRAF2activates the apoptosis-signaling kinase 1 (ASK1) lead-ing to activation of JNK and downstream mitochon-dria/Apaf-1-dependent caspase activation [39]. Inaddition in rodents but not in humans, caspase-12 isactivated which activates downstream caspase-9 andcaspase-3 without the need for mitochondrial amplifica-tion [40,41]. Caspase-4 has been suggested to fulfill thisrole in human [42]. Another death-signaling pathwayactivated by ER stress is mediated by transcriptionalactivation of CHOP, a b-ZIP transcription factor thatpotentiates apoptosis, possibly through repressingexpression of anti-apoptotic Bcl2 and Bcl-XL and induc-tion of ER oxidase 1a which generates reactive oxygenspecies and depletes GSH [43]. Although both theIRE1–XBP1 and the ATF6 pathways can up-regulateCHOP, the PERK-eIF2a pathway predominatesthrough selective upregulation of translation of ATF4,a transcription factor which subsequently activates tran-scription of CHOP and other genes involved in aminoacid metabolism and transport, and oxidation–reduc-tion reactions [44,45].

In addition, prolonged ER stress is associated withrelease of ER Ca2+ stores which can perturb mitochon-dria, triggering oxidative stress. Ca2+-induced oxidativestress can induce both cell death and activate NF-jB sig-naling (ER overload response), contributing to inflam-mation [10,11,42]. Ca2+ chelators and antioxidantsblock NF-jB activation [45]. Increased cytosol Ca2+

also activates calpains which proteolytically cleave Bcl-XL (inactivation) and caspase 12 (activation). Apoptosisis rapidly initiated after ER-Ca2+ depletion in photody-namic therapy and strictly requires BAX/BAK at themitochondria [46]. ER stress induces expression andcleavage of CREBH, a hepatocyte-specific bZip tran-scription factor that is structurally similar to ATF6[47]. Activated CREBH and ATF6 synergistically trig-ger an acute phase response (reactive protein and serumamyloid P expression) which occurs in response to ERstress or pro-inflammatory cytokines [47–49].

Aside from cell death and inflammation, ER stresscontributes to intracellular lipid accumulation which ismediated by the ER-associated transmembrane sterol-response element-binding proteins (SREBP). SREBP1cand 2 are usually retained in the ER in a complex withthe polytopic sterol-sensing transmembrane proteinSCAP (SREBP cleavage-activating protein) [50,51].Upon ER stress, or cholesterol deprivation, theSREBP–SCAP complex dissociates from the ER reten-tion protein Insig and subsequently translocates to the

Golgi, where SREBP is cleaved and activated via theRIP [50–53]. Insig normally turns over rapidly, so ERstress-induced translational arrest may lead to a rapiddecline in Insig allowing SREBP to escape. Once activat-ed, SREBP1c and 2 act as transcription factors that reg-ulate the genes that control the synthesis of fatty acids/triglycerides and cholesterol, respectively, and cellularuptake of lipoproteins [53]. ER stress-induced overpro-duction of lipids can lead to fatty liver [54,55]. In addi-tion, ER stress is associated with proatherogenicchanges in lipoprotein metabolism including increasedVLDL and reduced HDL cholesterol levels which con-tribute to cardiovascular disease [56].

4. ER stress and liver disease

4.1. Viral infection

4.1.1. Hepatitis C virus (HCV) infection

In theory, the burden of producing viral protein invirus-infected cells may induce the UPR in response tothe high levels of viral proteins. The ER is the majorsubcellular organelle with which the HCV life cycle isassociated. The HCV RNA genome contains �9600nucleotides with a 5 0 and 3 0 non-coding region (NCR)surrounding a large open-reading frame (ORF) whichencodes a polyprotein. The 5 0 NCR contains an internalribosome entry site (IRES) that directs the translation ofthe HCV polyprotein [57,58]. The polyprotein is pro-cessed by both viral and host proteases producing struc-tural proteins (C, E1 and E2) and non-structuralproteins (p7, NS2, NS3, NS4A, NS4B, NS5A andNS5B) that are associated with the ER membrane[59–61].

The UPR triggered by the burden of viral proteinscan be viewed as a two edged sword. On the one hand,the UPR can promote cell survival which impairs viraleradication. On the other hand, the UPR-inducedPERK-mediated translation inhibition could suppressviral protein synthesis. Although HCV induces compo-nents of the UPR, a variety of evidence indicates thatindividual HCV proteins modulate the UPR and ERstress response which can result mainly in increased viralreplication and failure to eliminate infected cells. Forinstance, NS3 and NS5B direct viral replication from aribonucleoprotein (RNP) replication complex associatedwith an ER-derived membrane [62]. NS4B inducesATF6 and IRE1 to favor the HCV subreplicon andHCV viral replication [63]. HCV gene expression corre-lates with the translocation of ATF6 cytoplasmicdomain to the nucleus of cells expressing HCV subge-nomic replicons. Aside from inducing chaperones,ATF6 activates the IRE1–XBP1 pathway of the UPRby upregulating the transcription of XBP1. AlthoughXBP1 spliced mRNA and sXBP1 protein are elevated


in HCV replicon-expressing cells [64], the transactivat-ing activity of sXBP1 is somehow repressed in theHCV-infected cells which prevents transcriptionalinduction of the ER degradation-enhancing-mannosi-dase-like protein (EDEM) [65,66]. Thus the protectiveUPR in HCV-infected cells may not unload excessiveHCV proteins allowing increased virus production.

Another way in which HCV alters the typical courseof the UPR is to interfere with the PERK/PKR path-way. In virus-infected cells, the double-stranded RNA-activated protein kinase (PKR) phosphorylates eIF2awhich leads to a general attenuation of protein transla-tion. However, HCV E2 and NS5A contain PKR-bind-ing domains which enable them bind to and inhibit PKR[61,67–69]. Cells that express HCV E2 or HCV NS5Ashow elevated levels of overall protein synthesis. PKRactivation is an important effect of interferon treatmentso the resistance to interferon may be partly due to theinhibition of PKR by HCV. PKR and PERK share asignificant amount of homology and both are activatedby ER stress and phosphorylate eIF2a [70,71]. It is verylikely that HCV infection also modulates protein synthe-sis through PERK. Indeed, HCV E2 suppresses PERKactivity [72]. E1 and/or E2 expression also induce com-ponents of ER stress as indicated by upregulation ofCHOP, sXBP-1, and the ERAD [73,74]. It should benoted that contradictory effects of expression of individ-ual HCV genes versus the entire genome have beenobserved. For example HCV replicons suppress theERAD whereas E1 or E2 alone enhances the ERAD.

Aside from modulating the UPR to favor viral persis-tence, HCV proteins may promote ER stress-inducedinjury. HCV gene expression elevates intracellular levelsof cholesterol which leads to the release of Ca2+ fromthe ER [75]. Expression of constructs encoding 191 aareference HCV core in Huh7 or HepG2 and in transgen-ic mice triggered hyperexpression of GRP78, GRP94,and calreticulin which was followed by Ca2+ depletion[75]. HCV core-induced CHOP, BAX translocation tomitochondria, cytochrome c release, caspase 3 andPARP cleavage. Reversal of the HCV core-inducedER Ca2+ depletion by transfection of the sarco/ER cal-cium ATPase abolished all these effects except CHOP.Furthermore, the uptake of Ca2+ in the mitochondriainduces ROS which activates multiple signaling path-ways, including NF-jB, modulating apoptosis andinflammation, and STAT-3, a transcription factor thatcontrols cellular processes for cell survival, proliferation,differentiation and oncogenesis [76]. Activation of NF-jB and STAT-3 by HCV is associated with chronic liverdisease [77]. In addition, HCV-induced ER stress resultsin reduced protein glycosylation which disrupts theproper protein folding and assembly of MHC class Imolecules. Cells expressing HCV subgenomic repliconshave lower MHC class I cell surface expression [78,79].HCV-infected cells may thus go undetected by the

immune system by suppressing MHC class I antigen pre-sentation to cytotoxic T lymphocytes and the persistenceand pathogenesis of HCV may depend upon the ERstress-mediated interference of MHC class I assemblyand cell surface expression.

