CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) has been shown to be a key molecule in endoplasmic reticulum (ER) stress-mediated apoptosis. ER oxidoreductin 1-α (ERO1α), a target of CHOP, is an important oxidizing enzyme that regulates reactive oxygen species (ROS), which play a prominent role in hepatocellular death during acute liver failure (ALF). However, little is known about how CHOP facilitates ROS-induced hepatocellular injury. The present study was designed to investigate the roles and molecular mechanisms of CHOP in ALF. In the liver tissues from ALF patients, the expression of CHOP was significantly increased, which was accompanied by increased expression of dsRNA-dependent protein kinase (PKR)-like ER kinase (PERK) signalling, activating transcription factor 4 (ATF6) signalling, inositol-requiring enzyme-1 (IRE1) signalling and ERO1α, as compared with healthy controls. In the mouse model of galactosamine (GaIN)/lipopolysaccharide (LPS)-induced ALF, the hepatocellular injury was accompanied by up-regulated PERK signalling, ATF6 signalling, IRE1 signalling, CHOP and ERO1α. In contrast, CHOP deficiency decreased hepatocellular apoptosis/necrosis and increased animal survival. Furthermore, disruption of CHOP decreased ERO1α expression leading to reducing ROS-induced cell death in vivo and in vitro. Interestingly, ERO1α overexpression restored GaIN/LPS-induced hepatocellular injury in CHOP-deficient mice. Our studies demonstrate for the first time that CHOP promotes liver damage during ALF through activation of ERO1α, a key mediator to link ER stress and ROS. Therefore, targeting CHOP/ERO1α signalling could be a novel therapeutic approach during ALF.

INTRODUCTION

Acute liver failure (ALF) is defined as a sudden onset of severe hepatocellular dysfunction. It is associated with a poor prognosis and with massive hepatocellular death. ALF can be caused by hepatitis virus infection, the action of drugs or toxins or hepatic ischaemia–reperfusion injury. Previous studies have suggested that endoplasmic reticulum (ER) stress is a major contributor to the development of various hepatic disorders, including ALF, steatohepatitis and hepatic fibrosis [13]. Prolonged or excessive ER stress may initiate multiple signalling pathways leading to cell death [4]. In fact, ER stress-induced cell death involves sequential steps of dsRNA-dependent protein kinase (PKR)-like ER kinase (PERK)-mediated eukaryotic initiation factor α (eIF2α) phosphorylation, the preferential translation of activating transcription factor 4 (ATF4)/cAMP response element binding protein 2 (CREB-2) messenger RNA, and the induction of CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP)/growth-arrest and DNA-damage-inducible protein 153 (GADD153) [1,5]. Moreover, CHOP deletion in mice demonstrated that CHOP is required for ER stress-mediated cell death in response to a variety of pathological conditions [6,7]. Evidence from previous studies indicates that CHOP is an important regulator of oxidative damage in various types of cells [8,9].

During ALF, oxidative stress, such as H2O2 and superoxide, is considered to play a prominent causative role in the development of ALF [10,11]. Liver oxidative stress is induced by increased production of reactive oxygen and nitrogen species (ROS and RNS), which are generated by the causal agents of ALF and exceed the antioxidative capacity of hepatocytes. ROS can activate various types of apoptotic signalling pathways, including Jun N-terminal kinase (JNK), Forkhead box protein O1 (FOXO1) and Fas/Fas-L pathways [1214]. Although antioxidants are effective in limiting liver injury by inhibiting ROS [15], and CHOP deletion can reduce oxidative damage, improve β-cell function and promote cell survival in multiple mouse models of diabetes [9], the role of CHOP in the development of ALF remains unknown.

In the present study, we reveal the crucial roles and the potential mechanisms of CHOP in the development of ALF. We demonstrate that increased expression of CHOP and ER oxidoreductin 1-α (ERO1α) in human liver tissues from ALF patients and/or from mouse livers in a mouse model of ALF. CHOP deficiency decreased ROS-induced hepatocellular apoptosis/necrosis in vivo and in vitro. Disruption of CHOP resulted in a decrease in the expression of ERO1α, which can generate ROS by oxidizing protein disulfide isomerase (PDI). Importantly, ERO1α overexprssion restored galactosamine (GaIN)/lipopolysaccharide (LPS)-induced liver injury in CHOP-deficient mice by adenoviral transfection. Our data demonstrate that CHOP, an ER stress-induced molecule, up-regulates ROS leading to increasing hepatocellular apoptosis by promoting ERO1α signalling during ALF.

