Targeting endoplasmic reticulum stress in liver disease

EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY, 2016 http://dx.doi.org/10.1080/17474124.2016.1179575

REVIEW

Targeting endoplasmic reticulum stress in liver disease Fa-Ling Wua,b, Wen-Yue Liu and Ming-Hua Zheng a,b

c

, Sven Van Poucke

d

, Martin Braddocke, Wei-Min Jinf, Jian Xiaog,h, Xiao-Kun Lig,h

Downloaded by [Wenzhou Medical College] at 16:28 08 May 2016

a Department of Hepatology, Liver Research Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; bInstitute of Hepatology, Wenzhou Medical University, Wenzhou, China; cDepartment of Endocrinology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; dDepartment of Anesthesiology, Intensive Care, Emergency Medicine and Pain Therapy, Ziekenhuis Oost-Limburg, Genk, Belgium; eGlobal Medicines Development, AstraZeneca R&D, Alderley Park, UK; fDepartment of Infection Diseases, People Hospital of Wencheng County, Wenzhou, China; gInstitute of Biology Science, Wenzhou University, Wenzhou, China; hSchool of Pharmacy, Wenzhou Medical University, Wenzhou, China

ABSTRACT

ARTICLE HISTORY

Introduction: The accumulation of unfolded protein in the endoplasmic reticulum (ER) initiates an unfolded protein response (UPR) via three signal transduction cascades, which involve protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme-1α (IRE1α) and activating transcription factor-6α (ATF6α). An ER stress response is observed in nearly all physiologies related to acute and chronic liver disease and therapeutic targeting of the mechanisms implicated in UPR signaling have attracted considerable attention. Areas covered: This review focuses on the correlation between ER stress and liver disease and the possible targets which may drive the potential for novel therapeutic intervention. Expert Commentary: We describe pathways which are involved in UPR signaling and their potential correlation with various liver diseases and underlying mechanisms which may present opportunities for novel therapeutic strategies are discussed.

Received 8 January 2016 Accepted 13 April 2016 Published online 29 April 2016

1. Introduction The endoplasmic reticulum (ER) is the intracellular organelle responsible for the synthesis and posttranslational modification of secreted and membrane proteins, lipid synthesis, drug metabolism, and homeostasis of intracellular Ca2+ [1]. In molecular biology, a chaperone is defined as a protein that assists noncovalent folding and assembly of other large structures. Physiologically, ER homeostasis maintains a dynamic equilibrium between the folding capacity and the influx of proteins. Three transmembrane sensors, including protein kinase RNAlike ER kinase (PERK), inositol requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α), bind to the luminal protein chaperone heavy-chain binding protein (BiP)/glucoseregulated protein of 78 kD (GRP78) in an inactive state [2,3]. The sensors are differently represented in different cell types. There are two IRE1 paralogs and multiple genes encoding ATF6 family members in mammalian cells [4]. BiP is also an important Ca2+-binding protein and a Ca2+ buffer in the lumen of the ER [5]. Depletion of the Ca2+ store leads to a rapid accumulation of unfolded proteins [6]. As the ER-folding capacity mismatches the accumulation of unfolded proteins (termed ER stress), the unfolded proteins bind to BiP/GRP78 by competing with the sensors. The dissociation of BiP/GRP78 activates three integral signal transduction pathways mediated by the sensors, defined as the unfolded protein response (UPR) [7].

KEYWORDS

Endoplasmic reticulum stress; alcoholic liver disease; nonalcoholic fatty liver disease; drug-induced liver injury; acute-on-chronic liver failure; hepatocellular carcinoma; therapeutic target

Activated PERK increases phosphorylation of eukaryotic initiation factor 2α (eIF2α), leading to attenuation of protein translation and an elevation of activating transcription factor 4 (ATF4) level. ATF4 translocates to the nucleus, inducing the expression of several UPR target genes, including chaperones, ER-associated protein degradation (ERAD), C/EBP-homologous protein (CHOP) and growth arrest and DNA damage-inducible 34 (GADD34). ERAD genes are associated with protein degradation. CHOP is linked with apoptosis, possibly through the transcriptional upregulation of several proapoptotic proteins of the BCL-2 family and the downregulation of BCL-2. GADD34 participates in a feedback loop to dephosphorylate eIF2α by interacting with protein phosphatase 1C, which restores protein synthesis [8]. Activated IRE1α splices XBP1 mRNA, which upregulates expression of chaperones, ERAD, and GADD34 genes in the nucleus. Expression of GADD34 may sensitize hepatocytes to cell death by resuming protein synthesis in stressed cells [9]. In addition, IRE1α activates the apoptosis signal-regulating kinase 1 (ASK1) and JUN N-terminal kinase (JNK) [10,11], and sustained regulated IRE1-dependent decay (RIDD) [12]. Activated ATF6α translocates to the Golgi apparatus for proteolytic cleavage and cleaved ATF6α transactivates ERAD and CHOP genes. ER stress is also involved in the process of inflammation through the activation of reactive oxygen species [13] and the transcription factors nuclear factor-κB (NF-κB) and JNK [14,15], as well as the induction of the acute-phase response [16].

CONTACT Ming-Hua Zheng [email protected] Department of Hepatology, Liver Research Center, the First Affiliated Hospital of Wenzhou Medical [email protected] University, Wenzhou, China; Institute of Hepatology, Wenzhou Medical University, No. 2 Fuxue Lane, Wenzhou, China; Xiao-Kun Li Institute of Biology Science, Wenzhou University, Wenzhou, China; School of Pharmacy, Wenzhou Medical University, Wenzhou, China © 2016 Informa UK Limited, trading as Taylor & Francis Group


2

F.-L. WU ET AL.

The UPR leads to the production of basic leucine zipper transcription factors, which work alone or together to activate UPR target genes to increase the protein-folding capacity in the ER. Besides, activation of PERK (via eIF2α phosphorylation) and IRE1α (via RIDD) also decreases the load of proteins entering the ER [4]. Each arm of the UPR may be responsive to different components of the ER lumen, depending on the nature of stress conditions and both outcomes may function as negative feedback loops among different signal pathways that attenuate ER stress. Mild UPR is beneficial for the recovery of protein folding homeostasis in the ER. If an ER stress is severe or chronic, the UPR will be not capable of repairing the protein folding defect, resulting in an inflammatory response, necrosis, and apoptosis in secretory cells such as hepatocytes [17,18]. In this review, we summarize recent data of the impact of ER stress in liver disease and highlight potential therapeutic strategies targeting the UPR signal transduction network.

2. ER stress in liver diseases In the past few decades, the possible contribution of ER stress to the development of various liver diseases has been extensively explored. It is generally accepted that the three UPR signaling pathways play independent roles in ER stress and may present opportunities for targeted therapies (Figure 1). Though the exact relationship between ER stress and different

cell injuries in liver disease remains uncertain, it is clear that there is a complex interplay between the cause, the stress response and the effect (Figure 2). We will now review their potential cross-talk in more detail.

2.1. Alcoholic liver disease Alcohol metabolism is the potential mechanisms that triggers the alcoholic ER stress response, including toxic acetaldehyde and homocysteine, oxidative stress, perturbations of calcium or iron homeostasis [19], resulting in direct impairment of ER structure and function in the hepatocyte [20]. The alterations of selected ER stress markers were associated with severe steatosis, scattered apoptosis, and inflammasome activation. In human chronic progressive alcoholic liver disease (ALD), high levels of IRE1α, PERK, Bip/GRP7, Ca2+, and their downstream products CHOP, ER oxidoreductin 1-α (ERO1α), Bcl-2associated X protein (BAX), and calnexin were found in the ER. IRE1α, CHOP, and Ca2+ have been associated with the apoptotic response to ER stress and ERO1α plays a role in cellular apoptosis via the CHOP pathway [21]. Increased expression of BAX and calnexin may disrupt Ca2+ homeostasis in the ER [22], and IRE1α and PERK were shown to promote inflammation in ALD via the downstream gene products XBP1 and ATF4, respectively [23]. Sterol-regulatory element binding protein (SREBP) is member of the family of transcription factors playing a role in the control of fatty acid, triglyceride (TG), and

Figure 1. The signaling pathways of UPR and their targeted therapies. Accumulation of unfolded proteins activates the three endoplasmic reticulum stress sensors PERK, IRE1α and ATF6 via dissociation from BiP. Activated PERK increases phosphorylation of eIF2α, leading to attenuation of protein translation and an elevation of ATF4 level. ATF4 translocates to the nucleus, inducing the expression of several UPR target genes, including chaperones, ERAD and CHOP. ERAD genes are associated to protein degradation, whereas CHOP is linked with apoptosis. Activated IRE1α splices XBP1 mRNA, which up-regulates expression of chaperons and ERAD genes in nucleus. Activated ATF6 translocates to the Golgi apparatus for proteolytic cleavage. Cleaved ATF6 transactivates ERAD and CHOP genes. Recently, a number of small molecular compounds have been identified to treat liver diseases via targeting the above signals. Most activators/inhibitors target specific UPR components, whereas chaperone modulators effect in a nonspecific manner. (UPR, unfolded protein response; PERK, kinase RNA-like ER kinase; IRE1α, inositol requiring enzyme1α; ATF6, activating transcription factor-6).


EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY

3

Figure 2. Endoplasmic reticulum stress in liver diseases. Transient and mild stimulus results in adaptation by activation of the UPR to eliminate stress. Chronic and severe stimuli trigger endoplasmic reticulum stress, playing a role in nearly all acute and chronic liver diseases.

cholesterol synthesis. Elevated levels of SREBP in the ERstressed cell disrupt lipid metabolism, resulting in an enhanced production of cholesterol and TGs [24]. Taken together, these studies suggest that ER stress is able to promote hepatic steatosis in fatty liver. Similar results have been observed in animals exposed to ethanol [25,26]. In models of ALD, elevated gene expression was detected in the ER for BiP, SREBP, CHOP, and caspase12 by use of microarray gene expression profiling [27], supporting the relationship between alcohol and ER stress. SREBP-1c knockout mice were protected against TG accumulation after administration of alcohol [28,29]. Stimulator of interferon genes (STING) is an ER adaptor and interferon regulatory factor 3 (IRF3) is a transcription factor regulating the innate immune responses. Ethanol-induced ER stress may trigger early hepatocyte apoptosis by activation of the STING-IRF3 pathway. The association of STING and IRF3 may determine the survival of hepatocytes, suggesting that innate immunity could be a major contributor in early-stage disease [30]. In progressive ALD, like cirrhosis, ethanol exposure resulted in an increased expression of markers of ER stress response in the liver triggered by lipopolysaccharide. Autophagy was also correlated with the progression of ethanol-exposed liver

disease [31]. Activation of the IRE1α pathway significantly increased autophagic activity in a p38 MAPK-dependent manner, leading to an upregulated fibrogenic response [32], probably by activating the endothelial-to-mesenchymal transition (EndMT). As shown in studies with human umbilical vein endothelial cells (HUVEC) under ER stress, the expression of the endothelial marker CD31 significantly decreased while mesenchymal markers α-SMA, vimentin, and collagen 1 increased [33]. Moreover, HUVEC cultures adopted a fibroblast-like appearance with the activation of Smad2 and Src kinase pathway. Further studies on the ER stress-mediated EndMT in the development of hepatic fibrosis are needed.

2.2. Nonalcoholic fatty liver disease In the field of liver disease, induction of ER stress has been well documented in patients with nonalcoholic steatohepatitis [34–36]. The role of ER stress in nonalcoholic fatty liver disease (NAFLD) has become a much studied area in recent years [37– 39]. The accumulation of lipids in the cytoplasm of hepatocytes in NAFLD derived from dietary chylomicron remnants, de novo lipogenesis excess, free fatty acids released from the


4

F.-L. WU ET AL.

lipolysis of adipose tissue, diminished very-low-density lipoprotein (VLDL) secretion and reduced the oxidation of fatty acids [40–42]. The bilateral interaction of ER stress in lipogenesis, lipotoxicity, and insulin resistance has been shown to play a central role in the pathogenesis of hepatosteatosis in NAFLD [43]. All the three pathways have been reported to regulate lipogenesis and hepatic steatosis. PERK-dependent signaling contributes to lipogenic differentiation in the mammary epithelium, and knockout of PERK attenuates the sustained expression of the lipogenic enzymes fatty acid synthesis [44]. Increased expression of GADD34 inhibited eIF2α phosphorylation, consequently, improving steatosis and glucose tolerance in rats fed a high-fat diet [45]. The IRE1α-XBP1 pathway has been associated with liver steatosis playing a vital role in the assembly and secretion of VLDL [46]. In hepatocytes, XBP1 regulates hepatic lipogenesis by directly binding to the promoters of a subset of lipogenic genes and activating their expression [47]. Selective genetic deletion of IRE1α in the liver resulted in a lower expression of hepatosteatosis with chronic fasting or ketogenic conditions. Liver-specific restoration of XBP1 reversed the defects in IRE1α-null mice. A point mutation in protein translocation channel Sec61α1 disrupted the ER secretory pathway in mice, increasing the susceptibility to hepatosteatosis [48]. ATF6α-deficient mice develop hepatic steatosis and glucose intolerance via reduced fatty acid β-oxidation and attenuated VLDL formation [49], in association with increased expression of SREBP-1c [50]. Mice lacking the Sirtuin 1 gene, when fed a high-fat diet exhibited an NAFLD phenotype characterized by severe hepatic steatosis. This was shown to involve activation of the lipogenic pathway through SREBP-1 [51]. GRP78 overexpression has been shown to inhibit ER stress and SREBP activation in ob/ob mice attenuates the severity of NAFLD [52]. Hepatic insulin resistance (IR) is a vital process of the pathogenesis of NAFLD. Increasing evidence indicates an intense correlation between ER stress and IR. A study suggests that ER stress response promotes IR via JNKmediated suppression on the insulin receptor substrate, resulting in hepatic steatosis [53]. IRE1α-mediated activation of JNK impairs insulin signaling through the serine phosphorylation of insulin receptor substrate-1 [35]. Furthermore, inhibition of Akt/PKB signaling and PERK-mediated FOXO phosphorylation are also involved in ER stress-induced IR [54,55]. We have previously proposed that fibroblast growth factor 19 (FGF19) and fibroblast growth factor 21 (FGF21) are important endogenous regulators involved in the pathogenesis of NAFLD via activation of the PERK-ATF4-CHOP signal [56,57]. In addition, hepatic iron accumulation may also induce ER stress-related oxidative stress in NAFLD [58,59].

2.3. Viral hepatitis Numerous viruses including hepatitis B virus (HBV) and hepatitis C virus (HCV) are causally related to ER stress in the liver [60–62]. The multifunctional regulatory protein of HBV (HBx protein) has been shown to be an inducer of UPR pathways [63]. Stable expression of HBx in Hep3B cells activates the IRE1α-XBP1 and ATF6α pathways and this observation was replicated in HepG2.2.15 cells that supported the replication

of the intact HBV genome. Lower levels of products of the IRE1α-XBP1 and ATF6α pathways were observed in cells transfected with a plasmid expressing a siRNA with null HBx expression [64]. HBx-mediated activation of these pathways is likely to promote HBV replication and expression in hepatocytes. It has been demonstrated that pre-S mutant in large HBV surface antigens initiates ER stress and induces oxidative DNA damage and genomic instability, triggering dysplasia of hepatocyte. Both the mature structural (core) and nonstructural (NS5A, NS4B) products of HCV have been demonstrated to induce UPR in hepatocyte. HCV core triggered apoptosis in liver cells by inducing ER stress and ER Ca2+ depletion [65]. NS5A protein altered intracellular Ca2+ levels, induced oxidative stress, and activated STAT-3 and NF-kappa B [66]. NS4B induced both XBP1 mRNA splicing and ATF6α cleavage, as well as perturbing intracellular Ca2+ homeostasis [67]. A recent study found that HCV induced significant increase in apoptosis (cleavage of caspase-3), autophagy, and UPR (increase in GRP78 expression) in HCV-infected cells [68]. However, HCV co-opts the cellular protein folding machinery in order to synthesize viral proteins. Autophagy interferes with different steps of the HCV life cycle to establish a persistent viral infection [69–71]. Moreover, HCV-induced autophagy has been shown to repress cellular apoptosis [72] and reduce the innate immune response in HCV-infected cells [73], thus promoting cell survival. Therefore, though intimately related to the ER in addition to the formation of lipid droplets, cell survival is the usual outcome in this setting [74]. These studies provide an important insight into the effect of ER stress on HCV-associated liver diseases via UPR pathways.