HCV gene products appear to modulate the UPR inexperimental cell systems, based upon a number of dis-parate observations. The composite evidence supportsthe interpretation that HCV burden favors inductionof the UPR but individual HCV proteins inhibit theresponses which would suppress viral protein produc-tion. It remains to be determined if these observationsare reflective of what occurs in vivo in HCV-infected liv-er and if an inadequate UPR contributes to the chronic-ity of viral infection or if the HCV-induced ER stressresponse promotes the progression to apoptosis andinflammation.

4.1.2. Hepatitis B virus (HBV) infection

Many HBV carriers are asymptomatic and have min-imal liver injury, despite extensive and ongoing intrahe-patic replication of the virus [80]. That implies that theHBV replication cycle is not directly cytotoxic to cells.The HBV surface antigen (HBsAg) consists of threestructurally related large, middle, and small envelopeproteins. The large form is translated from transcriptsspecified by a preS1 promoter, while the middle andsmall forms are translated from transcripts specified bya pre-S2 promoter. Overexpression of the large surfaceprotein of HBV in Huh7 cells results in a blockage ofsecretion of HBsAg which leads to an accumulation ofHBsAg in the ER lumen, which in turn induces expres-sion of GRP78 and GRP94 [81,82]. In transgenic mice,the intracellular retention of HBsAg in hepatocytescan cause pleiotropic physiological changes, groundglass morphology [83,84], and hypersensitivity to inflam-matory cytokines [85]. The retention of HBsAg in ERcan actually occur in natural infection. Two mutanttypes of the large HBV surface antigens with deletionsover the pre-S1 and pre-S2 regions, respectively, wereidentified [86,87]. It has been reported that expressionof the mutant proteins interferes with proper proteinfolding activity in the ER. Expression of the mutantHBV large surface proteins induced eIF2a phosphoryla-tion, nuclear translocation of NF-jB, activation of p38MAPK, and enhanced the expression of COX-2 in ML-1 and HuH-7 cells [88]. Higher expression of COX-2protein was detected in liver and kidney tissue of trans-genic mice expressing the mutant HBV large surfaceprotein in vivo. Expression of COX-2 mRNA was alsoobserved in human hepatocellular carcinoma tissueexpressing mutant HBV large surface proteins. Theselines of evidence provide important insights into cellularinflammation and carcinogenesis that are associatedwith latent ER stress due to HBV infection and chroniccarriage. As with HCV, it is uncertain if the UPR


protects against virus eradication and the role of ERstress in liver injury and inflammation needs to be betterdefined. Furthermore, immunosuppression in HIV co-infected patients and post-OLT is associated withenhanced viral replication of both HCV and HBV anda more severe, rapidly progressive liver disease. It istempting to speculate that ER stress contributes to thisoutcome.

4.2. Diabetes, obesity and hepatic metabolic disorder

Glucose metabolism and homeostasis are maintainedat the levels of insulin synthesis and secretion in the pan-creatic b-cell and peripheral utilization regulated byinsulin. Intracellular levels of glucose influence boththe secretion of insulin and insulin production [89–91].Although UPR signaling is a physiological mechanismto sustain insulin secretion, chronic ER stress not onlycontributes to the attrition of b-cell function but alsoto the impaired glucose homeostasis in diabetes[92–94]. The UPR signaling activates genes encodingglucose-regulated proteins in response to glucose/energydeprivation [95,96]. ER stress increases expression andactivity of glucose-6-phosphatase, one of the keyenzymes of gluconeogenesis, which modulates thecapacity for glucose release and glucose cycling in pri-mary rat hepatocytes and H4IIE liver cells [90]. TheUPR is required for survival of pancreatic b-cells duringintermittent fluctuations in blood glucose [97–99]. Dele-tion of PERK in humans and mice results in a pancreat-ic b-cell dysfunction and development of infancy-onsetdiabetes. Site-directed mutation of mouse eIF2 at thePERK phosphorylation site leads to a b-cell loss in ute-ro, suggesting that the PERK-eIF2a pathway of UPRsignaling is essential for proinsulin translation, b-cellfunction and survival. During the development of diabe-tes in Akita mice, both the transcription factor CHOPand the molecular chaperone GRP78 in the ER areinduced in the pancreas, and targeted disruption of theCHOP gene improved the glucose intolerance of hetero-zygous Akita mice indicative of the importance of ER ininsulin secretion in b-cells of the pancreas [100]. Thus,UPR is an important physiological component of insu-lin secretion. Physiological UPR promotes survival ofpancreatic b-cells while severe and prolonged ER stressin Akita mice promotes apoptosis of b-cells.

The ER also plays a crucial role in the regulation ofcellular responses to insulin. ER stress is increased inthe liver under diabetic conditions. GRP78 proteinwas increased in the liver of mice with high-fat diet-in-duced and genetic (ob/ob) obesity [98]. PERK and eIF2aphosphorylation were increased in the liver of obesemice compared with lean controls. ER stress in obesemice also led to suppression of insulin receptor signaling(insulin resistance) which was mediated by activation ofJNK [101,102]. Insulin receptor substrate 1 (IRS-1) and

Akt are substrates of JNK and are key molecules forinsulin signaling. Tunicamycin or thapsigargin inducedER stress and significantly inhibited insulin-stimulatedtyrosine phosphorylation of IRS-1. Tunicamycin pre-treatment also suppressed insulin-induced Akt phos-phorylation [101]. Inhibition of JNK activity with thesynthetic inhibitor SP600125 reversed the ER stress-in-duced serine phosphorylation of IRS-1. Pretreatmentof Fao cells with a highly specific inhibitory peptidederived from the JNK-binding protein, JIP, also pre-served insulin receptor signaling in cells exposed totunicamycin. Similar results were obtained with the syn-thetic JNK inhibitor, SP600125. The ER-resident oxy-gen-regulated protein 150 (ORP150) has been shownto protect cells from ER stress [102–105]. Overexpres-sion of ORP150 markedly improves insulin resistanceand ameliorates glucose tolerance in diabetic animalswhereas suppression of ORP150 in the liver of normalmice decreases insulin sensitivity [104]. The phosphory-lation state of IRS-1 and Akt as well as the expressionlevels of the enzymes of gluconeogenesis such as phos-phoenolpyruvate carboxykinase and glucose-6-phospha-tase were also altered by ORP150 overexpression. Theseresults indicate that ER stress promotes a JNK-depen-dent serine phosphorylation of IRS-1, which in turninhibits insulin receptor signaling [101]. Mice withheterozygous deficiency in XBP1 fed a high fat diet(obesity) develop insulin resistance and dysregulatedphosphorylation of IRS-1, suggesting the IREla–XBPlUPR pathway is critical for preventing insulin resis-tance. XBP-1 +/� mice fed high fat diet exhibited great-er PERK and JNK activation as a result of decreasedUPR protection. Thus, interfering with the UPR-in-duced production of chaperones worsens ER stress. Atpresent the triggering mechanism for hepatocellularER stress in diabetes/obesity is not certain but its devel-opment appears to be a major contributor to insulinresistance. Whether ER stress also directly contributesto the pathology of NASH is uncertain but possible.