MATERIALS AND METHODS

Study subjects and clinical assessment

Serum and liver samples were obtained from 12 randomly consecutive ALF patients that have been confirmed by clinical, biochemical, radiological and histological diagnosis. A total of eight age- and gender-matched healthy subjects served as controls. The baseline characteristics of ALF patients are summarized in Table 1. Informed consent was obtained from all participants, and the study was approved by the local ethics committee of Nanjing Medical University. Research was carried out in accordance with the Declaration of Helsinki.

Table 1
Patient characteristics
Variables ALF patients Controls 
N 12 
Sex (male/female) 7/5 4/4 
Age (years) INR 40±8.9 37±6.8 
PT (s) 3.4±0.7 0.9±0.2 
TBIL (μmol/l) 37.9±7.6 12±2.7 
Aetiology of diseases 442.3±78.6 6.7±2.1 
Viral (HBV and/or HCV)  
Alcohol  
Drugs  
Variables ALF patients Controls 
N 12 
Sex (male/female) 7/5 4/4 
Age (years) INR 40±8.9 37±6.8 
PT (s) 3.4±0.7 0.9±0.2 
TBIL (μmol/l) 37.9±7.6 12±2.7 
Aetiology of diseases 442.3±78.6 6.7±2.1 
Viral (HBV and/or HCV)  
Alcohol  
Drugs  

INR, international normalized ratio; PT, prothrombin time; TBIL, total bilirubin; HBV, hepatitis B virus; HCV, hepatitis C virus.

Animal studies

The CHOP gene knockout (Chop−/−) mice (C57BL/6 background) were purchased from Jackson Laboratories. Male wild-type (WT) and Chop−/− mice aged 6–8 weeks were used in the present study. Procedures were carried out in accordance with the guidelines for the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. ALF was induced with GaIN/LPS (GaIN, 700 mg/kg of body weight; LPS, 35 μg/kg of body weight intraperitoneally), and control animals were treated with sterile PBS. After treatment with GaIN/LPS, the animal survival rates were assessed (n=15 mice/group). In some experiments, mice were administered a butylated hydroxyanisole (BHA) diet (100 mg/kg of body weight per day) for 3 days or injected with Ad-HO-1 or Ad-LacZ (2.5×109 pfu, intravenously) 24 h prior to GaIN/LPS treatment.

Serum biochemical examination

The serum levels of alanine aminotransferase (sALT) and aspartate aminotransferase (sAST) were measured with an automated chemical analyser (Olympus Automated Chemistry Analyzer AU5400).

Histopathological study

The liver specimens were fixed with 10% neutral formaldehyde and then embedded in paraffin. The specimens were sectioned in 4-μm slices and stained with haematoxylin and eosin (H&E). CHOP was also detected in human liver specimens using immunohistochemistry staining, as described previously [16].

MPO, caspase-3, GSH and MDA activities

Myeloperoxidase (MPO), caspase-3, GSH and malondialdehyde (MDA) activities were assessed in liver tissues 6 h after GaIN/LPS treatment. The activities were measured using MPO, caspase-3, GSH or MDA assay kits (Jiancheng Biotechnology) according to the manufacturer's instructions.

Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling staining

Paraffin sections (4 μm) were stained by terminal deoxynucleotidyltransferase-mediated dUTP nick-end (TUNEL) staining using a commercially available kit (in situ cell death detection kit, Roche-Boehringer Mannheim).

Western blot analysis

The protein concentration was determined by the BCA method [17]. Proteins (30 μg/sample) from the cell cultures or the livers were subjected to SDS/PAGE (12% gels) and transferred to a nitrocellulose membrane (Bio-Rad) Laboratories. Primary antibodies detecting p-PERK, p-eIF2α, ATF4, CHOP, caspase-3, p-JNK, β-actin (Cell Signaling Technology), inositol-requiring enzyme-1 (IRE1), ATF6, X-box-binding protein 1 (XBP1) (Abcam) and ERO1α (Santa Cruz Biotechnology) were used. The relative quantities of the proteins were determined with a densitometer and expressed in absorbance units (AU).

Quantitative real-time PCR

Quantitative real-time PCR was performed using the DNA Engine with a Chromo 4 Detector (MJ Research). In a final reaction volume of 25 μl, the following were added: 1 × SuperMix (Platinum SYBR Green qPCR Kit; Invitrogen), cDNA and 2.5 μM of each primer. The amplification conditions were as follows: 50°C (2 min), 95°C (5 min), followed by 50 cycles at 95°C (15 s) and 60°C (30 s). The primer sequences for human PERK, ATF4, CHOP, ERO1α and hypoxanthine phosphoribosyltransferase (HPRT) and mouse PERK, ATF4, CHOP, tumour necrosis factor-α (TNF-α), interleukin 6 (IL-6), superoxide dismutase (SOD), SOD2, glutathione peroxidase 1 (GPX1), ERO1α and HPRT are shown in Supplementary Table S1. The expression of target genes was calculated based on the ratio of targeting gene to the housekeeping gene HPRT.