2.4. Drug-induced liver injury Acetaminophen (APAP) overdose is the major cause of acute drug-induced liver injury. It has been demonstrated that APAP toxicity results in elevated expression of eIF2α, JNK, and induction of CHOP in the livers from studies conducted in vitro [75]. In animal studies, murine ATF6α, GADD153/CHOP, and caspase-12 were shown to be activated by APAP administration [76]. In contrast, APAP bound to ER chaperones directly impaired the folding process and induced ER stress [77]. A rapid initiation of above pathways induced a significant intraluminal redox imbalance, disturbance of Ca2+-mediated mitochondrial permeability transition, and APAP-induced necrosis, finally resulting in the occurrence of acute hepatitis, even acute liver failure [78]. Recently, serotonin has been supported to attenuate APAP-induced hepatotoxicity mainly through inhibiting ER stress-induced hepatocyte inflammation and apoptosis and promotion of liver regeneration [79]. Other drugs such as arylating quinones, cyclosporin A, and human immunodeficiency virus protease inhibitors have been implicated in causing ER stress via different pathways. Arylating quinones have been shown to induce phosphorylation of PERK and eIF2α, as well as induction of ATF4 and CHOP by the formation of a Michael adduct [80]. The relationship between cyclosporin A-induced cholestasis and ER stress has been further strengthened by building a network including UPR-associated-genes encoding protein disulfide isomerase family A and microRNA mmu-miR-182-5p [81]. The protease


inhibitors have been shown to increase the SREBP levels and activate UPR [82].


2.5. Autoimmune liver disease and liver fibrosis Recent studies have suggested that ER stress may play a role in the pathogenesis of deregulated autophagy and cellular apoptosis in biliary epithelial lesions in primary biliary cirrhosis [83]. The expression of the ER stress markers GRP78 and protein disulfide isomerases was significantly increased in cultured biliary epithelial cells, compared with those in normal livers [84,85]. In another genetic model of intra-hepatic cholestasis, the accumulation of bile acids in the liver was associated with elevated mRNA expression of CHOP and BiP [86]. Furthermore, the disruption of protein folding in the ER might be related with the transgenic expression of the mutant Z allele of α1-antitrypsin (AAT) in the bile duct-ligated mice, resulting in increasing liver injury and fibrosis [87]. ER stress induced the expression of fibrogenic genes in hepatic stellate cells via phosphorylation of PERK and eIF2α, associated with significant CHOP upregulation [88]. Hepatic fibrosis is a common representation of activation of hepatic stellate cells, with AAT deficiency as the key factor as published in several studies [89,90]. Besides AAT, cannabidiol was another activator of ER stress in hepatic stellate cells [91]. Cannabidiol-mediated ER stress caused apoptosis in activated but not quiescent stellate cells.

2.6. Acute-on-chronic liver failure There are few studies in the field of ER stress response in the progression of acute-on-chronic liver failure (ACLF). A genetic analysis revealed that ER stress, or downstream pathwayrelated genes, such as BiP, ATF4, and PERK, were differentially expressed in patients with ACLF [92]. Expression of genes related to ER stress, apoptosis and inflammation were upregulated in ACLF, as iron-regulatory genes and autophagy pathways were markedly downregulated. Serum iron and ferritin levels were dramatically elevated and hepcidin levels declined [93]. The underlying link between the activation of ER stress and the iron homeostasis remains uncertain. The activation of ER stress was demonstrated in another study of ACLF caused by acute exacerbation of chronic hepatitis B [94]. The degree of liver injury contributed to the altered signaling pathways induced by ER stress. In patients with chronic hepatitis B, all three pathways of UPR were activated. In ACLF subjects, the expression of the ATF6α pathway was activated, whereas the expression of PERK and IRE1α was not activated. The expression of ER stress-related apoptosis molecules such as CHOP, caspase-4, Bax were activated in the progression of acute exacerbation of CHB, whereas GRP78 and GRP94 were gradually decreased. In this context, more studies are necessary to fully understand the effect of ER stress in the progression of ACLF.

5

in particular, in the early stage of disease. A gradual increase in the expression of PERK, IRE1α, ATF6α, and GRP78, as well as CHOP, eIF2α, and TRAF2 or caspase-12, was demonstrated during early liver carcinogenesis. In the late stage of disease, the expression level reached a peak [95]. Bip/GRP78 has been proposed to play a role in carcinogenesis by promoting malignant transformation in numerous tissues including the liver [96]. The UPR promoted adaptation to hypoxia, extending the survival of the hepatoma to hypoxic conditions [97]. The expression levels of BiP/GRP78 and ATF6α were considered to positively correlate with the histological grade of the tumors. A study of wild-type and MUP-uPA transgenic mice fed with high-fat diet showed that both NAFLD and obesitydriven HCC were dependent on TNF-α produced by inflammatory macrophages in response to hepatocyte ER stress. The MUP-uPA transgenic mice exhibited more liver damage, more immune infiltration, and increased lipogenesis. In these animals, classical histopathological indicators of steatohepatosis developed progressing to typical steatohepatitic HCC [98].

3. Targeting ER stress in liver diseases Based on the various mechanisms of ER stress-related liver diseases, novel therapeutic strategies have generated intense interest over the past few years. Several compounds were considered for the treatment of disease via their effects on signal transduction pathways, such as SNX-2112 and bortezomib in HCC [99–101]. In this review, we focus on the molecules that target a single signal transduction pathway or chaperone to regulate protein folding, induce autophagy, and promote ER proteostasis (Table 1).

3.1. PERK signaling Salubrinal has been tested in various models of disease and is a small molecule that selectively inhibits the PERK signaling pathway [107]. In human hepatoma, salubrinal showed protective effects against ER stress by reducing the overloading of misfolded proteins [102]. It also inhibited to some degree inflammation and necrosis of hepatosteatosis induced by fat accumulation, but not alcohol administration [108]. Sinulariolide is another compound that may induce hepatoma apoptosis through activation of PERK pathway in HA22T cells [103]. PKR is a kinase of the PERK-eIF2α-CHOP signal transduction pathway that phosphorylates Ser51 in response to ER stress, as well as the key antiviral component of the interferon response. A small molecule inhibitor of PKR named C16 was proposed to prevent ER stress-associated cell death in NAFLD. However, as ceramide accumulates in the livers of patients with advanced ALD, C16 has been proposed to contribute deterioration of the liver [21]. FGF19 and FGF21 were recently identified to play important roles in the UPR signal [104,105]. A study on tunicamycin-induced hepatic steatosis showed that administration of recombinant FGF 21 alleviated the steatosis, in parallel with reduced expression of the eIF2α-ATF4-CHOP signal [106].

2.7. Hepatocellular carcinoma

3.2. IRE1α signaling

Present studies suggest that ER stress is of significant importance in the development of hepatocellular carcinoma (HCC),

Structure–activity studies and mutational analysis of contact residues of IRE1α have shown that the N-terminal domain is

6

F.-L. WU ET AL.