4.3. Others: a1-antitrypsin deficiency and injury of

ischemia and reperfusion

a1-Antitrypsin (a1-AT) deficiency occurring in the liv-er is an example of a disorder associated with aberrantprotein accumulation in tissues and cellular compart-ments. The disease inducing form of a1-AT deficiencyis caused by a point mutation encoding a substitutionof lysine for glutamate-342 referred to as a1-ATZ orthe Z mutant [106]. The a1-ATZ is retained and accumu-lates in ER which results in its reduced secretion into theblood and body fluids. The reduction increases risk ofdeveloping emphysema in the lung because of inhibitionof connective tissue breakdown by neutrophil elastase,cathepsin G, and proteinase 3. In cell culture andtransgenic mice with a1-AT deficiency the ER retention


of a1-ATZ induced a marked autophagic response, inwhich injured ER is sequestered from the rest of thecytoplasm and then degraded by fusion with lysosomes[107]. Fasting induced steatosis in the PiZ transgenicmice [108]. Both UPR and ER overloading response(EOR) were activated after treatment with additionalstresses such as thapsigargin or elevated temperature.UPR activation was accompanied by a marked increasein grp78 promoter activity and in GRP78 and GRP94protein expression in the PiZ transfected cell lines com-pared with control. EOR activation led to an increase inNF-jB activity and degradation of IjB which was cor-related with IL-6 and IL-8 protein production in thePiZ transfected cells [107]. In the PiZ transgenic mice,the 58-kDa protein disulfide isomerase (PDI), an impor-tant oxidoreductase and chaperone of the ER, hasrecently been found to form a complex with PiZ, result-ing in a decrease of protein disulfide reductase activity ofthe ER. PiZ transgenic mice have a shift toward a morereduced ER environment and an elevation of cytoplas-mic chaperones (Hsp90 and Hsp70) and antioxidantenzymes (thioredoxin). Therefore, the toxic effects ofPiZ aggregation caused by a1-AT deficiency may bedue to lower availability of PDI and a decreased proteindisulfide reductase activity in the ER along with a cyto-plasmic stress [109]. However, Hidvegi et al. recentlycompared the effect of several different naturally occur-ring PiZ mutants which are retained in the ER [110];mutants which do not polymerize caused the UPRwhereas polymerogenic a1-ATZ did not cause UPR.Conversely ER stress and overload responses as evi-denced by the cleavage of caspase12 and BAP31 andNF-jB activation, respectively, were observed in poly-merogenic mutant expressing transgenic mice but notin non-polymerogenic mutant transgenic mice. Thus, itappears that GRP78 and the UPR do not sense reten-tion of insoluble polymers so a protective response doesnot occur while injurious components of ER stress areactivated. This work provides evidence that pro-death/pro-inflammatory signaling in response to ER retentioncan be dissociated from the UPR in some circumstances.What happens in the human liver expressing PiZ will beof interest-perhaps both types of responses (UPR andnon-UPR) will occur in different hepatocytes dependingon the level of mutant protein and other factors. It istempting to speculate that the a1-ATZ-induced ERstress response and EOR promote cell death and inflam-mation, respectively, leading to fibrosis and cirrhosis.

Ischaemia occurs when the reduction of blood flowdecreases the delivery of oxygen and substrates to main-tain cellular energy leading to decrease in intracellularATP level. ATP deficiency impairs the function of theCa2+ channel, releases Ca2+ from the ER to cytosol,and promotes cell death [111]. Reduced ER Ca2+ alsoalters the expression and activity of GRP78 andGRP94, thereby initiating several signal transduction

pathways. The expression of GRP78, CHOP and theXBP-1 spliced form (sXBP-1) is increased during humanliver transplantation suggesting an activation of theIRE1 pathway [112–115]. The PERK pathway is alsoactivated upon reperfusion, leading to a reduction ofthe overall rate of translation through phosphorylationof eIF2a. Increased generation of reactive oxygen spe-cies during reperfusion may enhance ER stress inducedinflammation and cell death causing additional cell inju-ry [116,117]. Reduction in ER stress induced hepatocel-lular injury in mice can be achieved by theadministration of sodium 4-phenylbutyrate (PBA), alow molecular weight fatty acid that acts as a chemicalchaperone [115]. PBA-treated mice had reduced pykno-sis, parenchymal hemorrhage, and neutrophil infiltra-tion during IR. The reduced injury was associatedwith a greater than 45% reduction in apoptosis due toa significant reduction of CHOP expression, caspase-12 activation, and eIF2a phosphorylation compared tountreated mice. Bax inhibitor-1 (BI-1) is an evolution-arily conserved ER protein that suppresses ER stressinduced apoptosis [116,117]. BI-1 is abundant in bothliver and kidney. Hepatic IR injury induced BI-1 mRNAin mouse liver, indicating that BI-1 may provide adap-tive protection of the liver from ER stress and IR injury.In BI-1 knockout mice under IR, increased histologicalinjury, serum transaminases, and hepatic apoptosis werefound to be associated with greater elevations in caspaseactivity, more activation of ATF6, and greater increasesin expression of CHOP and the sXBP-1 suggesting thatBI-1 is required for limiting tissue injury. These observa-tions on the role of ER stress reveal potential strategiesfor organ preservation and protection against IR injury.

5. Alcoholism, hyperhomocysteinemia, and ER stress

Steatosis, inflammation, apoptosis and fibrosis arecharacteristics of alcohol-induced liver injury. Althoughmany mechanisms have been implicated in the patho-genesis of alcoholic liver disease [118], alcohol-inducedhyperhomocysteinemia (HHcy) and ER stress hasrecently emerged as a novel mechanism for alcoholicliver disease [55,119–121].

Alcohol-induced HHcy is often observed. Alcoholicpatients have elevated plasma homocysteine (Hcy) levelswhich range from 10 to 120 lM compared to normal5–15 lM [122–124]. Hcy but not total B12 and B6 levelscorrelated with folate levels and blood alcohol levels.Even ‘‘social’’ drinking (30 g/d · 6 weeks) caused 20%increased Hcy and decreased folate [122,125]. Rats fedethanol orally exhibit a doubling of plasma Hcy despitenormal levels of folate, pyridoxal-phosphate (PLP) andB12 [126]. We have observed a 5- to 10-fold increase ofplasma Hcy levels in mice fed alcohol intragastricallyfor 4 weeks [55,119,127]. Similar elevations of plasma


Hcy were observed in TNFR1 null mice fed ethanol sug-gesting a minimal contribution of TNFa to ethanol-in-duced HHcy [128]. In the cells, Hcy is derived frommethionine after transmethylation reactions that useS-adenosylmethionine (SAM) as the methyl donor(Fig. 3). Methionine intake and transmethylation activ-ity determine the input of Hcy into the system. A certainamount of total Hcy is catabolically eliminated by trans-sulfuration to cysteine initiated by cystathionine-b-syn-thase (CBS), but about 50% in humans or male rats isconserved by remethylation to methionine which is cat-alyzed by methionine synthase (MS) and betaine-homo-cysteine methyltransferase (BHMT) [129,130]. Chronicalcohol consumption may have an impact on the expres-sion or enzyme activity of BHMT, MS, and CBS. Theenzyme activity of BHMT was inhibited by high concen-trations of acetaldehyde [118] and mRNA of BHMTwas reduced in the liver of mice with intragastric alcoholinfusion [127]. Ethanol feeding lowered methionine syn-thase leading to increased accumulation of 5-methyl tet-rahydrofolate and Hcy and to decreased levels ofbetaine, a product of choline oxidation and the substrateof BHMT [130]. In micropigs fed ethanol for 12 monthswith adequate folate, MS activity decreased by 20%which was associated with slightly decreased plasmamethionine, 20% increased plasma Hcy, and increasedhepatic SAH but no change in SAM [131–133]. Chronicalcohol increases choline uptake [134] and mitochondri-al oxidation to betaine [135] suggesting compensationfor increased demand for betaine. However, depletion

MTHFR

MS BHMT

Fig. 3. Methionine metabolism. B6, vitamin B6; B12, vitamin B12; BHMT,

GSH, glutathione; MAT, methionine adenosyltransferase; MS, methionine synt

of methyltransferases; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethi

of betaine by chronic alcohol feeding may be a majorfactor contributing to the alcoholic HHcy [119].