ELISA

TNF-α and IL-6 secretion in cell culture supernatants were measured using ELISA kits according to the manufacturer's instructions (eBioscience). Absorbance was read on a Multiscan FC plate reader and analysed with SkanIt for Multiscan FC software (Thermo Scientific).

Hepatocyte isolation, culture and treatment

Mouse primary hepatocytes were isolated and harvested from WT and CHOP−/− mice as described previously [16]. The hepatocytes were seeded in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. In some experiments, the cells (8×105 cells/well in a six-well plate) were incubated with tunicamycin (TM, 10 μg/ml) for 48 h in the presence or absence of 100 mM BHA [18].

Hepatocyte cytotoxicity assay

Lactate dehydrogenase (LDH) activities in the culture media were measured with a LDH assay kit (Jiancheng Bioengineering) according to the manufacturer's instructions.

In vitro siRNA transfection

The siRNA sequence against mice ERO1α was generated by QIAGEN. The target sequence was 5′-CAGCTCTTCACTGGGAATAAA-3′. The non-specific siRNA (NS siRNA) sequence was 5′-CGAATCCACAAAGCGCGCTT-3′ and served as a control. Hepatocytes were grown and transiently transfected with ERO1α siRNA or NS siRNA using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer's instructions. Briefly, the cells were seeded at 8×105 cells/well in 1.5 ml of OPTI medium (Invitrogen) in a six-well plate. After 20 h, the cells were transfected with 30 nmol/ml ERO1α siRNA or NS siRNA. Approximately 6 h after transfection, the medium was changed and the cells were cultured for 24 h.

Adenoviral transfection

Adenoviruses containing the constructs for murine ERO1α or LacZ were purchased from Invitrogen. Hepatocytes were infected with the virus at a MOI of 500 in medium containing 2% serum for 1.5 h, and then DMEM containing 10% FBS and 20% L cell-conditioned medium was added. The cells were cultured for 24 h.

Statistical analysis

The data are presented as the mean ± S.E.M. from at least three independent experiments. An ANOVA, followed by Tukey's post-test (Prism 5; GraphPad Software), was used to determine statistical significance between the groups. All P values were two-sided, and P< 0.05 was considered to be statistically significant.

RESULTS

Liver expression of CHOP is increased in human ALF

Previous studies have demonstrated that the ER stress response increases apoptosis through the unfolded protein response (UPR) in many diseases [1,19]. Under ER stress conditions, CHOP expression is primarily regulated by the PERK-ATF4 pathway [20]. To determine the role of CHOP in human ALF, we collected liver tissue from 12 ALF patients and eight healthy subjects (Table 1). ALF was diagnosed based on aetiology, sALT, sAST, international normalized ratio, prothrombin time, total bilirubin and pathology results (Table 1 and Supplementary Figure S1). As shown in Figure 1(A), the mRNA expressions coding for PERK (0.55±0.07 and 3.55±0.36, P< 0.001), ATF4 (1.03±0.10 and 8.30±0.90, P< 0.001) and CHOP (0.40±0.07 and 3.35±0.40, P< 0.001) in patients with ALF were significantly increased, as compared with healthy controls. Moreover, consistent with the mRNA expression, we found the protein levels of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased in patients with ALF (Figure 1B). In addition, IRE1 signalling and ATF6 signalling were also up-regulated. To localize CHOP in the liver tissue, CHOP was detected in liver sections by immunohistochemistry staining. Indeed, enhanced expression of CHOP was observed in liver tissues from ALF patients (Figure 1C); the number of positive cells was significantly increased, as compared with control group (5.50±0.076 compared with 57.67±4.17, P< 0.0001) (Figure 1D).

Liver expression of CHOP is increased in human ALF

Figure 1
Liver expression of CHOP is increased in human ALF

Liver samples were harvested from 12 ALF patients and eight healthy volunteers. (A) Analysis of gene expression of PERK, ATF4 and CHOP in human livers. (B) Western blotting analysis of protein expression of p-IRE1, cATF6 (cleaved ATF6), sXBP1 (spliced XBP1), p-PERK, p-eIF2α, ATF4 and CHOP in human liver. (C) IHC staining with CHOP antibody of human liver sections (×100 and ×200) and (D) ratio of CHOP-positive cells (**P< 0.001 compared with Health-CON).