Table 1. Molecules to target the endoplasmic reticulum stress in liver diseases. Molecule Salubrinal Sinulariolide C16 FGF21 MKC-3946 STF-083010 4μ8C Hydroxy-arylaldehydes Mycotoxins Resveratrol Nucleobindin1

PERK PERK PERK PERK IRE1α IRE1α IRE1α IRE1α

Liver disease HCC, NAFLD HCC NAFLD Drug-induced liver injury HCC HCC HCC HCC

Preclinical, Preclinical, Preclinical, Preclinical, Preclinical, Preclinical, Preclinical, Preclinical,

IRE1α IRE1α ATF6

HCC HCC HCC

Preclinical, in vitro Preclinical, in vitro Preclinical, in vitro

Baicalein

ATF6


Apigenin Acetyl shikonin Berberine Geldanamycin 4-Phenylbutyrate Tauroursodeoxycholic acid Cyclosporin A

Target

Polymicrobial sepsis -induced liver injury, HCC ATF6 Drug-induced liver injury, NAFLD, and HCC Chaperon GRP78 HCC Chaperon ORP150 HCC Chaperon GRP94 HCC Unfolded protein Autoimmune liver disease, cystic fibrosis Unfolded protein Urea-cycle disorders, steatohepatosis Liver transplantation Ca2+ channel 2+

Dantrolene Rapamycin

Ca channel Autophagy

6-Shogaol Indole Carbamazepine

Autophagy Autophagy Autophagy

NAFLD Liver ischemia–reperfusion injury HCC CCL4-induced fibrosis α1-Antitrypsin deficiencyinduced liver fibrosis

Phase/Model in vivo in vitro in vitro in vitro in vitro in vitro in vitro in vitro

Preclinical, in vitro

Effect Activation of PERK activity Initiation of hepatoma apoptosis Inhibition of PKR expression Inhibition of eIF2α-ATF4-CHOP activity Inhibition of XBP1 mRNA splicing Inhibition of XBP1 mRNA splicing Inhibition of XBP1 mRNA splicing Inhibition of XBP1 mRNA splicing

Refs. [68,69] [70] [7] [73] [79] [79] [79] [78]

Inhibition of XBP1 mRNA splicing Inhibition of XBP1 DNA binding Inhibition of ATF6 cleavage from site1 protease Inhibition of mitogen-activated protein kinases, initiation of autophagy

[80] [81] [82,83] [85,86]

Preclinical, in vitro

Prevention of SREBP-2 translocation

[87–90]

Preclinical, in vitro Preclinical, in vitro Preclinical, in vitro US FDA approved for the former, preclinical for the latter FDA approved for the former, preclinical for the latter Preclinical, in vitro

[68] [94] [95] [97,101]

Preclinical, in vitro Preclinical, in vitro

Upregulation of BiP expression Downregulation of ORP150 expression Downregulation of GRP94 expression Promotion of protein transportation, attenuation of protein misfolding Promotion of protein transportation, attenuation of protein misfolding Inhibition of Ca2+ influx into the mitochondria Inhibition of Ca2+ efflux Restoration of autophagy

Preclinical, in vitro Preclinical, in vitro Preclinical, in vitro

Restoration of autophagy Inhibition of autophagy Inhibition of autophagy

[104] [105] [106]

[98,100] [102] [101] [103]

PERK: Protein kinase RNA-like ER kinase; FGF21: fibroblast growth factor 21; ATF6: activating transcription factor-6; IRE1α: inositol requiring enzyme-1α; GRP78: glucose-regulated protein of 78 kD; GRP94: glucose-regulated protein of 94 kD; HCC: hepatocellular carcinoma; NAFLD: nonalcoholic fatty liver disease; BiP: heavy-chain binding protein; CHOP: C/EBP-homologous protein; eIF2α: eukaryotic initiation factor 2α; SREBP-2: sterol-regulatory element binding protein.

the RNase active site able to detect ER stress [109]. Salicylaldimine analogs, such as MKC-3946, STF-083010, and 4u8C, as well as hydroxy-aryl-aldehydes (HAA) were shown to covalently attach to the lysine residue and to inhibit the RNase activity of IRE1α [110–113]. Salicylaldimine analogs have been demonstrated to suppress cell viability and proliferation because of their in vitro cytotoxicity in HepG2 cells [114]. The crystal structures of murine IRE1α in a complex with three HAA inhibitors were studied. HAA inhibitors selectively attached to the RNase-active sites by forming an essential Schiff base with Lys907 and a hydrogen bond with Tyr892, as well as pi-stacking interactions with His910 and Phe889. Protein disulfide isomerase A6 was proved to regulate insulin secretion by selectively inhibiting the RIDD activity of IRE1α, maintaining its signaling within a physiologically tolerable range [115]. Besides the above synthetic molecules, some natural compounds such as mycotoxins and resveratrol may modulate the IRE1α pathway [116]. Resveratrol promoted the necrosis and apoptosis of hepatoma by inhibiting the DNA-binding activity of XBP1 [117]. These studies may lay the foundation for the design of new small molecules that suppress the IRE1α signaling pathway in liver diseases.

and is located in the Golgi apparatus [118]. Overexpression of NUCB1 inhibits ATF6α cleavage from site1 protease during ER stress, whereas knockdown of NUCB1 by siRNA accelerates ATF6α cleavage. The key biochemical step in the export of NUCB1from the ER is the activation of the proline residue at the +2-position [119]. The natural compounds baicalein and apigenin have been reported to upregulate ATF6α expression in animal models of liver disease [120]. Treatment with baicalein was shown to protect against polymicrobial sepsisinduced liver injury via inhibition of ER stress-related inflammation and apoptosis in mice [121]. A recent study showed that baicalein effectively blocked the cell viability and colony formation of HepG2 cells via triggering of autophagy [122]. It has been identified that the apoptosis and autophagy was induced by activating the UPR pathway. Apigenin, a polyhydroxylated flavonoid, was shown to be effective in the treatment of acute liver injury induced by APAP [123], NAFLD [124], and HCC [125]. A recent study showed that apigenin prevented SREBP-2 translocation and reduced downstream HMGCR transcription [126].

3.4. Chaperone modulators 3.3. ATF6α signaling Nucleobindin1 (NUCB1) is the first-identified negative feedback regulator of site1 protease-mediated ATF6α activation

As described above, BiP/GRP78 is an essential chaperone involved in the activation of PERK and IRE1α and translation of ATF6α. BiP inducer X was considered a pharmacological



modulator able to upregulate the levels of BiP mRNA in the neural system [127], kidney [128], and bone [129]. Studies on the use of BiP inducer X in liver disease remained to be explored in the future. A study conducted in Hep3B cells has shown that acetyl shikonin may induce hepatoma apoptosis by increasing the expression of BiP via the production of reactive oxygen species [102]. N-acetylcysteine protected cells from apoptosis by acting as an antioxidant. ORP150 is a chaperone induced by ER stress under the hypoxia condition. Berberine was shown to decrease the expression of ORP150 in HCC [130]. GRP94 is another chaperone of ER stress acting in the regulation of UPR signaling and geldanamycin is an inhibitor which downregulates GRP94 expression. Geldanamycin and its analogs 17-allylamino17-demethoxygeldanamycin, 17-[2-(piperidinyl-1′-yl)-ethylamino]-17-demethoxygeldanamycin have been shown to inhibit cell division of cultured HepG2 cells [131].

3.5. Chemical chaperones Chemical chaperones modulate ER stress by attenuating protein misfolding and aggregation, as well as promoting protein transportation and secretion in cells in a nonspecific manner [132]. The most well-known chemical chaperones of ER stress are 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA) [133,134]. 4-PBA and TUDCA were primarily investigated for the treatment of primary biliary cirrhosis and ureacycle disorders. When tested in model systems of cystic fibrosis and steatohepatosis, 4-PBA and TUDCA promoted the folding and intracellular trafficking of the unfolded proteins [135,136]. In HUVEC, 4-PBA was shown to inhibit ER stress induced by tunicamycin and thapsigargin, and showed an increase in the mesenchymal markers α-SMA, vimentin, and collagen 1 [33]. In metabolic disorders such as obesity and diabetes, 4-PBA restored the impaired insulin signal transduction in Cystatin C-treated hepatocytes by alleviation of ER stress [137]. In Pelteobagrus fulvidraco, a yellow catfish model used to investigate the effect of Cu2+ exposure on ER stress and Ca2+ homeostasis, Cu2+-induced ER stress was shown to be involved in hepatic lipid metabolism [138]. 4-PBA rectified lipid metabolism through the attenuation of the mRNA expression of SREBP-1c, SCAP, ACC, FAS, GRP78/BiP, GRP94, eIF2α, and XBP-1. It is suggested that TUDCA binds to the hydrophobic regions of proteins and prevents their subsequent aggregation. This may stabilize unfolded proteins in the ER or facilitate their degradation through cellular degradation pathways [139]. TUDCA reversed abnormal autophagy, reduced ER stress, and restored insulin sensitivity in the liver of obese mice [140]. In a diethylnitrosamine-induced mouse HCC model, TUDCA reduced hepatocarcinogenesis by inhibiting carcinogen-induced ER stress-mediated cell death and inflammation [134]. TUDCA reduced eIF2α phosphorylation, CHOP, and caspase-12 processing, as well as increasing the expression of the NF-κB inhibitor, IκBα.