Alcohol-induced HHcy promotes ER stress, inflam-mation, and cell death. Excessive intracellular Hcy canbe converted by methionyl tRNA synthase to Hcy thio-lactone which has unique reactive properties. Hcy thio-lactone can cause homocysteinylation of lysine residuesand free amine groups of proteins resulting in malfold-ing and premature degradation [136]. Hcy also disruptsdisulfide bond formation and also can induce misfoldingof proteins traversing the ER leading to ER stress. Ele-vated levels of intracellular Hcy increase the expressionof several UPR genes, including GRP78, GRP94, Herpand RTP [32,54,55,121,137–141]. Hcy-induced ER stresscauses dysregulation of lipid biosynthesis by activatingthe SREBPs leading to increased hepatic biosynthesisand uptake of cholesterol and triglycerides [53–55,142].Hcy induces the expression of CHOP which is involvedin ER stress-induced cell death [143]. In both Chop nulland wild type mice fed alcohol, significantly increasedhepatomegaly, steatosis, and UPR indicated byincreased Grp78 mRNA were observed [128]. However,CHOP null mice exhibited the absence of hepatocellularapoptosis in response to alcohol feeding but no protec-tion against HHcy, steatosis and ER stress implying thatCHOP up-regulation occurs downstream of and con-tributes to ER stress-induced apoptosis.

Finally, Hcy-induced cell death is mimicked by otherER stress agents and is dependent on IRE-1 signaling.Activation of IRE-1 by Hcy leads to a rapid and

Homocysteine

MAT

CBS

betaine-homocysteine methyltransferase; CBS, cystathionine-b-synthase;

hase; MTHFR, 5, 10-methylenetetrahydrofolate reductase; R, substrates

onine; THF, tetrahydrofolate.


sustained activation of JNK [144–146], a result consis-tent with the finding that activation of JNK by ER stressinvolves binding of IRE-1 to TRAF2 [146]. Because per-sistent activation of JNK correlates with cell death[102,147], these studies provide further support for amechanism involving Hcy-induced programmed celldeath. In addition, feeding mice betaine to promotemethylation of Hcy to methionine ameliorated alco-hol-induced ER stress in mouse livers [55], indicatingthat Hcy-induced ER stress contributes to alcohol-in-duced liver injury. Hence, alcohol intake leads to HHcy,which in turn may elicit ER stress promoting alcohol-in-duced liver injury [119,123].

A feature common to HHcy and consequent ERstress induced by alcohol, MTHFR KO, CBS KO, orhigh methionine/low folate diet is the development offatty liver [53–55,148–150]. Activation and inductionof SREBP-1c and SREBP-2 as a consequence of ERstress accounts for the bulk of triglyceride and cholester-ol accumulation in these models. In the intragastric eth-anol infusion model, ER stress, up-regulation ofSREBPs, accumulation of triglycerides and cholesterol,as well as apoptosis and necroinflammation are attenu-ated by feeding quantities of betaine sufficient to lowerhomocysteine. Furthermore, a potential interactionbetween homocysteine and HCV-induced liver diseasehas been suggested by the correlation between HCV, ste-atosis and fibrosis in patients with an MTHFR poly-morphism (C667T) leading to HHcy [151]. It istempting to speculate that alcohol may interact withHCV in a similar fashion via HHcy and ER stress.

6. Conclusions

Much evidence is emerging that a critical determinantof liver disease is how the liver copes with stress. Stresscan emerge from exogenous sources through membranereceptors or internally from any organelle, i.e. nucleus,mitochondria, cytoskeleton, or ER. In each case, signalsare released which recruit built-in pathways which pro-mote a protective response or; when overwhelmed, apathological response. In the ER, perturbation in proteinload, protein folding, glycosylation, calcium sequestra-tion, or redox balance trigger a finely tuned response tocope, referred to as the UPR, which is vital to regulateand protect the ER by adjusting the levels of chaperones,protein degradative apparatus, protein synthesis, andmembrane lipid synthesis in an attempt to make moreER. However, when the hepatocytes cannot copebecause of the great extent or duration of ER stress,apoptosis and steatosis occur. It has been speculated thatthe apoptosis response to ER stress evolved as a mecha-nism for coping with viral infection by suicide of infectedcells. However, the exact role of the UPR and ER stressresponse in chronic HCV and HBV infection is unclear.

Evidence suggests that HCV may modify the UPR tosupport continued virus production. However, cell deathand inflammation in HCV and HBV infection may resultof ER stress response and EOR. In the case of insulinresistance as well as alcoholic liver disease, a potentiallyimportant contribution of ER stress has emerged. Manyquestions remain to be answered in this exciting new areaof research. A fundamental question is whether the liveris coping with protein overload or misfolded proteins bythe physiological UPR or whether the UPR cannot copeleading to ER stress induced apoptosis, inflammationand steatosis. The state of the art at present stronglypoints to the occurrence of ER stress in various modelsof liver disease but a great deal of research is needed tounderstand the contribution of impaired UPR orincreased ER stress response versus the myriad of othercellular and intercellular interactions in the pathogenesisof liver disease.

Acknowledgements

This work was supported in part by the NationalInstitute of Diabetes and Digestive and Kidney DiseasesP30DK048522-11 (C.J.) and by the U.S. NationalInstitute of Alcohol Abuse and Alcoholism R01AA014428-03 (N.K. and C.J.) and P50AA11999.

References

[1] Lindquist S. The heat-shock response. Annu Rev Biochem1986;55:1151–1191.

[2] Ma Y, Hendershot LM. The unfolding tale of the unfoldedprotein response. Cell 2001;107:827–830.

[3] Wiest DL, Burkhardt JK, Hester S, Hortsch M, Meyer DI,Argon Y. Membrane biogenesis during B cell differentiation:most endoplasmic reticulum proteins are expressed coordinately.J Cell Biol 1990;110:1501–1511.

[4] Iwakoshi NN, Lee AH, Vallabhajosyula P, Otipoby KL,Rajewsky K, Glimcher LH. Plasma cell differentiation and theunfolded protein response intersect at the transcription factorXBP-1. Nat Immunol 2003;4:321–329.

[5] Gunn KE, Gifford NM, Mori K, Brewer JW. A role for theunfolded protein response in optimizing antibody secretion. MolImmunol 2004;41:919–927.

[6] Kaufman RJ. Stress signaling from the lumen of the endoplasmicreticulum: coordination of gene transcriptional and translationalcontrols. Genes Dev 1999;13:1211–1233.

[7] Mori K. Tripartite management of unfolded proteins in theendoplasmic reticulum. Cell 2000;101:451–454.

[8] Patil C, Walter P. Intracellular signaling from the endoplasmicreticulum to the nucleus: the unfolded protein response in yeastand mammals. Curr Opin Cell Biol 2001;13:349–355.