Figure 1
Liver expression of CHOP is increased in human ALF

Liver samples were harvested from 12 ALF patients and eight healthy volunteers. (A) Analysis of gene expression of PERK, ATF4 and CHOP in human livers. (B) Western blotting analysis of protein expression of p-IRE1, cATF6 (cleaved ATF6), sXBP1 (spliced XBP1), p-PERK, p-eIF2α, ATF4 and CHOP in human liver. (C) IHC staining with CHOP antibody of human liver sections (×100 and ×200) and (D) ratio of CHOP-positive cells (**P< 0.001 compared with Health-CON).

GaIN/LPS-induced ALF up-regulates hepatic CHOP expression

To test whether CHOP expression was induced by GaIN/LPS in a mouse model of ALF, WT mice were injected with GaIN/LPS. We found that induction of ALF was associated with a time-dependent course, which showed mRNA expression coding for PERK, ATF4 and CHOP was up-regulated in GaIN/LPS-induced ALF. As shown in Figure 2(A), PERK expression was increased 2.5-fold after 1 h, 6.5-fold after 3 h and 6.3-fold after 6 h; ATF4 mRNA was increased 1.6-fold after 1 h, 3.5-fold after 3 h and 5.4-fold (peak expression) after 6 h; CHOP expression was increased 3.3-fold after 1 h, 9.1-fold after 3 h and 11-fold (peak expression) after 6 h. These results were consistent with that in human ALF patients (Figure 1B).

Liver expression of CHOP is increased in GaIN/LPS-induced mouse ALF

Figure 2
Liver expression of CHOP is increased in GaIN/LPS-induced mouse ALF

Liver tissues were obtained from WT mice that were either sham-treated or GaIN/LPS-treated, followed by various lengths of treatment (1, 3 and 6 h). (A) Kinetics of quantitative real-time PCR analysis of mRNA expression coding for PERK, ATF4 and CHOP in mouse livers after GaIN/LPS treatment. (B) Western blotting analysis of protein expression of p-IRE1, cATF6 (cleaved ATF6), sXBP1 (spliced XBP1), p-PERK, p-eIF2α, ATF4 and CHOP in mouse livers after GaIN/LPS treatment (n=6–8/time point, *P< 0.05, **P< 0.001 compared with Sham).

Figure 2
Liver expression of CHOP is increased in GaIN/LPS-induced mouse ALF

Liver tissues were obtained from WT mice that were either sham-treated or GaIN/LPS-treated, followed by various lengths of treatment (1, 3 and 6 h). (A) Kinetics of quantitative real-time PCR analysis of mRNA expression coding for PERK, ATF4 and CHOP in mouse livers after GaIN/LPS treatment. (B) Western blotting analysis of protein expression of p-IRE1, cATF6 (cleaved ATF6), sXBP1 (spliced XBP1), p-PERK, p-eIF2α, ATF4 and CHOP in mouse livers after GaIN/LPS treatment (n=6–8/time point, *P< 0.05, **P< 0.001 compared with Sham).

CHOP is critical for the development of ALF

To investigate whether CHOP mediates GaIN/LPS-induced ALF, we used CHOP−/− and WT mice to analyse the functional role of CHOP in our animal model. Four animal groups were set up: (1) WT sham group; (2) CHOP−/− sham group; (3) WT mice + GaIN/LPS group; and (4) CHOP−/− mice + GaIN/LPS group. Hepatocellular damage was determined by sALT or sAST levels 6 h after the GaIN/LPS challenge. As shown in Figure 3(A), sALT (3853.0±264.0 and 1248.0±173.5, P< 0.05) and sAST (6853.0±264.0 and 2374±500.0, P< 0.05) levels were markedly decreased in CHOP−/− mice compared with the WT mice. Histology data showed that WT groups had exacerbated liver damage, whereas liver lesions were decreased in CHOP−/− mice (Figure 3B). Moreover, using TUNEL staining (Figure 3C), we found CHOP deletion markedly reduced the frequency of TUNEL-positive cells in the liver tissues in CHOP−/− mice (75.5±3.7 and 7.3±1.1, P< 0.0001) compared with WT controls. To characterize the inflammatory infiltration, we used MPO activity assay and found decreased neutrophil accumulation in the CHOP−/− mice, as compared with the WT groups (7.1±0.6 units/g compared with 2.8±0.2 units/g, P< 0.001) (Figure 3D). As ALF is characterized by the stimulus-dependent activation of TNF-receptor signalling pathways leading to triggering massive apoptotic and/or necrotic cell death [21,22], we also analysed the expression of TNF-α and IL-6 in liver tissues by quantitative real-time-PCR. As shown in Figure 3(E) and Supplementary Figure S2, the expressions of TNF-α and IL-6 were significantly decreased in the CHOP−/− mice, as compared with the WT mice. Importantly, the GaIN/LPS-induced mortality rate was 100% in the WT mice within 12 h, whereas all CHOP−/− mice survived within 48 h (Figure 3F). These data indicate that CHOP plays a key role in GaIN/LPS-induced ALF.