3.6. Regulators of Ca2+ homeostasis Ca2+ homeostasis is critical for lipid and glucose metabolism in the liver as it modulates protein folding, modification, and maturation in the ER. During ER stress, Ca2+ leak from the ER to the

7

mitochondria occurs under the regulation of cyclophilin subsets, including cyclophilin B, C, and D [141]. As an inhibitor of cyclophilin, cyclosporin A was shown effective in suppressing Ca2+ influx into the mitochondria [142]. The application of cyclosporin A therapy in liver disease is limited due to its high risk of druginduced liver injury. Dantrolene, a muscle relaxant, functions as a Ca2+ channel blocker by inhibiting Ca2+ efflux and has been shown to prevent Cu2+-induced intracellular Ca2+ elevation, which may have utility in the correction of dysregulated lipid metabolism [138].

3.7. Inducers of autophagy Autophagy is an intracellular self-digesting pathway responsible for the removal of unfolded/misfolded proteins accumulating in the ER. The relation between autophagy and liver ischemia– reperfusion injury (IRI) has been well studied. A study in a murine liver model found that transient ischemia activated autophagy in order to protect the liver from IRI, whereas prolonged ischemia had the opposite effect [143]. After transient ischemia, both the chemical chaperon 4-PBA and the autophagy inhibitor 3-methyladenine (3-MA) disrupted autophagy flux and increased liver IRI. After prolonged ischemia, treatment with 4PBA was found to protect the liver by restoring autophagy flux, and 3-MA abrogated its liver protective effect [133]. Although both types of ischemia activated 5′-adenosine monophosphateactivated protein kinase and inactivated protein kinase B, prolonged ischemia also resulted in downregulation of autophagyrelated genes in ischemic livers. These results indicate a potential time or intensity-dependent effect of ER stress response in liver IRI via its regulation of autophagy. Rapamycin enhanced hepatic autophagy during the process of reperfusion [144]. A recent study has shown that other autophagy inducers may have an effect on various liver diseases. 6-Shogaol induced apoptosis, modulated cyclin expression, and activated UPR signaling pathways via inducing autophagy in HepG2 cells [145]. Another study demonstrated that melatonin inhibited autophagy and ER stress in mice in which fibrosis was induced with carbon tetrachloride. Administration of indole resulted in a significant inhibition of the autophagic flux and UPR expression [146]. Carbamazepine could also induce autophagy in the liver and may have future therapeutic potential in the treatment of AAT-induced liver fibrosis [147]. However, it should be recognized that most of these molecules are at the nonclinical testing phase and more studies are needed to understand their potentials as drugs to deliver acceptable standards of safety and efficacy.

4. Conclusion Protein misfolding and aggregation is the basis of ER stress and a common feature of many acute and chronic liver diseases. We propose that a direct measure of the accumulated misfolded proteins over the time course of liver disease may provide valuable target validation data which could further support the potential for drug discovery. The UPR sensors respond to ER stress in different manners: IRE1α activation by XBP1 splicing, PERK activation by co-phosphorylation with eIF2α, and ATF6α by its nuclear translocation. Published

8

F.-L. WU ET AL.

studies focus on potential therapeutic modalities in ER stress and draw on a wide range of molecules from biosynthesized large compounds to small molecules, from animal models to human clinical studies. To date, most intervention studies have considered their potential adverse effects on normal tissues. Further studies are needed for a better understanding of the efficacy and safety of the novel pharmacologic strategies targeting ER stress in the field of liver disease.


5. Expert commentary and 5-year view Different injuries in the liver result in a variety of pathophysiological changes, although the same ER stress may activate different signaling pathways. In each pathway, a variety of target sites and sensors can be described. Based on the development of proteomic techniques and recombinant gene technology, gene knockout and chimeras have been developed in specialized murine models, where hepatocyte pathology and biochemistry have and can be extensively studied. ATF6αablation mice were prone to suffer from hepatosteatosis and death with ER stress in the liver [148]. CHOP-null mice were free from ER stress-induced apoptosis [149]. CHOP deficiency dampened cholestasis-induced liver fibrosis by reduction of hepatocyte injury [86]. The genetic deletion of IRE1α led to early embryonic lethality, resulting in fetal liver hypoplasia in embryos [150]. A further study demonstrated that embryonic lethality associated with XBP1 deletion could be treated with a transgene encoding spliced XBP1 [151]. Interestingly, the expression of BiP/GRP78 and ATF6α has a positive correlation with the histologic grade of the tumors. The levels of BiP/ GRP78 and ATF6α could be tested from blood rather than more invasive techniques, such as tissue biopsy. Although recent research is limited to nonclinical studies, we believe that with a growing evidence base, rigorous pharmacological testing, and further development of tissue microarray techniques, precise intervention of liver diseases targeting ER stress may be an achievable goal in the future.

Key issues ● The UPR is activated by three ER transmembrane sensors: PERK, IRE1α and ATF6α. ● Prolonged ER stress may result in progression of the majority of acute and chronic liver diseases. ● A wide range of compounds targeting the UPR components have been applied to treat liver diseases in non-clinical in vitro and in vivo models. ● This emerging area may be a candidate for precision medicine approaches which are urgently needed for exploration and integration in the clinical practice treatment of patients with liver disease.

Financial & competing interests disclosure This work was supported by grants from National Natural Science Foundation of China (81500665), Health Bureau of Zhejiang Province (2010KYB070), Research Foundation of Education Bureau of Zhejiang Province (Y201009942) and Project of New Century 551 Talent Nurturing in Wenzhou. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or

financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

ORCID Wen-Yue Liu http://orcid.org/0000-0003-1454-7077 Sven Van Poucke http://orcid.org/0000-0001-8070-8786 Ming-Hua Zheng http://orcid.org/0000-0003-4984-2631

References Papers of special note have been highlighted as: • of interest •• of considerable interest 1. Anelli T, Sitia R. Protein quality control in the early secretory pathway. EMBO J. 2008;27(2):315–327. 2. Bertolotti A, Zhang Y, Hendershot LM, et al. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000;2(6):326–332. • Key paper confirming BiP/GRP78 as the luminal protein chaperon inactively binding to the three transmembrane sensors before UPR. 3. Shen J, Chen X, Hendershot L, et al. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev Cell. 2002;3(1):99–111. 4. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334(6059):1081– 1086. 5. Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol. 2007;18 (6):716–731. 6. Soboloff J, Rothberg BS, Madesh M, et al. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 2012;13 (9):549–565. 7. Oikawa D, Kimata Y, Kohno K, et al. Activation of mammalian IRE1alpha upon ER stress depends on dissociation of BiP rather than on direct interaction with unfolded proteins. Exp Cell Res. 2009;315(15):2496–2504. 8. Novoa I, Zeng H, Harding HP, et al. Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. J Cell Biol. 2001;153(5):1011–1022. 9. Marciniak SJ, Yun CY, Oyadomari S, et al. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev. 2004;18(24):3066–3077. 10. Urano F, Wang X, Bertolotti A, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000;287(5453):664–666. 11. Kim I, Shu CW, Xu W, et al. Chemical biology investigation of cell death pathways activated by endoplasmic reticulum stress reveals cytoprotective modulators of ASK1. J Biol Chem. 2009;284(3):1593– 1603. 12. Han D, Lerner AG, Vande Walle L, et al. IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates. Cell. 2009;138(3):562–575. 13. Santos CXC, Tanaka LY, Wosniak J, et al. Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox Signal. 2009;11(10):2409–2427. 14. Yamazaki H, Hiramatsu N, Hayakawa K, et al. Activation of the AktNF- B pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response. J Immunol. 2009;183(2):1480–1487. 15. Jiang HY, Wek SA, McGrath BC, et al. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses. Mol Cell Biol. 2003;23(16):5651–5663. 16. Zhang K, Shen X, Wu J, et al. Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell. 2006;124(3):587–599.



17. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012;13 (2):89–102. • The author describes the intact signal-conducting pathways and the outcomes of effected cells. 18. Dara L, Ji C, Kaplowitz N. The contribution of endoplasmic reticulum stress to liver diseases. Hepatology. 2011;53(5):1752–1763. 19. Ji C. Mechanisms of alcohol-induced endoplasmic reticulum stress and organ injuries. Biochem Res Int. 2012;2012:216450. 20. Howarth DL, Vacaru AM, Tsedensodnom O, et al. Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes. Alcohol Clin Exp Res. 2012;36(1):14–23. 21. Longato L, Ripp K, Setshedi M, et al. Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease. Oxid Med Cell Longev. 2012; 2012:479348. 22. Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11(4):381–389. 23. Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease. J Hepatol. 2011;54(4):795–809. •• Key paper presenting the effects of ER stress on various liver diseases. 24. Werstuck GH, Lentz SR, Dayal S, et al. Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways. J Clin Invest. 2001;107 (10):1263–1273. 25. Esfandiari F, Villanueva JA, Wong DH, et al. Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs. Am J Physiol Gastrointest Liver Physiol. 2005;289(1):G54–G63. 26. Ramirez T, Longato L, Dostalek M, et al. Insulin resistance, ceramide accumulation and endoplasmic reticulum stress in experimental chronic alcohol-induced steatohepatitis. Alcohol Alcohol. 2013;48 (1):39–52. 27. Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology. 2003;124(5):1488–1499. 28. Ji C, Chan C, Kaplowitz N. Predominant role of sterol response element binding proteins (SREBP) lipogenic pathways in hepatic steatosis in the murine intragastric ethanol feeding model. J Hepatol. 2006;45(5):717–724. 29. Ji C, Kaplowitz N. ER stress: can the liver cope? J Hepatol. 2006;45 (2):321–333. 30. Petrasek J, Iracheta-Vellve A, Csak T, et al. STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease. Proc Natl Acad Sci U S A. 2013;110 (41):16544–16549. 31. Wang LR, Zhu GQ, Shi KQ, et al. Autophagy in ethanol-exposed liver disease. Expert Rev Gastroenterol Hepatol. 2015;9(8):1031– 1037. • Presents the interrelation of autophagy, ER stress, and alcoholic liver diseases. 32. Hernandez-Gea V, Hilscher M, Rozenfeld R, et al. Endoplasmic reticulum stress induces fibrogenic activity in hepatic stellate cells through autophagy. J Hepatol. 2013;59(1):98–104. 33. Ying R, Wang XQ, Yang Y, et al. Hydrogen sulfide suppresses endoplasmic reticulum stress-induced endothelial-to-mesenchymal transition through Src pathway. Life Sci. 2016;144:208–217. 34. Puri P, Mirshahi F, Cheung O, et al. Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease. Gastroenterology. 2008;134(2):568–576. 35. Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science. 2004;306 (5695):457–461. •• Evidence is presented that bilateral interaction of the ER stress in lipogenesis, lipotoxicity, and insulin resistance was the key factor in the pathogenetic process of hepatosteatosis in NAFLD. 36. Gregor MF, Yang L, Fabbrini E, et al. Endoplasmic reticulum stress is reduced in tissues of obese subjects after weight loss. Diabetes. 2009;58(3):693–700.

9

37. Mosinski JD, Pagadala MR, Mulya A, et al. Gastric bypass surgery is protective from high-fat diet induced nonalcoholic fatty liver disease and hepatic endoplasmic reticulum stress. Acta Physiol. 2015. doi:10.1111/apha.12640. [Epub ahead of print] 38. Li JS, Wang WJ, Sun Y, et al. Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress. Food Funct. 2015;6(5):1643–1651. 39. Zhang N, Lu Y, Shen X, et al. Fenofibrate treatment attenuated chronic endoplasmic reticulum stress in the liver of nonalcoholic fatty liver disease mice. Pharmacology. 2015;95(3–4):173–180. 40. Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology. 2012;142(4):711–725.e6. 41. Sozio MS, Liangpunsakul S, Crabb D. The role of lipid metabolism in the pathogenesis of alcoholic and nonalcoholic hepatic steatosis. Semin Liver Dis. 2010;30(4):378–390. 42. Reddy JK, Rao MS. Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol. 2006;290(5):G852–G858. 43. Ji C, Shinohara M, Kuhlenkamp J, et al. Mechanisms of protection by the betaine-homocysteine methyltransferase/betaine system in HepG2 cells and primary mouse hepatocytes. Hepatology. 2007;46 (5):1586–1596. 44. Bobrovnikova-Marjon E, Hatzivassiliou G, Grigoriadou C, et al. PERKdependent regulation of lipogenesis during mouse mammary gland development and adipocyte differentiation. Proc Natl Acad Sci U S A. 2008;105(42):16314–16319. 45. Yoshiuchi K, Kaneto H, Matsuoka TA, et al. Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice. Biochem Biophys Res Commun. 2008;366(2):545–550. 46. Boden G, Duan X, Homko C, et al. Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals. Diabetes. 2008;57(9):2438–2444. 47. Lee AH, Scapa EF, Cohen DE, et al. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science. 2008;320 (5882):1492–1496. 48. Wang D, Wei Y, Pagliassotti MJ. Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. Endocrinology. 2006;147(2):943–951. 49. Yamamoto K, Takahara K, Oyadomari S, et al. Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress. Mol Biol Cell. 2010;21(17):2975–2986. 50. Usui M, Yamaguchi S, Tanji Y, et al. Atf6alpha-null mice are glucose intolerant due to pancreatic beta-cell failure on a high-fat diet but partially resistant to diet-induced insulin resistance. Metabolism. 2012;61(8):1118–1128. 51. Kim KE, Kim H, Heo RW, et al. Myeloid-specific SIRT1 deletion aggravates hepatic inflammation and steatosis in high-fat diet-fed mice. Korean J Physiol Pharmacol. 2015;19(5):451–460. 52. Kammoun HL, Chabanon H, Hainault I, et al. GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice. J Clin Invest. 2009;119(5):1201–1215. 53. Hirosumi J, Tuncman G, Chang L, et al. A central role for JNK in obesity and insulin resistance. Nature. 2002;420(6913):333–336. 54. Du K, Herzig S, Kulkarni RN, et al. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science. 2003;300 (5625):1574–1577. 55. Nakae J, Biggs WH 3rd, Kitamura T, et al. Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1. Nat Genet. 2002;32(2):245–253. 56. Liu WY, Xie DM, Zhu GQ, et al. Targeting fibroblast growth factor 19 in liver disease: a potential biomarker and therapeutic target. Expert Opin Ther Targets. 2015;19(5):675–685. • The authors provide evidence that FGF19 could be a therapeutic target in liver diseases. 57. Liu WY, Huang S, Shi KQ, et al. The role of fibroblast growth factor 21 in the pathogenesis of liver disease: a novel predictor and

10

F.-L. WU ET AL.

• 58.

59.

60.

61.

62.


63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76. 77.

78.

