ER stress: Can the liver cope?

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1 Journal of Hepatology 45 (2006) 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 signaling. 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 protein response (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 consequences including hepatic fat accumulation, inflammation and cell death which can lead to liver disease or worsen underlying causes of liver injury, such as viral or diabetes-obesity-related liver disease. Ó 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 turned over inside cells. When subjected to stress from denatured or malfunctioning proteins, eukaryotic cells apparently use two mechanisms to maintain protein stability. One is heat-shock response which occurs in the Available online 15 June 2006 * Corresponding author. Tel.: ; fax: address: chengji@usc.edu (C. Ji). Abbreviations: ASK1, apoptosis signaling kinase 1; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine-b-synthase; CHOP, C/EBP homologous protein; eif2a, eukaryotic initiation factor-2a; EOR, ER overloading response; ERAD, ER-associated degradation; 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, homocysteine-induced protein; Hcy, homocysteine; HHcy, hyperhomocysteinemia; HSP, heat-shock proteins; IRE1a, type-i ER transmembrane protein 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 proteolysis; SAH,S-adenosylhomocysteine; SAM, S-adenosylmethionine; SREBP, sterol regulatory element-binding protein; UPR, unfolded protein response; UPRE, UPR response elements; XBP-1, X-box DNAbinding 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 most eukaryotic cells, the endoplasmic reticulum (ER), and is characterized by upregulation of molecular chaperones which are a class of proteins that are highly conserved in all life forms and help other polypeptides reach a proper conformation or cellular location without themselves becoming part of the final structure. Unlike the heat-shock-response which is exclusively a stress response, the UPR is a normal physiological process required for organelle expansion to promote more protein folding and secretion during differentiation of specialized secretory cells such as mature B cells and those in the liver or pancreas [2 5] and also for safeguarding protein synthesis, post-translational modifications, folding and secretion, calcium storage and signaling, and lipid biosynthesis [6 11]. Under normal conditions the ER maintains high concentrations of resident calcium-dependent chaperone proteins, such as glucose-regulated protein-78 (GRP78 also known as BiP) and GRP94 [12,13], a high level of calcium and an oxidized environment. Only properly folded proteins are allowed to reach their final destination, whereas unfolded and misfolded proteins are exported or /$32.00 Ó 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 322 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) dislocated from the ER and degraded by cytoplasmic proteasomes. Perturbations, such as elevated secretory protein synthesis, over-expression and/or accumulation of mutant proteins, glucose deprivation, altered glycosylation, ER calcium depletion, shifting of redox status to 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 signaling pathways mediated by three ER-resident sensors in mammalian cells: the type-i ER transmembrane protein kinase (IRE1) [14,15], the activating transcription factor 6 (ATF6) and the PKR like ER kinase (PERK) (Fig. 1) [16,17]. The ER lumenal domains of these sensor proteins are usually associated with or bound to intralumenal GRP78 in the absence of ER stress. As unfolded proteins accumulate, engaging more GRP78 in the ER, GRP78 dissociates from IRE1, ATF6, and PERK liberating these signal transducers to promote a compensatory protective response [18 21]. IRE1 dimerizes and is autophosphorylated, which allows it to act as an endoribonuclease in the alternative splicing of the mrna of the X-box DNA-binding protein 1 (XBP-1) [22], which removes a 26 base pair intron and results in a translation frameshift that permits XBP-1 to act as a transcriptional activator (sxbp-1). Bcl-2 family members BAX and BAK may modulate the expression of XBP-1 by a direct interaction with IRE1a [23]. sxbp-1 upregulates genes such as GRP78, GRP94, and calreticulin, all of which contain a common motif of upstream ER stress response elements (ERSE) within their promoter regions [24]. Concomitantly, ATF6 is transported to the Golgi where its cytosolic transactivation domain is Fig. 1. The protective responses of the unfolded protein response (UPR). During early UPR, unfolded proteins cause dissociation of molecular chaperones such as Bip/GRP78 from ER-resident kinases-ire1a (type-i ER transmembrane protein kinase) and PERK (PKR like ER kinase) and transcription factor-atf-6. Activated PERK phosphorylates eif2a (eukaryotic initiation factor-2a) resulting in translational attenuation. Activated IRE1a and ATF6 up-regulate genes encoding ER chaperones leading to increased capacity for protein-folding. ERAD, ER-associated degradation; ERSE, ER stress response elements; GRP78, glucose-regulated protein 78 also known as BiP; Herp, homocysteine-induced protein; natf-6, activated ATF6; Auto-P, autophosphorylation; eif-2a-p, phosphorylated eif-2a. nrf-2, NF-E2-related factor-2; RIP, regulated intramembrane proteolysis; UPRE, UPR response elements; XBP-1, X-box DNA-binding protein 1; sxbp-1, alternative spliced XBP-1.