CHOP is critical for the development of ALF

Figure 3
CHOP is critical for the development of ALF

WT and CHOP−/− mice were treated with vehicle or GaIN/LPS. Liver samples and serum were harvested at 6 h post-treatment. (A) Serum ALT and AST levels. (B) H&E staining of mouse liver sections (×100): (a) WT sham, (b) WT+GaIN/LPS, (c) CHOP−/− sham and (d) CHOP−/− +GaIN/LPS. (C) TUNEL staining of liver sections (×200): (a) WT sham, (b) WT+GaIN/LPS, (c) CHOP−/− sham and (d) CHOP−/− +GaIN/LPS, as well as apoptotic cell count. (D) MPO activity. (E) quantitative real-time-PCR analysis of liver TNF-α and IL-6. (F) Survival curves (n=6–8/group, **P< 0.001 compared with WT).

Figure 3
CHOP is critical for the development of ALF

WT and CHOP−/− mice were treated with vehicle or GaIN/LPS. Liver samples and serum were harvested at 6 h post-treatment. (A) Serum ALT and AST levels. (B) H&E staining of mouse liver sections (×100): (a) WT sham, (b) WT+GaIN/LPS, (c) CHOP−/− sham and (d) CHOP−/− +GaIN/LPS. (C) TUNEL staining of liver sections (×200): (a) WT sham, (b) WT+GaIN/LPS, (c) CHOP−/− sham and (d) CHOP−/− +GaIN/LPS, as well as apoptotic cell count. (D) MPO activity. (E) quantitative real-time-PCR analysis of liver TNF-α and IL-6. (F) Survival curves (n=6–8/group, **P< 0.001 compared with WT).

CHOP deletion attenuates ROS-induced pro-apoptotic signalling in ALF

As it has been shown above that GaIN/LPS-induced liver injury is characterized by the apoptosis of hepatocytes, we then next analysed whether CHOP deletion enhances protection by inhibiting cell death. As shown in the Figure 4(A), GaIN/LPS challenge resulted in dramatically activating caspase-3 (46.0±4.0 and 5.5±0.6, P< 0.001) and its cleavage (Supplementary Figure S3). However, the decreased caspase-3 activity was observed in CHOP-deficient mice. Similar results were also observed in the liver-tissue sections by TUNEL staining (Figure 3C). In the GaIN/LPS-induced ALF model, ROS are the major mediators leading to apoptotic liver injury [22]. CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis [9]. To determine whether CHOP may mediate ROS accumulation in our animal model, we measured the levels of GSH and MDA in liver tissues. As expected, CHOP deletion significantly increased the levels of GSH after GaIN/LPS treatment, as compared with the WT mice (2.6±0.2 compared with 8.5±0.9, P< 0.001) (Figure 4B). However, MDA, an index of lipid peroxidation that reflects oxidative stress levels, was significantly decreased in the CHOP−/− groups compared with the WT group (569.3±76.8 compared with 132.5±13.7, P< 0.005) (Figure 4B). Moreover, in contrast with control groups, sALT was decreased in the GaIN/LPS-treated mice after feeding with antioxidant BHA-supplemented diet (3598.0±297.4 and 1346.0±165.0, P< 0.005) (Figure 4C), which is in line with the histological changes (Figure 4D). Consistent with this result, ROS-induced JNK activation was also decreased in the CHOP-deficient mice (Figure 4E). Indeed, CHOP-deficient mice had increased expression of the genes encoding antioxidative stress functions [9]. As shown in Figure 4(F), the expressions of antioxidant SOD1, SOD2 and GPX1 were markedly increased after GaIN/LPS treatment in CHOP−/− mice, as compared with WT mice. These data indicate that the CHOP deletion may enhance its anti-apoptotic activity by inhibiting oxidative stress and JNK signalling.