[9] Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcrip-tional and translational control in the Mammalian unfoldedprotein response. Annu Rev Cell Dev Biol 2002;18:575–599.

[10] Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress:cell life and death decisions. J Clin Invest 2005;115:2656–2664.

[11] Zhang K, Kaufman RJ. The unfolded protein response: a stresssignaling pathway critical for health and disease. Neurology2006;66:S102–S109.


[12] Lee AS, Delegeane AM, Baker V, Chow PC. Transcriptionalregulation of two genes specifically induced by glucose starvationin a hamster mutant fibroblast cell line. J Biol Chem1983;258:597–603.

[13] Munro S, Pelham HR. An Hsp70-like protein in the ER: identitywith the 78 kd glucose-regulated protein and immunoglobulinheavy chain binding protein. Cell 1986;46:291–300.

[14] Tirasophon W, Welihinda AA, Kaufman RJ. A stress responsepathway from the endoplasmic reticulum to the nucleus requiresa novel bifunctional protein kinase/endoribonuclease (Ire1p) inmammalian cells. Genes Dev 1998;12:1812–1824.

[15] Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda M,Ron D. Cloning of mammalian Ire1 reveals diversity in the ERstress responses. EMBO J 1998;17:5708–5717.

[16] Haze K, Yoshida H, Yanagi H, Yura T, Mori K. Mammaliantranscription factor ATF6 is synthesized as a transmembraneprotein and activated by proteolysis in response to endoplasmicreticulum stress. Mol Biol Cell 1999;10:3787–3799.

[17] Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, et al.Identification and characterization of pancreatic eukaryoticinitiation factor 2 alpha-subunit kinase, PERK, involved intranslational control. Mol Cell Biol 1998;18:7499–7509.

[18] Dorner AJ, Wasley LC, Kaufman RJ. Overexpression of GRP78mitigates stress induction of glucose regulated proteins andblocks secretion of selective proteins in Chinese hamster ovarycells. Eur Mol Biol Org J 1992;11:1563–1571.

[19] Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D.Dynamic interaction of BiP and ER stress transducers in theunfolded-protein response. Nat Cell Biol 2000;2:326–332.

[20] Shen J, Chen X, Hendershot L, Prywes R. ER stress regulationof ATF6 localization by dissociation of BiP/GRP78 binding andunmasking of Golgi localization signals. Dev Cell 2002;3:99–111.

[21] Liu CY, Wong HN, Schauerte JA, Kaufman RJ. The proteinkinase/endoribonuclease IRE1alpha that signals the unfoldedprotein response has a luminal N-terminal ligand-independentdimerization domain. J Biol Chem 2002;277:18346–18356.

[22] Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP,et al. IRE1 couples endoplasmic reticulum load to secretorycapacity by processing the XBP-1 mRNA. Nature2002;415:92–96.

[23] Hetz C, Bernasconi P, Fisher J, Lee AH, Bassik MC, AntonssonB, et al. Proapoptotic BAX and BAK modulate the unfoldedprotein response by a direct interaction with IRE1alpha. Science2006;312:572–576.

[24] Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subsetof endoplasmic reticulum resident chaperone genes in theunfolded protein response. Mol Cell Biol 2003;23:7448–7459.

[25] Brown MS, Ye J, Rawson RB, Goldstein JL. Regulatedintramembrane proteolysis: a control mechanism conserved frombacteria to humans. Cell 2000;1:391–398.

[26] Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R,et al. ER stress induces cleavage of membrane-bound ATF6 bythe same proteases that process SREBPs. Mol Cell2000;6:1355–1364.

[27] Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M,et al. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for themammalian unfolded protein response. Mol Cell Biol2000;20:6755–6767.

[28] Li M, Baumeister P, Roy B, Phan T, Foti D, Luo S, et al. ATF6as a transcription activator of the endoplasmic reticulum stresselement: thapsigargin stress-induced changes and synergisticinteractions with NF-Y and YY1. Mol Cell Biol2000;20:5096–5106.

[29] Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M,et al. Endoplasmic reticulum stress-induced formation of tran-scription factor complex ERSF including NF-Y (CBF) and

activating transcription factors 6alpha and 6beta that activatesthe mammalian unfolded protein response. Mol Cell Biol2001;21:1239–1248.

[30] Kokame K, Kato H, Miyata T. Identification of ERSE-II, a newcis-acting element responsible for the ATF6-dependent mamma-lian unfolded protein response. J Biol Chem 2001;276:9199–9205.

[31] Parker R, Phan T, Baumeister P, Roy B, Cheriyath V, Roy AL,et al. Identification of TFII-I as the endoplasmic reticulum stressresponse element binding factor ERSF: its autoregulation by stressand interaction with ATF6. Mol Cell Biol 2001;21:3220–3233.

[32] Ma Y, Hendershot LM. Herp is dually regulated by both theendoplasmic reticulum stress-specific branch of the unfoldedprotein response and a branch that is shared with other cellularstress pathways. J Biol Chem 2004;279:13792–13799.

[33] Yamamoto K, Yoshida H, Kokame K, Kaufman RJ, Mori K.Differential contributions of ATF6 and XBP1 to the activation ofendoplasmic reticulum stress-responsive cis-acting elementsERSE, UPRE and ERSE-II. J Biochem (Tokyo)2004;136:343–350.

[34] Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y,Jungreis R, et al. CHOP induces death by promoting proteinsynthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev 2004;18:3066–3077.

[35] Werner ED, Brodsky JL, McCracken AA. Proteasome-depen-dent endoplasmic reticulum-associated protein degradation: anunconventional route to a familiar fate. Proc Natl Acad Sci USA1996;93:13797–13801.

[36] Dorner AJ, Wasley LC, Kaufman RJ. Protein dissociation fromGRP78 and secretion are blocked by depletion of cellular ATPlevels. Proc Natl Acad Sci USA 1990;87:7429–7432.

[37] Leppa S, Bohmann D. Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene1999;18:6158–6162.

[38] Yoneda T, Imaizumi K, Oono K, et al. Activation of caspase-12,an endoplastic reticulum (ER) resident caspase, through tumornecrosis factor receptor-associated factor 2-dependent mecha-nism in response to the ER stress. J Biol Chem2001;276:13935–13940.

[39] Nishitoh H, Matsuzawa A, Tobiume K, et al. ASK1 is essentialfor endoplasmic reticulum stress-induced neuronal cell deathtriggered by expanded polyglutamine repeats. Genes Dev2002;16:1345–1355.

[40] Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediatesendoplasmic-reticulum-specific apoptosis and cytotoxicity byamyloid-beta. Nature 2000;403:98–103.

[41] Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasu-hiko Y. An ER stress-specific caspase cascade in apoptosis:cytochrome c-independent activation of caspase-9 by caspase-12.J Biol Chem 2002;3:3.

[42] Boyce M, Yuan J. Cellular response to endoplasmic reticulumstress: a matter of life or death. Cell Death Differ2006;13:363–373.

[43] Ma Y, Brewer JW, Diehl JA, Hendershot LM. Two distinctstress signaling pathways converge upon the CHOP promoterduring the mammalian unfolded protein response. J Mol Biol2002;318:1351–1365.

[44] Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M,et al. An integrated stress response regulates amino acidmetabolism and resistance to oxidative stress. Mol Cell2003;11:619–633.

[45] Pahl HL, Baeuerle PA. The ER-overload response: activation ofNF-kappa B. Trends Biochem Sci 1997;22:63–67.

[46] Buytaert E, Callewaert G, Hendrickx N, Scorrano L, HartmannD, Missiaen L, et al. Role of endoplasmic reticulum depletionand multidomain proapoptotic BAX and BAK proteins inshaping cell death after hypericin-mediated photodynamic ther-apy. FASEB J 2006;20:756–758.