therapeutic target. Expert Opin Ther Targets. 2014;18(11):1305– 1313. The authors provide evidence that FGF21 could be a novel predictor and therapeutic target in liver diseases. Cheong H, Lee SS, Lee JS, et al. Phagocytic function of Kupffer cells in mouse nonalcoholic fatty liver disease models: evaluation with superparamagnetic iron oxide. J Magn Reson Imaging. 2015;41 (5):1218–1227. Feldman A, Aigner E, Weghuber D, et al. The potential role of iron and copper in pediatric obesity and nonalcoholic fatty liver disease. Biomed Res Int. 2015;2015:287401. Okamoto K, Moriishi K, Miyamura T, et al. Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein. J Virol. 2004;78(12):6370–6380. Awe K, Lambert C, Prange R. Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein. FEBS Lett. 2008;582(21–22):3179–3184. Sir D, Chen WL, Choi J, et al. Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response. Hepatology. 2008;48(4):1054–1061. Wang HC, Wu HC, Chen CF, et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol. 2003;163(6):2441–2449. Li B, Gao B, Ye L, et al. Hepatitis B virus X protein (HBx) activates ATF6 and IRE1-XBP1 pathways of unfolded protein response. Virus Res. 2007;124(1–2):44–49. Benali-Furet NL, Chami M, Houel L, et al. Hepatitis C virus core triggers apoptosis in liver cells by inducing ER stress and ER calcium depletion. Oncogene. 2005;24(31):4921–4933. Gong G, Waris G, Tanveer R, et al. Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B. Proc Natl Acad Sci U S A. 2001;98(17):9599–9604. Zheng Y, Gao B, Ye L, et al. Hepatitis C virus non-structural protein NS4B can modulate an unfolded protein response. J Microbiol. 2005;43(6):529–536. Yeganeh B, Rezaei Moghadam A, Alizadeh J, et al. Hepatitis B and C virus-induced hepatitis: apoptosis, autophagy, and unfolded protein response. World J Gastroenterol. 2015;21(47):13225–13239. Dreux M, Gastaminza P, Wieland SF, et al. The autophagy machinery is required to initiate hepatitis C virus replication. Proc Natl Acad Sci U S A. 2009;106(33):14046–14051. Sir D, Kuo CF, Tian Y, et al. Replication of hepatitis C virus RNA on autophagosomal membranes. J Biol Chem. 2012;287(22):18036– 18043. Tanida I, Fukasawa M, Ueno T, et al. Knockdown of autophagyrelated gene decreases the production of infectious hepatitis C virus particles. Autophagy. 2009;5(7):937–945. Vescovo T, Refolo G, Romagnoli A, et al. Autophagy in HCV infection: keeping fat and inflammation at bay. Biomed Res Int. 2014;2014:265353. Shrivastava S, Raychoudhuri A, Steele R, et al. Knockdown of autophagy enhances the innate immune response in hepatitis C virus-infected hepatocytes. Hepatology. 2011;53(2):406–414. Gregoire IP, Rabourdin-Combe C, Faure M. Autophagy and RNA virus interactomes reveal IRGM as a common target. Autophagy. 2012;8(7):1136–1137. Nagy G, Szarka A, Lotz G, et al. BGP-15 inhibits caspase-independent programmed cell death in acetaminophen-induced liver injury. Toxicol Appl Pharmacol. 2010;243(1):96–103. Lorz C, Justo P, Sanz A, et al. Paracetamol-induced renal tubular injury: a role for ER stress. J Am Soc Nephrol. 2004;15(2):380–389. Nagy G, Kardon T, Wunderlich L, et al. Acetaminophen induces ER dependent signaling in mouse liver. Arch Biochem Biophys. 2007;459(2):273–279. James LP, Letzig L, Simpson PM, et al. Pharmacokinetics of acetaminophen-protein adducts in adults with acetaminophen overdose and acute liver failure. Drug Metab Dispos. 2009;37 (8):1779–1784.

79. Zhang J, Song S, Pang Q, et al. Corrigendum: serotonin deficiency exacerbates acetaminophen-induced liver toxicity in mice. Sci Rep. 2015;5:12184. 80. Wang X, Thomas B, Sachdeva R, et al. Mechanism of arylating quinone toxicity involving Michael adduct formation and induction of endoplasmic reticulum stress. Proc Natl Acad Sci U S A. 2006;103 (10):3604–3609. 81. Van den Hof WF, Van Summeren A, Lommen A, et al. Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity. Toxicology. 2014;324:18–26. 82. Chen L, Jarujaron S, Wu X, et al. HIV protease inhibitor lopinavirinduced TNF-alpha and IL-6 expression is coupled to the unfolded protein response and ERK signaling pathways in macrophages. Biochem Pharmacol. 2009;78(1):70–77. 83. Bochkis IM, Rubins NE, White P, et al. Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress. Nat Med. 2008;14(8):828–836. 84. Sasaki M, Yoshimura-Miyakoshi M, Sato Y, et al. A possible involvement of endoplasmic reticulum stress in biliary epithelial autophagy and senescence in primary biliary cirrhosis. J Gastroenterol. 2015;50(9):984–995. 85. Adachi T, Kaminaga T, Yasuda H, et al. The involvement of endoplasmic reticulum stress in bile acid-induced hepatocellular injury. J Clin Biochem Nutr. 2014;54(2):129–135. 86. Tamaki N, Hatano E, Taura K, et al. CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury. Am J Physiol Gastrointest Liver Physiol. 2008;294(2):G498–505. • Present the role of CHOP in the pathogenesis of liver fibrosis. 87. Mencin A, Seki E, Osawa Y, et al. Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis. Hepatology. 2007;46(5):1443–1452. 88. Borkham-Kamphorst E, Steffen BT, Van de Leur E, et al. CCN1/ CYR61 overexpression in hepatic stellate cells induces ER stressrelated apoptosis. Cell Signal. 2016;28(1):34–42. 89. Perlmutter DH, Silverman GA. Hepatic fibrosis and carcinogenesis in alpha1-antitrypsin deficiency: a prototype for chronic tissue damage in gain-of-function disorders. Cold Spring Harb Perspect Biol. 2011;3(3):a005801. 90. Koo JH, Lee HJ, Kim W, et al. Endoplasmic reticulum stress in hepatic stellate cells promotes liver fibrosis via PERK-mediated degradation of HNRNPA1 and up-regulation of SMAD2. Gastroenterology. 2016;150(1):181–193. 91. Lim MP, Devi LA, Rozenfeld R. Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis. Cell Death Dis. 2011;2:e170. 92. Jin L, Wang K, Liu H, et al. Genomewide histone H3 lysine 9 acetylation profiling in CD4+ T cells revealed endoplasmic reticulum stress deficiency in patients with acute-on-chronic liver failure. Scand J Immunol. 2015;82(5):452–459. 93. Maras JS, Maiwall R, Harsha HC, et al. Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute-on-chronic liver failure. Hepatology. 2015;61 (4):1306–1320. 94. Ren F, Shi H, Zhang L, et al. The dysregulation of endoplasmic reticulum stress response in acute-on-chronic liver failure patients caused by acute exacerbation of chronic hepatitis B. J Viral Hepat. 2016;23(1):23–31. 95. Shuda M, Kondoh N, Imazeki N, et al. Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis. J Hepatol. 2003;38(5):605–614. •• Key article confirming the UPR genes involved in hepatocarcinogenesis. 96. Jiang X, Kanda T, Nakamoto S, et al. Involvement of androgen receptor and glucose-regulated protein 78 kDa in human hepatocarcinogenesis. Exp Cell Res. 2014;323(2):326–336. 97. Romero-Ramirez L, Cao H, Nelson D, et al. XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Res. 2004;64(17):5943–5947.



98. Nakagawa H, Umemura A, Taniguchi K, et al. ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development. Cancer Cell. 2014;26(3):331–343. 99. Wang X, Wang S, Liu Y, et al. The Hsp90 inhibitor SNX-2112 induces apoptosis of human hepatocellular carcinoma cells: the role of ER stress. Biochem Biophys Res Commun. 2014;446(1):160–166. 100. Fribley A, Wang CY. Proteasome inhibitor induces apoptosis through induction of endoplasmic reticulum stress. Cancer Biol Ther. 2006;5(7):745–748. 101. Jang MK, Han YR, Nam JS, et al. Protective effects of alisma orientale extract against hepatic steatosis via inhibition of endoplasmic reticulum stress. Int J Mol Sci. 2015;16(11):26151–26165. 102. Moon J, Koh SS, Malilas W, et al. Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress. Eur J Pharmacol. 2014;735:132–140. 103. Chen YJ, Su JH, Tsao CY, et al. Sinulariolide induced hepatocellular carcinoma apoptosis through activation of mitochondrial-related apoptotic and PERK/eIF2alpha/ATF4/CHOP pathway. Molecules. 2013;18(9):10146–10161. 104. Shimizu M, Morimoto H, Maruyama R, et al. Selective regulation of FGF19 and FGF21 expression by cellular and nutritional stress. J Nutr Sci Vitaminol. 2015;61(2):154–160. 105. Lin Z, Pan X, Wu F, et al. Fibroblast growth factor 21 prevents atherosclerosis by suppression of hepatic sterol regulatory element-binding protein-2 and induction of adiponectin in mice. Circulation. 2015;131(21):1861–1871. • Describes FGF21 as an important modulator of lipid metabolism. 106. Jiang S, Yan C, Fang QC, et al. Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis. J Biol Chem. 2014;289(43):29751–29765. 107. Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat Rev Drug Discov. 2013;12(9):703–719. •• Key paper presents the molecular basis of the therapeutic strategies targeting UPR. 108. Kuo TF, Tatsukawa H, Matsuura T, et al. Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways. J Cell Physiol. 2012;227 (3):1130–1137. 109. Yoshida H, Matsui T, Yamamoto A, et al. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 2001;107(7):881–891. 110. Volkmann K, Lucas JL, Vuga D, et al. Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease. J Biol Chem. 2011;286(14):12743–12755. 111. Mimura N, Fulciniti M, Gorgun G, et al. Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma. Blood. 2012;119(24):5772–5781. 112. Papandreou I, Denko NC, Olson M, et al. Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma. Blood. 2011;117(4):1311–1314. 113. Sanches M, Duffy NM, Talukdar M, et al. Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors. Nat Commun. 2014;5:4202. 114. Rahman FU, Ali A, Guo R, et al. Synthesis and anticancer activities of a novel class of mono- and di-metallic Pt(II)(salicylaldiminato) (DMSO or Picolino)Cl complexes. Dalton Trans. 2015;44(5):2166– 2175. 115. Eletto D, Eletto D, Boyle S, et al. PDIA6 regulates insulin secretion by selectively inhibiting the RIDD activity of IRE1. FASEB J. 2016;30 (2):653–665. 116. Fribley AM, Cruz PG, Miller JR, et al. Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response. J Biomol Screen. 2011;16(8):825–835. 117. Rojas C, Pan-Castillo B, Valls C, et al. Resveratrol enhances palmitate-induced ER stress and apoptosis in cancer cells. Plos One. 2014;9(12):e113929.