3 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) cleaved from the membrane by specific proteases (S1P and S2P); a process termed regulated intramembrane proteolysis (RIP) [25,26]. The cleaved ATF6 (natf6) localizes to the nucleus where it interacts with the constitutively expressed transcription factor NF-Y and with ERSE, thereby activating transcription of UPR-responsive genes, including GRP78, XBP-1, ERp72, and Hcy-induced ER protein (Herp) [27 32]. Therefore, activation of ATF6 and IRE1 as well as downstream XBP-1 (IRE1 XBP-1) increases the expression of ER-resident chaperones. Comparing ATF6 and IRE1, ATF6 regulates genes encoding ER-resident chaperones and folding enzymes via ERSE, whereas the IRE1 XBP-1 pathway also regulates the expression of ER-resident chaperones that are essential for protein folding and maturation via ERSE as well as the expression of genes involved in ER-associated degradation (ERAD) via a distinct unfolded protein response element (UPRE) [33]. In addition to transcriptional regulation of a group of genes encoding ER-resident proteins and enzymes, the UPR also induces a rapid attenuation in protein synthesis which is mediated by PERK. Once dissociated from GRP78, PERK phosphorylates eukaryotic initiation factor-2a (eif-2a), which blocks global mrna translation initiation and helps reduce the protein burden on the ER [17]. At the same time the phosphoeif-2a increases expression of a subset of genes by selectively enhancing the translation of the transcription factor ATF4 and also leads to increased translation of GADD34 which associates with protein phosphatase 1 to act as an eif-2a phosphatase to reverse the suppression of translation [34]. Under physiological conditions, the latter is designed to turn-off the UPR so that protein synthesis can resume. PERK has recently been shown to enhance nrf-2 phosphorylation and translocation to the nucleus where this transcription factor up-regulates the expression of antioxidant genes. 3. ER stress response: pathological consequences of prolonged UPR The UPR deals with adverse effects of ER stress in a timely and efficient manner at the early stage and thus enhances cell survival. However, prolonged ER stress has severe consequences, including apoptosis, and is referred to as the ER stress response (Fig. 2). For example, to resolve ER stress, sustained UPR consumes Fig. 2. Major mechanisms of injury mediated by ER stress response. Prolonged ER stress leads to interactions of IRE1a with TRAF2 (tumor necrosis factor receptor associated factor 2) which activates ASK1 (apoptosis signaling kinase 1), caspases, JNK and p38mapk, modulating cell death. Upregulation of CHOP (C/EBP homologous protein) by ATF4 represses members of Bcl2 family which promotes cell death and increases ERO1a (oxidative stress) and GADD34 (reversal of translation inhibition leading to more ER stress). Reactive oxygen species (ROS) resulting from interaction of cytosolic Ca 2+ and activation of NF-jB lead to inflammation. Activation of SREBP (sterol regulatory element-binding protein) increases fat accumulation. ERO1a, ER oxidase 1a; PP1, phosphatase 1; eif ptase, eif phosphatase; EOR, ER overload response; nsrebp, activated SREBP; GADD34, growth arrest and DNA damage-inducible protein 34.