[47] Omori Y, Imai J, Watanabe M, Komatsu T, Suzuki Y, KataokaK, et al. CREB-H: a novel mammalian transcription factorbelonging to the CREB/ATF family and functioning via the box-B element with a liver-specific expression. Nucleic Acids Res2001:2154–2162.

[48] Yoo JY, Desiderio S. Innate and acquired immunity intersect ina global view of the acute-phase response. Proc Natl Acad SciUSA 2003:1157–1162.

[49] Zhang K, Shen X, Wu J, Sakaki K, Saunders T, Rutkowski DT,et al. Endoplasmic reticulum stress activates cleavage of CRE-BH to induce a systemic inflammatory response. Cell2006;124:587–599.

[50] Sakai J, Nohturfft A, Cheng D, Ho YK, Brown MS, GoldsteinJL. Identification of complexes between the COOH-terminaldomains of sterol regulatory element-binding proteins (SREBPs)and SREBP cleavage-activating protein. J Biol Chem1997;272:20213–20221.

[51] Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors formembrane sterols. Cell 2006;124:35–46.

[52] Brown MS, Goldstein JL. The SREBP pathway: regulation ofcholesterol metabolism by proteolysis of a membrane-boundtranscription factor. Cell 1997;89:331–340.

[53] Ron D, Oyadomari S. Lipid phase perturbations and theunfolded protein response. Dev Cell 2004;7:287–288.

[54] Werstuck GH, Lentz SR, Dayal S, Hossain GS, Sood SK, ShiYY, et al. Homocysteine-induced endoplasmic reticulum stresscauses dysregulation of the cholesterol and triglyceride biosyn-thetic pathways. J Clin Invest 2001;107:1263–1273.

[55] Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia,endoplasmic reticulum stress, and liver injury in alcohol-fedmice. Gastroenterology 2003;124:1488–1499.

[56] Hansson GK. Inflammation, atherosclerosis, and coronaryartery disease. N Engl J Med 2005;352:1685–1695.

[57] Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A.Internal ribosome entry site within hepatitis C virus RNA. JVirol 1992:1476–1483.

[58] Wang C, Sarnow P, Siddiqui A. Translation of human hepatitisC virus RNA is cultured cells is mediated by an internalribosome-binding mechanism. J Virol 1993:3338–3344.

[59] Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM.Expression and identification of hepatitis C virus polyproteincleavage products. J Virol 1993:1385–1395.

[60] Gong G, Waris G, Tanveer R, Siddiqui A. Human hepatitis Cvirus NS5A protein alters intracellular calcium levels, inducesoxidative stress, and activates STAT-3 and NF-jB. Proc NatlAcad Sci USA 2001:9599–9604.

[61] Tardif Keith D, Mori Kazutoshi, Siddiqui Aleem. Hepatitis Cvirus subgenomic replicons induce endoplasmic reticulum stressactivating an intracellular signaling pathway. J Virol2002:7453–7459.

[62] Egger Denise, Wolk Benno, Gosert Rainer, Bianchi Leonardo,Blum Hubert E, Moradpour Darius, et al. Expression ofhepatitis C virus proteins induces distinct membrane alterationsincluding a candidate viral replication complex. J Virol2002:5974–5984.

[63] Zheng Y, Gao B, Ye L, Kong L, Jing W, Yang X, et al.Hepatitis C virus non-structural protein NS4B can modulate anunfolded protein response. J Microbiol 2005;43:529–536.

[64] Tardif KD, Mori K, Kaufman RJ, Siddiqui A. Hepatitis C virussuppresses the IRE1–XBP1 pathway of the unfolded proteinresponse. J Biol Chem 2004:17158–17164.

[65] Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay LO,Herscovics A, et al. A novel ER alpha-mannosidase-like proteinaccelerates ER-associated degradation. EMBO Rep2001;2:415–422.

[66] Tardif KD, Waris G, Siddiqui A. Hepatitis C virus, ER stress,and oxidative stress. Trends Microbiol 2005;13:159–163.

[67] Michael Jr J, Gale Marcus J, Korth Norina M, Tang Seng-LaiTan, Deborah A, Hopkins Thomas E, et al. Evidence thathepatitis C virus resistance to interferon is mediated throughrepression of the PKR protein kinase by the nonstructuralprotein 5A. Virology 1997:217–227.

[68] Taylor DR, Shi ST, Romano PR, Barber GN, Lai MM.Inhibition of the interferon-inducible protein kinase PKR byHCV E2 protein. Science 1999:107–110.

[69] He B. Viruses, endoplasmic reticulum stress, and interferonresponses. Cell Death Differ 2006;13:393–403.

[70] Harding HP, Yuhong Zhang, Ron D. Protein translation andfolding are coupled by an endoplasmic-reticulum-resident kinase.Nature 1999:271–274.

[71] Prostko CR, Dholakia JN, Brostrom MA, Brostrom CO.Activation of the double-stranded RNA-regulated protein kinaseby depletion of endoplasmic reticular calcium stores. J BiolChem 1995:6211–6215.

[72] Pavio N, Romano PR, Graczyk TM, Feinstone SM, Taylor DR.Protein synthesis and endoplasmic reticulum stress can bemodulated by the hepatitis C virus envelope protein E2 throughthe eukaryotic initiation factor 2alpha kinase PERK. J Virol2003;77:3578–3585.

[73] Chan SW, Egan PA. Hepatitis C virus envelope proteins regulateCHOP via induction of the unfolded protein response. FASEB J2005;19:1510–1512.

[74] Liberman E, Fong YL, Selby MJ, Choo QL, Cousens L, HoughtonM, et al. Activation of the grp78 and grp94 promoters by hepatitisC virus E2 envelope protein. J Virol 1999;73:3718–3722.

[75] Benali-Furet NL, Chami M, Houel L, De Giorgi F, Vernejoul F,Lagorce D, et al. Hepatitis C virus core triggers apoptosis inliver cells by inducing ER stress and ER calcium depletion.Oncogene 2005;24:4921–4933.

[76] Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis.Oncogene 2000;19:2474–2488.

[77] Waris G, Turkson J, Hassanein T, Siddiqui A. Hepatitis C virus(HCV) constitutively activates STAT-3 via oxidative stress:roleof STAT-3 in HCV replication. J Virol 2005;79:1569–1580.

[78] Tardif KD, Siddiqui A. Cell surface expression of majorhistocompatibility complex class I molecules is reduced inhepatitis C virus subgenomic replicon-expressing cells. J Virol2003;77:11644–11650.

[79] McKiernan SM, Hagan R, Curry M, McDonald GS, Kelly A,Nolan N, et al. Distinct MHC class I and II alleles are associatedwith hepatitis C viral clearance, originating from a single source.Hepatology 2004;40:108–114.

[80] Ganem D, Prince AM. Hepatitis B virus infection – natural historyand clinical consequences. N Engl J Med 2004;350:1118–1129.

[81] Xu Z, Jensen G, Yen TS. Activation of hepatitis B virus Spromoter by the viral large surface protein via induction of stressin the endoplasmic reticulum. J Virol 1997;71:7387–7392.

[82] Chua PK, Wang RY, Lin MH, Masuda T, Suk FM, Shih C.Reduced secretion of virions and hepatitis B virus (HBV) surfaceantigen of a naturally occurring HBV variant correlates with theaccumulation of the small S envelope protein in the endoplasmicreticulum and Golgi apparatus. J Virol 2005;79:13483–13496.