CHOP deletion attenuates ROS-induced pro-apoptotic signalling in ALF

Figure 4
CHOP deletion attenuates ROS-induced pro-apoptotic signalling in ALF

(A) Caspase-3 activity. (B) GSH and MDA levels in mouse livers. (C) sALT levels after BHA treatment in GaIN/LPS-challenged WT mice. (D) Liver histology after BHA treatment in GaIN/LPS-challenged WT mice (×100): (a) Control (Ctrl) (b) BHA treatment, (c) GaIN/LPS, (d) BHA + GaIN/LPS. (E) Western blot analysis of p-JNK and β-actin in liver tissues. (F) quantitative real-time-PCR analysis of mRNA expression coding for SOD1, SOD2 and GXP1 (n=6–8/group, *P< 0.05, **P< 0.001 compared with WT).

Figure 4
CHOP deletion attenuates ROS-induced pro-apoptotic signalling in ALF

(A) Caspase-3 activity. (B) GSH and MDA levels in mouse livers. (C) sALT levels after BHA treatment in GaIN/LPS-challenged WT mice. (D) Liver histology after BHA treatment in GaIN/LPS-challenged WT mice (×100): (a) Control (Ctrl) (b) BHA treatment, (c) GaIN/LPS, (d) BHA + GaIN/LPS. (E) Western blot analysis of p-JNK and β-actin in liver tissues. (F) quantitative real-time-PCR analysis of mRNA expression coding for SOD1, SOD2 and GXP1 (n=6–8/group, *P< 0.05, **P< 0.001 compared with WT).

Liver expression of ERO1α is increased in human ALF

ERO1-α, an ER stress protein (a target of CHOP), is an oxidizing enzyme that exists in the ER, and its expression is augmented under hypoxia or ER stress [23]. In human ALF, ERO1-α mRNAs were significantly elevated compared with healthy subjects (1.5±0.2 compared with 6.2±0.7, P< 0.005) (Figure 5A). Consistent with this, the protein levels of ERO1-α were substantially increased in ALF patients (2.7±0.4 compared with 1.0±0.2, P< 0.05), as compared with healthy controls (Figures 5B and 5C).

Liver expression of ERO1α is increased in human ALF

Figure 5
Liver expression of ERO1α is increased in human ALF

Liver tissues were harvested from 12 ALF patients and eight healthy volunteers. (A) quantitative real-time-PCR and (B and C) Western blot analysis of hepatic ERO1α expression in human livers (*P< 0.05; **P< 0.005 compared with Health-CON).

Figure 5
Liver expression of ERO1α is increased in human ALF

Liver tissues were harvested from 12 ALF patients and eight healthy volunteers. (A) quantitative real-time-PCR and (B and C) Western blot analysis of hepatic ERO1α expression in human livers (*P< 0.05; **P< 0.005 compared with Health-CON).

ERO1α is required for CHOP-mediated ROS production in hepatocytes

In the GaIN/LPS-induced ALF model, we found an approximate 2.5-fold increase in ERO1α mRNA and a 1.5-fold increase in ERO1α protein. In the CHOP-deficient mice, both ERO1α mRNA and protein at the basal levels were decreased after GaIN/LPS challenge (Figure 6A). Indeed, ER stress via TM treatment or overexpression of ATF4 and CHOP increases ROS levels [24,25]. We then investigate whether CHOP plays a role in ROS-induced hepatocyte death after TM treatment. As indicated by Figure 6(B), CHOP deletion had an effect similar to that of BHA treatment. This result indicates that oxidative stress is critical for ER stress-induced cell death. Next, we determined the role of ERO1α in CHOP-mediated cell death. We found that ERO1α protein expression was decreased after transfection of ERO1α siRNA (Figure 6C). ERO1α knockdown resulted in a significant reduction of ROS-induced cell death after TM treatment (Figure 6C, 39.61±3.4 compared with 18.2±1.6, P< 0.05). Consistent with the cytotoxicity assay, the level of the cleaved caspase-3 protein was significantly decreased in the ERO1α-silenced hepatocytes after TM treatment (Figure 6D). To further elucidate the role of ERO1α in CHOP-dependent cell death, hepatocytes from the CHOP−/− mice were transduced with an adenovirus containing mouse ERO1α cDNA to restore the ERO1α expression (Figure 6E). The ROS-induced cell death was significantly diminished in TM-treated CHOP−/− hepatocytes after transfection of Ad-LacZ. In contrast, the cytotoxicity was restored in TM-treated CHOP−/− hepatocytes transfected with Ad-ERO1α (Figure 6E). These data demonstrate that ERO1α is a key mediator in CHOP-mediated cell death.