4 324 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) energy in retrotranslocating unfolded or misfolded proteins retained in the ER to the cytoplasm for ubiquitination and the ERAD [35]. Energy depletion can contribute to programmed cell death [36]. During ER stress, the activated IRE1 interacts with the c-jun-n-terminal inhibitory kinase (JIK) which recruits cytosolic adapter TRAF2 to the ER membrane [37,38]. TRAF2 activates the apoptosis-signaling kinase 1 (ASK1) leading to activation of JNK and downstream mitochondria/apaf-1-dependent caspase activation [39]. In addition in rodents but not in humans, caspase-12 is activated which activates downstream caspase-9 and caspase-3 without the need for mitochondrial amplification [40,41]. Caspase-4 has been suggested to fulfill this role in human [42]. Another death-signaling pathway activated by ER stress is mediated by transcriptional activation of CHOP, a b-zip transcription factor that potentiates apoptosis, possibly through repressing expression of anti-apoptotic Bcl2 and Bcl-X L and induction of ER oxidase 1a which generates reactive oxygen species and depletes GSH [43]. Although both the IRE1 XBP1 and the ATF6 pathways can up-regulate CHOP, the PERK-eIF2a pathway predominates through selective upregulation of translation of ATF4, a transcription factor which subsequently activates transcription of CHOP and other genes involved in amino acid metabolism and transport, and oxidation reduction reactions [44,45]. In addition, prolonged ER stress is associated with release of ER Ca 2+ stores which can perturb mitochondria, triggering oxidative stress. Ca 2+ -induced oxidative stress can induce both cell death and activate NF-jB signaling (ER overload response), contributing to inflammation [10,11,42]. Ca 2+ chelators and antioxidants block NF-jB activation [45]. Increased cytosol Ca 2+ also activates calpains which proteolytically cleave Bcl- X L (inactivation) and caspase 12 (activation). Apoptosis is rapidly initiated after ER-Ca 2+ depletion in photodynamic therapy and strictly requires BAX/BAK at the mitochondria [46]. ER stress induces expression and cleavage of CREBH, a hepatocyte-specific bzip transcription factor that is structurally similar to ATF6 [47]. Activated CREBH and ATF6 synergistically trigger an acute phase response (reactive protein and serum amyloid P expression) which occurs in response to ER stress or pro-inflammatory cytokines [47 49]. Aside from cell death and inflammation, ER stress contributes to intracellular lipid accumulation which is mediated by the ER-associated transmembrane sterolresponse element-binding proteins (SREBP). SREBP1c and 2 are usually retained in the ER in a complex with the polytopic sterol-sensing transmembrane protein SCAP (SREBP cleavage-activating protein) [50,51]. Upon ER stress, or cholesterol deprivation, the SREBP SCAP complex dissociates from the ER retention protein Insig and subsequently translocates to the Golgi, where SREBP is cleaved and activated via the RIP [50 53]. Insig normally turns over rapidly, so ER stress-induced translational arrest may lead to a rapid decline in Insig allowing SREBP to escape. Once activated, SREBP1c and 2 act as transcription factors that regulate the genes that control the synthesis of fatty acids/ triglycerides and cholesterol, respectively, and cellular uptake of lipoproteins [53]. ER stress-induced overproduction of lipids can lead to fatty liver [54,55]. In addition, ER stress is associated with proatherogenic changes in lipoprotein metabolism including increased VLDL and reduced HDL cholesterol levels which contribute to cardiovascular disease [56]. 4. ER stress and liver disease 4.1. Viral infection Hepatitis C virus (HCV) infection In theory, the burden of producing viral protein in virus-infected cells may induce the UPR in response to the high levels of viral proteins. The ER is the major subcellular organelle with which the HCV life cycle is associated. The HCV RNA genome contains 9600 nucleotides with a 5 0 and 3 0 non-coding region (NCR) surrounding a large open-reading frame (ORF) which encodes a polyprotein. The 5 0 NCR contains an internal ribosome entry site (IRES) that directs the translation of the HCV polyprotein [57,58]. The polyprotein is processed by both viral and host proteases producing structural proteins (C, E1 and E2) and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) that are associated with the ER membrane [59 61]. The UPR triggered by the burden of viral proteins can be viewed as a two edged sword. On the one hand, the UPR can promote cell survival which impairs viral eradication. On the other hand, the UPR-induced PERK-mediated translation inhibition could suppress viral protein synthesis. Although HCV induces components of the UPR, a variety of evidence indicates that individual HCV proteins modulate the UPR and ER stress response which can result mainly in increased viral replication and failure to eliminate infected cells. For instance, NS3 and NS5B direct viral replication from a ribonucleoprotein (RNP) replication complex associated with an ER-derived membrane [62]. NS4B induces ATF6 and IRE1 to favor the HCV subreplicon and HCV viral replication [63]. HCV gene expression correlates with the translocation of ATF6 cytoplasmic domain to the nucleus of cells expressing HCV subgenomic replicons. Aside from inducing chaperones, ATF6 activates the IRE1 XBP1 pathway of the UPR by upregulating the transcription of XBP1. Although XBP1 spliced mrna and sxbp1 protein are elevated