[83] Chisari FV, Filippi P, McLachlan A, Milich DR, Riggs M, LeeS. Expression of hepatitis B virus large envelope polypeptideinhibits hepatitis B surface antigen secretion in transgenic mice. JVirol 1986;60:880–887.

[84] Chisari FV, Filippi P, Buras J, McLachlan A, Popper H, PinkertCA, et al. Structural and pathological effects of synthesis ofhepatitis B virus large envelope polypeptide in transgenic mice.Proc Natl Acad Sci USA 1987;84:6909–6913.

[85] Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, ChisariFV. HBsAg retention sensitizes the hepatocyte to injury byphysiological concentrations of interferon-gamma. Hepatology1992;16:655–663.


[86] Wang HC, Chang WT, Chang WW, Wu HC, Huang W, Lei HY,et al. Hepatitis B virus pre-S2 mutant upregulates cyclin Aexpression and induces nodular proliferation of hepatocytes.Hepatology 2005;41:761–770.

[87] Hsieh YH, Su IJ, Wang HC, Chang WW, Lei HY, Lai MD,et al. Pre-S mutant surface antigens in chronic hepatitis B virusinfection induce oxidative stress and DNA damage. Carcinogen-esis 2004;25:2023–2032.

[88] Hung JH, Su IJ, Lei HY, Wang HC, Lin WC, Chang WT, et al.Endoplasmic reticulum stress stimulates the expression of cyclo-oxygenase-2 through activation of NF-kappaB and pp38 mito-gen-activated protein kinase. J Biol Chem2004;279:46384–46392.

[89] Hotamisligil GS. Role of endoplasmic reticulum stress and c-JunNH2-terminal kinase pathways in inflammation and origin ofobesity and diabetes. Diabetes 2005;54:S73–S78.

[90] Itoh N, Okamoto H. Translational control of proinsulinsynthesis by glucose. Nature 1980;283:100–102.

[91] Lipson KL, Fonseca SG, Urano F. Endoplasmic reticulumstress-induced apoptosis and auto-immunity in diabetes. CurrMol Med 2006;6:71–77.

[92] Day CP, James O. Steatohepatitis: a tale of two ‘‘hits’’?Gastroenterology 1998;114:842–845.

[93] Listenberger LL, Ory DS, Schaffer JE. Palmitate-induced apop-tosis can occur through a ceramide-independent pathway. J BiolChem 2001;276:14890–14895.

[94] Wang D, Wei Y, Pagliassotti MJ. Saturated fatty acids promoteendoplasmic reticulum stress and liver injury in rats with hepaticsteatosis. Endocrinology 2006;147:943–951.

[95] Pouyssegur J, Shiu R, Pastan I. Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a blockin glycoprotein synthesis or glucose deprivation. Cell1977;11:941–947.

[96] Wang D, Wei Y, Schmoll D, Maclean KN, Pagliassotti MJ.Endoplasmic reticulum stress increases glucose-6-phosphataseand glucose cycling in liver cells. Endocrinology2006;147:350–358.

[97] Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P,et al. Translational control is required for the unfolded proteinresponse and in vivo glucose homeostasis. Mol Cell2001;7:1165–1176.

[98] Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, Plesken H,et al. Diabetes mellitus and exocrine pancreatic dysfunction inperk�/� mice reveals a role for translational control in secretorycell survival. Mol Cell 2001;7:1153–1163.

[99] Delepine M, Nicolino M, Barrett T, Golamaully M, MarkLa-throp G, Julier C. EIF2AK3, encoding translation initiationfactor 2-alpha kinase 3, is mutated in patients with wolcott-rallison syndrome. Nat Genet 2000;25:406–409.

[100] Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulumstress pathway on pancreatic beta-cells and diabetes mellitus. ExpBiol Med (Maywood) 2003;228:1213–1217.

[101] Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E,et al. Endoplasmic reticulum stress links obesity, insulin action,and type 2 diabetes. Science 2004;306:457–461.

[102] Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, HardingHP, et al. Coupling of stress in the ER to activation of JNKprotein kinases by transmembrane protein kinase IRE1. Science2000:664–666.

[103] Tamatani M, Matsuyama T, Yamaguchi A, Mitsuda N,Tsukamoto Y, Taniguchi M, et al. ORP150 protects againsthypoxia/ischemia-induced neuronal death. Nat Med2001;7:317–323.

[104] Nakatani Y, Kaneto H, Hatazaki M, Yoshiuchi K, KawamoriD, Sakamoto K, et al. Increased stress protein ORP150 auto-antibody production in Type 1 diabetic patients. Diabet Med2006;23:216–219.

[105] Tsukamoto Y, Kuwabara K, Hirota S, et al. 150-kDa oxygen-regulated protein is expressed in human atherosclerotic plaquesand allows mononuclear phagocytes to withstand cellular stresson exposure to hypoxia and modified low density lipoprotein. JClin Invest 1996;98:1930–1941.

[106] Teckman JH, Lindblad D. Alpha-1-antitrypsin deficiency: diag-nosis, pathophysiology, and management. Curr GastroenterolRep 2006;8:14–20.

[107] Lawless MW, Greene CM, Mulgrew A, Taggart CC, O’Neill SJ,McElvaney NG. Activation of endoplasmic reticulum-specificstress responses associated with the conformational disease Zalpha 1-antitrypsin deficiency. J Immunol 2004;172:5722–5726.

[108] Teckman JH, An JK, Loethen S, Perlmutter DH. Fasting inalpha1-antitrypsin deficient liver: constitutive activation ofautophagy. Am J Physiol Gastrointest Liver Physiol2002;283:G1156–G1165.

[109] Papp E, Szaraz1 P, Korcsmaros T, Csermely P. Changes ofendoplasmic reticulum chaperone complexes, redox state, andimpaired protein disulfide reductase activity in misfolding{ralpha}1-antitrypsin transgenic mice. FASEB J 2006.

[110] Hidvegi T, Schmidt BZ, Hale P, Perlmutter DH. Accumulationof mutant alpha1-antitrypsin Z in the endoplasmic reticulumactivates caspases-4 and -12, NFkappaB, and BAP31 but not theunfolded protein response. J Biol Chem 2005;280:39002–39015.

[111] Sakon M, Ariyoshi H, Umeshita K, Monden M. Ischemia–reperfusion injury of the liver with special reference to calcium-dependent mechanisms. Surg Today 2002;32:1–12.

[112] Li C, Jackson RM. Reactive species mechanisms of cellularhypoxia-reoxygenation injury. Am J Physiol Cell Physiol2002;282:C227–C241.

[113] Tilg H, Ceska M, Vogel W, Herold M, Margreiter R, Huber C.Interleukin-8 serum concentrations after liver transplantation.Transplantation 1992;53:800–803.

[114] Emadali A, Nguyen DT, Rochon C, Tzimas GN, Metrakos PP,Chevet E. Distinct endoplasmic reticulum stress responses aretriggered during human liver transplantation. J Pathol2005;207:111–118.

[115] Vilatoba M, Eckstein C, Bilbao G, Smyth CA, Jenkins S,Thompson JA, et al. Sodium 4-phenylbutyrate protectsagainst liver ischemia reperfusion injury by inhibition ofendoplasmic reticulum-stress mediated apoptosis. Surgery2005;138:342–351.

[116] Bailly-Maitre B, Fondevila C, Kaldas F, Droin N, Luciano F,Ricci JE, et al. Cytoprotective gene bi-1 is required for intrinsicprotection from endoplasmic reticulum stress and ischemia–reperfusion injury. Proc Natl Acad Sci USA 2006.

[117] Chae HJ, Kim HR, Xu C, Bailly-Maitre B, Krajewska M,Krajewski S, et al. BI-1 regulates an apoptosis pathway linked toendoplasmic reticulum stress. Mol Cell 2004;15:355–366.