ERO1α is required for CHOP-mediated ROS production in hepatocytes

Figure 6
ERO1α is required for CHOP-mediated ROS production in hepatocytes

(A) qRT-PCR and Western blot analysis of hepatic ERO1α expression in mice livers (n=6–8/group, *P< 0.05; **P< 0.001 compared with WT). (B) Hepatocytes were treated with vehicle, TM or BHA combined with TM. At the end of the 48-h incubation period, the cytotoxicity was measured by LDH assay. Values were means ± S.D. of three independent experiments (*P< 0.05; **P< 0.001 compared with WT). (C) Hepatocytes were transfected with ERO1α siRNA or NS siRNA; ERO1α protein was detected by immunoblotting. The transfected hepatocytes were incubated with vehicle, TM or BHA combined with TM and then analysed for cytotoxicity. Values were means ± S.D. of three independent experiments (**P< 0.001 compared with NS siRNA). (D) Caspase-3 protein was detected in the hepatocytes transfected with ERO1α siRNA or NS siRNA after TM treatment for 12 h. (E) Hepatocytes from CHOP−/− mice were transfected with Ad-LacZ or Ad-ERO1α. At 24 h after incubation with the virus, one set of cells was harvested for ERO1α protein analysis, and another set was incubated with vehicle, TM or BHA combined with TM for 48 h and then assayed for cytotoxicity. Values were means ± S.D. of three independent experiments (*P< 0.05; **P< 0.001).

Figure 6
ERO1α is required for CHOP-mediated ROS production in hepatocytes

(A) qRT-PCR and Western blot analysis of hepatic ERO1α expression in mice livers (n=6–8/group, *P< 0.05; **P< 0.001 compared with WT). (B) Hepatocytes were treated with vehicle, TM or BHA combined with TM. At the end of the 48-h incubation period, the cytotoxicity was measured by LDH assay. Values were means ± S.D. of three independent experiments (*P< 0.05; **P< 0.001 compared with WT). (C) Hepatocytes were transfected with ERO1α siRNA or NS siRNA; ERO1α protein was detected by immunoblotting. The transfected hepatocytes were incubated with vehicle, TM or BHA combined with TM and then analysed for cytotoxicity. Values were means ± S.D. of three independent experiments (**P< 0.001 compared with NS siRNA). (D) Caspase-3 protein was detected in the hepatocytes transfected with ERO1α siRNA or NS siRNA after TM treatment for 12 h. (E) Hepatocytes from CHOP−/− mice were transfected with Ad-LacZ or Ad-ERO1α. At 24 h after incubation with the virus, one set of cells was harvested for ERO1α protein analysis, and another set was incubated with vehicle, TM or BHA combined with TM for 48 h and then assayed for cytotoxicity. Values were means ± S.D. of three independent experiments (*P< 0.05; **P< 0.001).

Ad-ERO1α restores GaIN/LPS-induced hepatocyte death in CHOP−/− mice

On the basis of our in vitro findings, we next investigated whether ERO1α may affect CHOP-mediated cell death in vivo. We injected Ad-ERO1α to restore the ERO1α expression prior to injection of GaIN/LPS in CHOP−/− mice. As showed by Figure 7(A), Ad-ERO1α significantly increased the serum ALT level after GaIN/LPS treatment compared with the Ad-LacZ group in CHOP−/− mice (615.7±256.9 and 3276.0±597.1, P< 0.001). Consistent with this result, severe hepatic lobule distortion, sinusoidal congestion, apparent oedema and massive necrosis were shown in the Ad-ERO1α treatment group (Figure 7B). Moreover, Ad-ERO1α significantly enhanced caspase-3 activity, as compared with the Ad-LacZ-treated group (34.0±5.7 compared with 9.7±1.2, P< 0.001). Similar results were also observed in the liver-tissue sections by TUNEL assay (Figure 7D). These data indicate that ERO1α over-expression restores GaIN/LPS-induced liver injury, suggesting ERO1α is essential for CHOP-mediated cell death during GaIN/LPS-induced ALF.

ERO1α overexpression restores GaIN/LPS-induced liver injury in CHOP−/− mice

Figure 7
ERO1α overexpression restores GaIN/LPS-induced liver injury in CHOP−/− mice

(A) sALT level of mice (n=6–8/group, **P< 0.001). (B) H&E staining of mice liver sections (×100): (a) CHOP−/−+Ad-LacZ+GaIN/LPS and (b) CHOP−/−+Ad-ERO1α+GaIN/LPS; (C) caspase-3 activity (n=6–8/group, **P< 0.001). (D) TUNEL staining of liver sections (×400): (a) CHOP−/−+Ad-LacZ+GaIN/LPS and (b) CHOP−/−+ Ad-ERO1α+GaIN/LPS.