5 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) in HCV replicon-expressing cells [64], the transactivating activity of sxbp1 is somehow repressed in the HCV-infected cells which prevents transcriptional induction of the ER degradation-enhancing-mannosidase-like protein (EDEM) [65,66]. Thus the protective UPR in HCV-infected cells may not unload excessive HCV proteins allowing increased virus production. Another way in which HCV alters the typical course of the UPR is to interfere with the PERK/PKR pathway. In virus-infected cells, the double-stranded RNAactivated protein kinase (PKR) phosphorylates eif2a which leads to a general attenuation of protein translation. However, HCV E2 and NS5A contain PKR-binding domains which enable them bind to and inhibit PKR [61,67 69]. Cells that express HCV E2 or HCV NS5A show elevated levels of overall protein synthesis. PKR activation is an important effect of interferon treatment so the resistance to interferon may be partly due to the inhibition of PKR by HCV. PKR and PERK share a significant amount of homology and both are activated by ER stress and phosphorylate eif2a [70,71]. It is very likely that HCV infection also modulates protein synthesis through PERK. Indeed, HCV E2 suppresses PERK activity [72]. E1 and/or E2 expression also induce components of ER stress as indicated by upregulation of CHOP, sxbp-1, and the ERAD [73,74]. It should be noted that contradictory effects of expression of individual HCV genes versus the entire genome have been observed. For example HCV replicons suppress the ERAD whereas E1 or E2 alone enhances the ERAD. Aside from modulating the UPR to favor viral persistence, HCV proteins may promote ER stress-induced injury. HCV gene expression elevates intracellular levels of cholesterol which leads to the release of Ca 2+ from the ER [75]. Expression of constructs encoding 191 aa reference HCV core in Huh7 or HepG2 and in transgenic mice triggered hyperexpression of GRP78, GRP94, and calreticulin which was followed by Ca 2+ depletion [75]. HCV core-induced CHOP, BAX translocation to mitochondria, cytochrome c release, caspase 3 and PARP cleavage. Reversal of the HCV core-induced ER Ca 2+ depletion by transfection of the sarco/er calcium ATPase abolished all these effects except CHOP. Furthermore, the uptake of Ca 2+ in the mitochondria induces ROS which activates multiple signaling pathways, including NF-jB, modulating apoptosis and inflammation, and STAT-3, a transcription factor that controls cellular processes for cell survival, proliferation, differentiation and oncogenesis [76]. Activation of NFjB and STAT-3 by HCV is associated with chronic liver disease [77]. In addition, HCV-induced ER stress results in reduced protein glycosylation which disrupts the proper protein folding and assembly of MHC class I molecules. Cells expressing HCV subgenomic replicons have 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 presentation to cytotoxic T lymphocytes and the persistence and pathogenesis of HCV may depend upon the ER stress-mediated interference of MHC class I assembly and cell surface expression. HCV gene products appear to modulate the UPR in experimental cell systems, based upon a number of disparate observations. The composite evidence supports the interpretation that HCV burden favors induction of the UPR but individual HCV proteins inhibit the responses which would suppress viral protein production. It remains to be determined if these observations are reflective of what occurs in vivo in HCV-infected liver and if an inadequate UPR contributes to the chronicity of viral infection or if the HCV-induced ER stress response promotes the progression to apoptosis and inflammation Hepatitis B virus (HBV) infection Many HBV carriers are asymptomatic and have minimal liver injury, despite extensive and ongoing intrahepatic replication of the virus [80]. That implies that the HBV replication cycle is not directly cytotoxic to cells. The HBV surface antigen (HBsAg) consists of three structurally related large, middle, and small envelope proteins. The large form is translated from transcripts specified by a pres1 promoter, while the middle and small forms are translated from transcripts specified by a pre-s2 promoter. Overexpression of the large surface protein of HBV in Huh7 cells results in a blockage of secretion of HBsAg which