[118] Lieber CS. Pathogenesis and treatment of alcoholic liver disease:progress over the last 50 years. Rocz Akad Med Bialymst2005;50:7–20.

[119] Ji C, Kaplowitz N. Hyperhomocysteinemia, endoplasmic retic-ulum stress, and alcoholic liver injury. World J Gastroenterol2004;10:1699–1708.

[120] Coll O, Colell A, Garcia-Ruiz C, Kaplowitz N, Fernandez-ChecaJC. Sensitivity of the 2-oxoglutarate carrier to alcohol intakecontributes to mitochondrial glutathione depletion. Hepatology2003;38:692–702.

[121] Esfandiari F, Villanueva JA, Wong DH, French SW, HalstedCH. Chronic ethanol feeding and folate deficiency activatehepatic endoplasmic reticulum stress pathway in micropigs. Am JPhysiol Gastrointest Liver Physiol 2005;289:G54–G63.

[122] Bleich S, Bleich K, Kropp S, Bittermann HJ, Degner D, SperlingW, et al. Moderate alcohol consumption in social drinkers raisesplasma homocysteine levels: a contradiction to the ‘FrenchParadox’? Alcohol Alcohol 2001;36:189–192.


[123] Carmel R, James SJ. Alcohol abuse: an important cause of severehyperhomocysteinemia. Nutr Rev 2002;60:215–221.

[124] Sakuta H, Suzuki T. Alcohol consumption and plasma homo-cysteine. Alcohol 2005;37:73–77.

[125] Bleich S, Bandelow B, Javaheripour K, Muller A, Degner D,Wilhelm J, et al. Hyperhomocysteinemia as a new risk factor forbrain shrinkage in patients with alcoholism. Neurosci Lett2003;335:179–182.

[126] Stickel F, Choi SW, Kim YI, Bagley PJ, Seitz HK, Russell RM,et al. Effect of chronic alcohol consumption on total plasmahomocysteine level in rats. Alcohol Clin Exp Res2000;24:259–264.

[127] Ji C, Mehrian-Shai R, Chan C, Hsu YH, Kaplowitz N. Role ofCHOP in hepatic apoptosis in the murine model of intragastricethanol feeding. Alcohol Clin Exp Res 2005;29:1496–1503.

[128] Ji C, Deng Q, Kaplowitz N. Role of TNF-alpha in ethanol-induced hyperhomocysteinemia and murine alcoholic liver inju-ry. Hepatology 2004;40:442–451.

[129] Kenyon SH, Nicolaou A, Gibbons WA. The effect of ethanoland its metabolites upon methionine synthase activity in vitro.Alcohol 1998;15:305–309.

[130] Barak AJ, Beckenhauer HC, Tuma DJ. Betaine, ethanol, and theliver: a review. Alcohol 1996;13:395–398.

[131] Halsted CH, Villanueva J, Chandler CJ, Stabler SP, Allen RH,Muskhelishvili L, et al. Ethanol feeding of micropigs altersmethionine metabolism and increases hepatocellular apoptosisand proliferation. Hepatology 1996;23:497–505.

[132] Halsted CH, Villanueva JA, Devlin AM, Niemela O, Parkkila S,Garrow TA, et al. Folate deficiency disturbs hepatic methioninemetabolism and promotes liver injury in the ethanol-fed micro-pig. Proc Natl Acad Sci USA 2002;99:10072–10077.

[133] Villanueva JA, Halsted CH. Hepatic transmethylation reactionsin micropigs with alcoholic liver disease. Hepatology2004;39:1303–1310.

[134] Tuma DJ, Keefer RC, Beckenhauer HC, Barak AJ. Effect ofethanol on uptake of choline by the isolated perfused rat liver.Biochim Biophys Acta 1970;218:141–147.

[135] Thompson JA, Reitz RC. Studies of the acute and chronic effectsof ethanol ingestion on choline oxidation. Ann N Y Acad Sci1976;273:194–204.

[136] Jakubowski H. Protein homocysteinylation: possible mechanismunderlying pathological consequences of elevated homocysteinelevels. FASEB J 1999;13:2277–2283.

[137] Outinen PA, Sood SK, Liaw PC, Sarge KD, Maeda N, Hirsh J,et al. Characterization of the stress-inducing effects of homo-cysteine. Biochem J 1998;332:213–221.

[138] Outinen PA, Sood SK, Pfeifer SI, Pamidi S, Podor TJ, Li J, et al.Homocysteine-induced endoplasmic reticulum stress and growtharrest leads to specific changes in gene expression in humanvascular endothelial cells. Blood 1999;94:959–967.

[139] Zhang C, Cai Y, Adachi MT, Oshiro S, Aso T, Kaufman RJ,et al. Homocysteine induces programmed cell death in humanvascular endothelial cells through activation of the unfoldedprotein response. J Biol Chem 2001;276:35867–35874.

[140] Agarwala KL, Kokame K, Kato H, Miyata T. Phosphorylationof RTP, an ER stress-responsive cytoplasmic protein. BiochemBiophys Res Commun 2000;272:641–647.

[141] Dimitrova KR, DeGroot K, Myers AK, Kim YD. Estrogen andhomocysteine. Cardiovasc Res 2002;53:577–588.

[142] Roybal CN, Yang S, Sun CW, Hurtado D, Vander Jagt DL,Townes TM, et al. Homocysteine increases the expression ofVEGF by a mechanism involving endoplasmic reticulum stressand transcription factor ATF4. J Biol Chem2004;279:14844–14852.

[143] Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A, Mi LJ,et al. Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein(CHOP/GADD153). Mol Cell Biol1996;16:4273–4280.

[144] Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S, et al.Homocysteine responsive ATF3 gene expression in human vascu-lar endothelial cells: activation of c-Jun NH(2)-terminal kinase andpromoter response element. Blood 2000;96:2140–2148.

[145] Zhang C, Kawauchi J, Adachi MT, Hashimoto Y, Oshiro S, AsoT, et al. Activation of JNK and transcriptional repressor ATF3/LRF1 through the IRE1/TRAF2 pathway is implicated inhuman vascular endothelial cell death by homocysteine. BiochemBiophys Res Commun 2001;289:718–724.

[146] Nishitani Y, Matsumoto H. Ethanol rapidly causes activation ofJNK associated with ER stress under inhibition of ADH. FEBSLett 2006;580:9–14.

[147] Chen YR, Meyer CF, Tan TH. Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis.J Biol Chem 1996;271:631–634.

[148] Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R,Malinow MR, et al. Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia.Proc Natl Acad Sci USA 1995;92:1585–1589.

[149] Chen Z, Karaplis AC, Ackerman SL, Pogribny IP, Melnyk S,Lussier-Cacan S, et al. Mice deficient in methylenetetrahydrof-olate reductase exhibit hyperhomocysteinemia and decreasedmethylation capacity, with neuropathology and aortic lipiddeposition. Hum Mol Genet 2001;10:433–443.

[150] Ji C, Chan C, Kaplowitz N. Predominant role of SREBPlipogenic pathways in hepatic steatosis in the murine intragastricethanol feeding model. J Hepatol 2006; doi:10.1016/j.jhep.2006.05.009.

[151] Luigi E Adinolfi, Diego Ingrosso, Giuseppe Cesaro, AmeliaCimmino, Maria D’Anto, et al. Hyperhomocysteinemia and theMTHFR C677T polymorphism promote steatosis and fibrosis inchronic hepatitis C patients. Hepatology 2005;41:995–1003.

Documents

ER stress: Can the liver cope?