11

118. Tsukumo Y, Tomida A, Kitahara O, et al. Nucleobindin 1 controls the unfolded protein response by inhibiting ATF6 activation. J Biol Chem. 2007;282(40):29264–29272. 119. Tsukumo Y, Tsukahara S, Saito S, et al. A novel endoplasmic reticulum export signal: proline at the +2-position from the signal peptide cleavage site. J Biol Chem. 2009;284(40):27500–27510. 120. Choi JH, Choi AY, Yoon H, et al. Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction. Exp Mol Med. 2010;42 (12):811–822. 121. Liu A, Wang W, Fang H, et al. Baicalein protects against polymicrobial sepsis-induced liver injury via inhibition of inflammation and apoptosis in mice. Eur J Pharmacol. 2015;748:45–53. 122. Wang YF, Li T, Tang ZH, et al. Baicalein triggers autophagy and inhibits the protein kinase B/mammalian target of rapamycin pathway in hepatocellular carcinoma HepG2 cells. Phytother Res. 2015;29(5):674–679. 123. Yang J, Wang XY, Xue J, et al. Protective effect of apigenin on mouse acute liver injury induced by acetaminophen is associated with increment of hepatic glutathione reductase activity. Food Funct. 2013;4(6):939–943. 124. Shi T, Zhuang R, Zhou H, et al. [Effect of apigenin on protein expressions of PPARs in liver tissues of rats with nonalcoholic steatohepatitis]. Zhonghua Gan Zang Bing Za Zhi. 2015; 23(2):124–129. 125. Hu XY, Liang JY, Guo XJ, et al. 5-Fluorouracil combined with apigenin enhances anticancer activity through mitochondrial membrane potential (ΔΨm)-mediated apoptosis in hepatocellular carcinoma. Clin Exp Pharmacol Physiol. 2015;42(2):146–153. 126. Wong TY, Lin SM, Leung LK. The flavone apigenin blocks nuclear translocation of sterol regulatory element-binding protein-2 in the hepatic cells WRL-68. Br J Nutr. 2015;113(12):1844–1852. 127. Nakanishi T, Shimazawa M, Sugitani S, et al. Role of endoplasmic reticulum stress in light-induced photoreceptor degeneration in mice. J Neurochem. 2013;125(1):111–124. 128. Prachasilchai W, Sonoda H, Yokota-Ikeda N, et al. The protective effect of a newly developed molecular chaperone-inducer against mouse ischemic acute kidney injury. J Pharmacol Sci. 2009;109 (2):311–314. 129. Hino S, Kondo S, Yoshinaga K, et al. Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis. J Bone Miner Metab. 2010;28(2):131–138. 130. Wang ZS, Lu FE, Xu LJ, et al. Berberine reduces endoplasmic reticulum stress and improves insulin signal transduction in Hep G2 cells. Acta Pharmacol Sin. 2010;31(5):578–584. 131. Li YP, Chen JJ, Shen JJ, et al. Synthesis and biological evaluation of geldanamycin analogs against human cancer cells. Cancer Chemother Pharmacol. 2015;75(4):773–782. 132. Ozcan U, Yilmaz E, Ozcan L, et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science. 2006;313(5790):1137–1140. • Evidence is presented that chemical chaperones influence glucose and lipid metabolism in a nonspecific manner. 133. Vilatoba M, Eckstein C, Bilbao G, et al. Sodium 4-phenylbutyrate protects against liver ischemia reperfusion injury by inhibition of endoplasmic reticulum-stress mediated apoptosis. Surgery. 2005;138(2):342–351. 134. Vandewynckel YP, Laukens D, Devisscher L, et al. Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure. Oncotarget. 2015;6(29):28011–28025. 135. Suaud L, Miller K, Panichelli AE, et al. 4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells. J Biol Chem. 2011;286 (52):45083–45092. 136. Cho EJ, Yoon JH, Kwak MS, et al. Tauroursodeoxycholic acid attenuates progression of steatohepatitis in mice fed a methionine-choline-deficient diet. Dig Dis Sci. 2014;59(7):1461–1474.


12

F.-L. WU ET AL.

137. Ji X, Yao L, Wang M, et al. Cystatin C attenuates insulin signaling transduction by promoting endoplasmic reticulum stress in hepatocytes. FEBS Lett. 2015;589(24 Pt B):3938–3944. 138. Song YF, Luo Z, Zhang LH, et al. Endoplasmic reticulum stress and disturbed calcium homeostasis are involved in copper-induced alteration in hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco. Chemosphere. 2015;144:2443–2453. 139. Gani AR, Uppala JK, Ramaiah KVA. Tauroursodeoxycholic acid prevents stress induced aggregation of proteins in vitro and promotes PERK activation in HepG2 cells. Arch Biochem Biophys. 2015;568:8–15. 140. Yang L, Li P, Fu S, et al. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab. 2010;11(6):467–478. 141. Stocki P, Chapman DC, Beach LA, et al. Depletion of cyclophilins B and C leads to dysregulation of endoplasmic reticulum redox homeostasis. J Biol Chem. 2014;289(33):23086–23096. 142. Korolczuk A, Maciejewski M, Czechowska G, et al. Ultrastructural examination of renal tubular epithelial cells and hepatocytes in the course of chronic cyclosporin A treatment–a possible link to oxidative stress. Ultrastruct Pathol. 2013;37(5):332–339. 143. Zhou H, Zhu J, Yue S, et al. The dichotomy of endoplasmic reticulum stress response in liver ischemia-reperfusion injury. Transplantation. 2016;100(2):365–372. • Presented the effect of ER stress on ischemia–reperfusion injury, providing basis for improving success rate of liver transplantation.

144. Zhu J, Lu T, Yue S, et al. Rapamycin protection of livers from ischemia and reperfusion injury is dependent on both autophagy induction and mammalian target of rapamycin complex 2-Akt activation. Transplantation. 2015;99(1):48–55. 145. Wu JJ, Omar HA, Lee YR, et al. 6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms. Eur J Pharmacol. 2015;762:449–458. 146. San-Miguel B, Crespo I, Sanchez DI, et al. Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis. J Pineal Res. 2015;59(2):151–162. 147. Kim JS, Wang JH, Biel TG, et al. Carbamazepine suppresses calpainmediated autophagy impairment after ischemia/reperfusion in mouse livers. Toxicol Appl Pharmacol. 2013;273(3):600–610. 148. Rutkowski DT, Wu J, Back SH, et al. UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev Cell. 2008;15(6):829– 840. 149. Zinszner H, Kuroda M, Wang X, et al. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev. 1998;12(7):982–995. 150. Zhang K, Wong HN, Song B, et al. The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis. J Clin Invest. 2005;115(2):268–281. 151. Lee AH, Chu GC, Iwakoshi NN, et al. XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J. 2005;24 (24):4368–4380.