leads to an accumulation of HBsAg in the ER lumen, which in turn induces expression of GRP78 and GRP94 [81,82]. In transgenic mice, the intracellular retention of HBsAg in hepatocytes can cause pleiotropic physiological changes, ground glass morphology [83,84], and hypersensitivity to inflammatory cytokines [85]. The retention of HBsAg in ER can actually occur in natural infection. Two mutant types of the large HBV surface antigens with deletions over the pre-s1 and pre-s2 regions, respectively, were identified [86,87]. It has been reported that expression of the mutant proteins interferes with proper protein folding activity in the ER. Expression of the mutant HBV large surface proteins induced eif2a phosphorylation, nuclear translocation of NF-jB, activation of p38 MAPK, and enhanced the expression of COX-2 in ML- 1 and HuH-7 cells [88]. Higher expression of COX-2 protein was detected in liver and kidney tissue of transgenic mice expressing the mutant HBV large surface protein in vivo. Expression of COX-2 mrna was also observed in human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins. These lines of evidence provide important insights into cellular inflammation and carcinogenesis that are associated with latent ER stress due to HBV infection and chronic carriage. As with HCV, it is uncertain if the UPR

6 326 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) protects against virus eradication and the role of ER stress in liver injury and inflammation needs to be better defined. Furthermore, immunosuppression in HIV coinfected patients and post-olt is associated with enhanced viral replication of both HCV and HBV and a more severe, rapidly progressive liver disease. It is tempting to speculate that ER stress contributes to this outcome Diabetes, obesity and hepatic metabolic disorder Glucose metabolism and homeostasis are maintained at the levels of insulin synthesis and secretion in the pancreatic b-cell and peripheral utilization regulated by insulin. Intracellular levels of glucose influence both the secretion of insulin and insulin production [89 91]. Although UPR signaling is a physiological mechanism to sustain insulin secretion, chronic ER stress not only contributes to the attrition of b-cell function but also to the impaired glucose homeostasis in diabetes [92 94]. The UPR signaling activates genes encoding glucose-regulated proteins in response to glucose/energy deprivation [95,96]. ER stress increases expression and activity of glucose-6-phosphatase, one of the key enzymes of gluconeogenesis, which modulates the capacity for glucose release and glucose cycling in primary rat hepatocytes and H4IIE liver cells [90]. The UPR is required for survival of pancreatic b-cells during intermittent fluctuations in blood glucose [97 99]. Deletion of PERK in humans and mice results in a pancreatic b-cell dysfunction and development of infancy-onset diabetes. Site-directed mutation of mouse eif2 at the PERK phosphorylation site leads to a b-cell loss in utero, suggesting that the PERK-eIF2a pathway of UPR signaling is essential for proinsulin translation, b-cell function and survival. During the development of diabetes in Akita mice, both the transcription factor CHOP and the molecular chaperone GRP78 in the ER are induced in the pancreas, and targeted disruption of the CHOP gene improved the glucose intolerance of heterozygous Akita mice indicative of the importance of ER in insulin secretion in b-cells of the pancreas [100]. Thus, UPR is an important physiological component of insulin secretion. Physiological UPR promotes survival of pancreatic b-cells while severe and prolonged ER stress in Akita mice promotes apoptosis of b-cells. The ER also plays a crucial role in the regulation of cellular responses to insulin. ER stress is increased in the liver under diabetic conditions. GRP78 protein was increased in the liver of mice with high-fat diet-induced and genetic (ob/ob) obesity [98]. PERK and eif2a phosphorylation were increased in the liver of obese mice compared with lean controls. ER stress in obese mice also led to suppression of insulin receptor signaling (insulin resistance) which was mediated by activation of JNK [101,102]. Insulin receptor substrate 1 (IRS-1) and Akt are substrates of JNK and are key molecules for insulin signaling. Tunicamycin or thapsigargin induced ER stress and significantly inhibited insulin-stimulated tyrosine phosphorylation of IRS-1. Tunicamycin pretreatment also suppressed insulin-induced Akt phosphorylation [101]. Inhibition of JNK activity with the synthetic inhibitor SP reversed the ER stress-induced serine phosphorylation of IRS-1. Pretreatment of Fao cells with a highly specific inhibitory peptide derived from the JNK-binding protein, JIP, also preserved insulin receptor signaling in cells exposed to tunicamycin. Similar results were obtained with the synthetic JNK inhibitor, SP The ER-resident oxygen-regulated protein 150 (ORP150) has been shown to protect cells from ER stress [ ]. Overexpression of ORP150 markedly improves insulin resistance and ameliorates glucose tolerance in diabetic animals whereas suppression of ORP150 in the liver of normal mice decreases insulin sensitivity [104]. The phosphorylation state of IRS-1 and Akt as well as the expression levels of the enzymes of gluconeogenesis such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were also altered by ORP150 overexpression. These results indicate that ER stress promotes a JNK-dependent serine phosphorylation of IRS-1, which in turn inhibits insulin receptor signaling [101]. Mice with heterozygous deficiency in XBP1 fed a high fat diet (obesity) develop insulin resistance and dysregulated phosphorylation of IRS-1, suggesting the IREla XBPl UPR pathway is critical for preventing insulin resistance. XBP-1 +/ mice fed high fat diet exhibited greater PERK and JNK activation as a result of decreased UPR protection. Thus, interfering with the UPR-induced production of chaperones worsens ER stress. At present the triggering mechanism for hepatocellular ER stress in diabetes/obesity is not certain but its development appears to be a major contributor to insulin resistance. Whether ER stress also directly contributes to the pathology of NASH is uncertain but possible Others: a 1 -antitrypsin deficiency and injury of ischemia and reperfusion a 1 -Antitrypsin (a 1 -AT) deficiency occurring in the liver is an example of a disorder associated with aberrant protein accumulation in tissues and cellular compartments. The disease inducing form of a 1 -AT deficiency is caused by a point mutation encoding a substitution of lysine for glutamate-342 referred to as a 1 -ATZ or the Z mutant [106]. The a 1 -ATZ is retained and accumulates in ER which results in its reduced secretion into the blood and body fluids. The reduction increases risk of developing emphysema in the lung because of inhibition of connective tissue breakdown by neutrophil elastase, cathepsin G, and proteinase 3. In cell culture and transgenic mice with a 1 -AT deficiency the ER retention

7 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) of a 1 -ATZ induced a marked autophagic response, in which injured ER is sequestered from the rest of the cytoplasm and then degraded by fusion with lysosomes [107]. Fasting induced steatosis in the PiZ transgenic mice [108]. Both UPR and ER overloading response (EOR) were activated after treatment with additional stresses such as thapsigargin or elevated temperature. UPR activation was accompanied by a marked increase in grp78 promoter activity and in GRP78 and GRP94 protein expression in the PiZ transfected cell lines compared with control. EOR activation led to an increase in NF-jB activity and degradation of IjB which was correlated with IL-6 and IL-8 protein production in the PiZ transfected cells [107]. In the PiZ transgenic mice, the 58-kDa protein disulfide isomerase (PDI), an important oxidoreductase and chaperone of the ER, has recently been found to form a complex with PiZ, resulting in a decrease of protein disulfide reductase activity of the ER. PiZ transgenic mice have a shift toward a more reduced ER environment and an elevation of cytoplasmic chaperones (Hsp90 and Hsp70) and antioxidant enzymes (thioredoxin). Therefore, the toxic effects of PiZ aggregation caused by a 1 -AT deficiency may be due to lower availability of PDI and a decreased protein disulfide reductase activity in the ER along with a cytoplasmic stress [109]. However, Hidvegi et al. recently compared the effect of several different naturally occurring PiZ mutants which are retained in the ER [110]; mutants which do not polymerize caused the UPR whereas polymerogenic a 1 -ATZ did not cause UPR. Conversely ER stress and overload responses as evidenced by the cleavage of caspase12 and BAP31 and NF-jB activation, respectively, were observed in polymerogenic mutant expressing transgenic mice but not in non-polymerogenic mutant transgenic mice. Thus, it appears that GRP78 and the UPR do not sense retention of insoluble polymers so a protective response does not occur while injurious components of ER stress are activated. This work provides evidence that pro-death/ pro-inflammatory signaling in response to ER retention can be dissociated from the UPR in some circumstances. What happens in the human liver expressing PiZ will be of interest-perhaps both types of responses (UPR and non-upr) will occur in different hepatocytes depending on the level of mutant protein and other factors. It is tempting to speculate that the a1-atz-induced ER stress response and EOR promote cell death and inflammation, respectively, leading to fibrosis and cirrhosis. Ischaemia occurs when the reduction of blood flow decreases the delivery of oxygen and substrates to maintain cellular energy leading to decrease in intracellular ATP level. ATP deficiency impairs the function of the Ca 2+ channel, releases Ca 2+ from the ER to cytosol, and promotes cell death [111]. Reduced ER Ca 2+ also alters the expression and activity of GRP78 and GRP94, thereby initiating several signal transduction pathways. The expression of GRP78, CHOP and the XBP-1 spliced form (sxbp-1) is increased during human liver transplantation suggesting an activation of the IRE1 pathway [ ]. The PERK pathway is also activated upon reperfusion, leading to a reduction of the overall rate of translation through phosphorylation of eif2a. Increased generation of reactive oxygen species during reperfusion may enhance ER stress induced inflammation and cell death causing additional cell injury [116,117]. Reduction in ER stress induced hepatocellular injury in mice can be achieved by the administration of sodium 4-phenylbutyrate (PBA), a low molecular weight fatty acid that acts as a chemical chaperone [115]. PBA-treated mice had reduced pyknosis, parenchymal hemorrhage, and neutrophil infiltration during IR. The reduced injury was associated with a greater than 45% reduction in apoptosis due to a significant reduction of CHOP expression, caspase- 12 activation, and eif2a phosphorylation compared to untreated mice. Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses ER stress induced apoptosis [116,117]. BI-1 is abundant in both liver and kidney. Hepatic IR injury induced BI-1 mrna in mouse liver, indicating that BI-1 may provide adaptive protection of the liver from ER stress and IR injury. In BI-1 knockout mice under IR, increased histological injury, serum transaminases, and hepatic apoptosis were found to be associated with greater elevations in caspase activity, more activation of ATF6, and greater increases in expression of CHOP and the sxbp-1 suggesting that BI-1 is required for limiting tissue injury. These observations on the role of ER stress reveal potential strategies for organ preservation and protection against IR injury. 5. Alcoholism, hyperhomocysteinemia, and ER stress Steatosis, inflammation, apoptosis and fibrosis are characteristics of alcohol-induced liver injury. Although many mechanisms have been implicated in the pathogenesis of alcoholic liver disease [118], alcohol-induced hyperhomocysteinemia (HHcy) and ER stress has recently emerged as a novel mechanism for alcoholic liver disease [55, ]. Alcohol-induced HHcy is often observed. Alcoholic patients have elevated plasma homocysteine (Hcy) levels which range from 10 to 120 lm compared to normal 5 15 lm [ ]. Hcy but not total B 12 and B 6 levels correlated 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 fed ethanol orally exhibit a doubling of plasma Hcy despite normal levels of folate, pyridoxal-phosphate (PLP) and B 12 [126]. We have observed a 5- to 10-fold increase of plasma Hcy levels in mice fed alcohol intragastrically for 4 weeks [55,119,127]. Similar elevations of plasma

8 328 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) Hcy were observed in TNFR1 null mice fed ethanol suggesting a minimal contribution of TNFa to ethanol-induced HHcy [128]. In the cells, Hcy is derived from methionine after transmethylation reactions that use S-adenosylmethionine (SAM) as the methyl donor (Fig. 3). Methionine intake and transmethylation activity determine the input of Hcy into the system. A certain amount of total Hcy is catabolically eliminated by transsulfuration to cysteine initiated by cystathionine-b-synthase (CBS), but about 50% in humans or male rats is conserved by remethylation to methionine which is catalyzed by methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) [129,130]. Chronic alcohol consumption may have an impact on the expression or enzyme activity of BHMT, MS, and CBS. The enzyme activity of BHMT was inhibited by high concentrations of acetaldehyde [118] and mrna of BHMT was reduced in the liver of mice with intragastric alcohol infusion [127]. Ethanol feeding lowered methionine synthase leading to increased accumulation of 5-methyl tetrahydrofolate and Hcy and to decreased levels of betaine, a product of choline oxidation and the substrate of BHMT [130]. In micropigs fed ethanol for 12 months with adequate folate, MS activity decreased by 20% which was associated with slightly decreased plasma methionine, 20% increased plasma Hcy, and increased hepatic SAH but no change in SAM [ ]. Chronic alcohol increases choline uptake [134] and mitochondrial oxidation to betaine [135] suggesting compensation for increased demand for betaine. However, depletion of betaine by chronic alcohol feeding may be a major factor contributing to the alcoholic HHcy [119]. Alcohol-induced HHcy promotes ER stress, inflammation, and cell death. Excessive intracellular Hcy can be converted by methionyl trna synthase to Hcy thiolactone which has unique reactive properties. Hcy thiolactone can cause homocysteinylation of lysine residues and free amine groups of proteins resulting in malfolding and premature degradation [136]. Hcy also disrupts disulfide bond formation and also can induce misfolding of proteins traversing the ER leading to ER stress. Elevated levels of intracellular Hcy increase the expression of several UPR genes, including GRP78, GRP94, Herp and RTP [32,54,55,121, ]. Hcy-induced ER stress causes dysregulation of lipid biosynthesis by activating the SREBPs leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides [53 55,142]. Hcy induces the expression of CHOP which is involved in ER stress-induced cell death [143]. In both Chop null and wild type mice fed alcohol, significantly increased hepatomegaly, steatosis, and UPR indicated by increased Grp78 mrna were observed [128]. However, CHOP null mice exhibited the absence of hepatocellular apoptosis in response to alcohol feeding but no protection against HHcy, steatosis and ER stress implying that CHOP up-regulation occurs downstream of and contributes to ER stress-induced apoptosis. Finally, Hcy-induced cell death is mimicked by other ER stress agents and is dependent on IRE-1 signaling. Activation of IRE-1 by Hcy leads to a rapid and MAT MS BHMT MTHFR Homocysteine CBS Fig. 3. Methionine metabolism. B6, vitamin B6; B12, vitamin B12; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine-b-synthase; GSH, glutathione; MAT, methionine adenosyltransferase; MS, methionine synthase; MTHFR, 5, 10-methylenetetrahydrofolate reductase; R, substrates of methyltransferases; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate.

9 C. Ji, N. Kaplowitz / Journal of Hepatology 45 (2006) sustained activation of JNK [ ], a result consistent with the finding that activation of JNK by ER stress involves binding of IRE-1 to TRAF2 [146]. Because persistent activation of JNK correlates with cell death [102,147], these studies provide further support for a mechanism involving Hcy-induced programmed cell death. In addition, feeding mice betaine to promote methylation of Hcy to methionine ameliorated alcohol-induced ER stress in mouse livers [55], indicating that Hcy-induced ER stress contributes to alcohol-induced liver injury. Hence, alcohol intake leads to HHcy, which in turn may elicit ER stress promoting alcohol-induced liver injury [119,123]. A feature common to HHcy and consequent ER stress induced by alcohol, MTHFR KO, CBS KO, or high methionine/low folate diet is the development of fatty liver [53 55, ]. Activation and induction of SREBP-1c and SREBP-2 as a consequence of ER stress accounts for the bulk of triglyceride and cholesterol accumulation in these models. In the intragastric ethanol infusion model, ER stress, up-regulation of SREBPs, accumulation of triglycerides and cholesterol, as well as apoptosis and necroinflammation are attenuated by feeding quantities of betaine sufficient to lower homocysteine. Furthermore, a potential interaction between homocysteine and HCV-induced liver disease has been suggested by the correlation between HCV, steatosis and fibrosis in patients with an MTHFR polymorphism (C667T) leading to HHcy [151]. It is tempting to speculate that alcohol may interact with HCV in a similar fashion via HHcy and ER stress. 6. Conclusions Much evidence is emerging that a critical determinant of liver disease is how the liver copes with stress. Stress can emerge from exogenous sources through membrane receptors or internally from any organelle, i.e. nucleus, mitochondria, cytoskeleton, or ER. In each case, signals are released which recruit built-in pathways which promote a protective response or; when overwhelmed, a pathological response. In the ER, perturbation in protein load, protein folding, glycosylation, calcium sequestration, or redox balance trigger a finely tuned response to cope, referred to as the UPR, which is vital to regulate and protect the ER by adjusting the levels of chaperones, protein degradative apparatus, protein synthesis, and membrane lipid synthesis in an attempt to make more ER. However, when the hepatocytes cannot cope because of the great extent or duration of ER stress, apoptosis and steatosis occur. It has been speculated that the apoptosis response to ER stress evolved as a mechanism for coping with viral infection by suicide of infected cells. However, the exact role of the UPR and ER stress response in chronic HCV and HBV infection is unclear. Evidence suggests that HCV may modify the UPR to support continued virus production. However, cell death and inflammation in HCV and HBV infection may result of ER stress response and EOR. In the case of insulin resistance as well as alcoholic liver disease, a potentially important contribution of ER stress has emerged. Many questions remain to be answered in this exciting new area of research. A fundamental question is whether the liver is coping with protein overload or misfolded proteins by the physiological UPR or whether the UPR cannot cope leading to ER stress induced apoptosis, inflammation and steatosis. 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