Figure 7
ERO1α overexpression restores GaIN/LPS-induced liver injury in CHOP−/− mice

(A) sALT level of mice (n=6–8/group, **P< 0.001). (B) H&E staining of mice liver sections (×100): (a) CHOP−/−+Ad-LacZ+GaIN/LPS and (b) CHOP−/−+Ad-ERO1α+GaIN/LPS; (C) caspase-3 activity (n=6–8/group, **P< 0.001). (D) TUNEL staining of liver sections (×400): (a) CHOP−/−+Ad-LacZ+GaIN/LPS and (b) CHOP−/−+ Ad-ERO1α+GaIN/LPS.

DISCUSSION

ALF is a severe liver damage associated with oxidative stress-mediated cell death. CHOP, a stress-induced transcription factor, has been shown to regulate cell apoptosis and cytokine production in a variety of diseases [1,2,5]. In the present study, we identified the critical role of CHOP in mediating liver damage during ALF. Indeed, in human ALF and GaIN/LPS-induced mouse ALF, CHOP expression was significantly up-regulated, whereas disruption of CHOP in mouse livers diminished hepatic damage by inhibiting ER stress-induced apoptosis during ALF. These data indicate that CHOP plays a key role in mediating liver damage in ALF.

The ER is a centrally located intracellular organelle of eukaryotic cells that performs critical functions in protein synthesis and folding, lipid and sterol synthesis and calcium homoeostasis. Perturbation of any of these major ER functions results in ER stress, including a variety of pathophysiological stimuli, such as hypoxia, glucose deprivation and calcium depletion from the lumen. ER stress is characterized by the accumulation of unfolded and misfolded proteins in the ER lumen that trigger the UPR. Previous studies from our group and others have demonstrated that acute liver damage induces ER stress and activates the UPR in macrophages and hepatocytes [1,5,16]. However, the exact link between the UPR and liver injury in ALF remains to be elucidated. Indeed, it has been shown that CHOP is an essential transcription factor involved in UPR-mediated apoptosis [16]. The present study demonstrated that the expression of CHOP, ATF4 and PERK was increased in both human ALF and GaIN/LPS-induced mouse ALF, suggesting that CHOP is a multifunctional factor, which interacts with ATF4 and PERK during ER stress-mediated cell death. These data are consistent with previous results, indicating that GaIN/LPS-induced liver injury was associated with UPR activation in mice [26].

The question arises as to whether CHOP is critical for the development of ALF. A previous report showed that CHOP deficiency significantly decreased hepatocellular apoptosis induced by alcohol intake [27]. CHOP-knockout mice were protected against acetaminophen (APAP)-induced liver damage leading to increasing survival [28]. The in vivo data from the present study demonstrated that CHOP deletion ameliorated liver injury and improved hepatocellular function, as shown by decreasing sALT and sAST levels, hepatocellular apoptosis and pro-inflammatory cytokine mediators. Indeed, ALF can activate TNF-receptors and/or the mitochondrial death signalling pathway, which triggers massive apoptotic and/or necrotic cell death [12]. CHOP deficiency decreased the expression of the pro-inflammatory cytokines TNF-α and IL-6, suggesting activation of pro-inflammatory cytokines is linked to ER stress and UPR activation in macrophages [16].

It has been shown that oxidative stress is a major cause of the induction of cell death and that CHOP is an important regulator of ROS [9]. The present study demonstrated that CHOP-mediated liver injury is mainly dependent on ROS accumulation during ALF. Disruption of CHOP or treatment with the antioxidant BHA decreased oxidative stress-induced MDA levels and caspase-3 activity, while increasing antioxidant GSH levels and the expression of antioxidants SOD1, SOD2 and GPX1 in livers after GaIN/LPS challenge. Indeed, oxidative stress may activate many apoptotic signalling pathways, including JNK, FOXO1 and Fas/Fas-L pathways [1214]. Studies have demonstrated that JNK phosphorylation followed by oxidative stress induces cell apoptosis activating a caspase-dependent mitochondrial pathway and c-JUN phosphorylation in livers. Consistent with this notion, we found that JNK activation was markedly decreased in CHOP-deficient livers. In addition, JNK phosphorylation was also inhibited after BHA treatment (results not shown). Taken together, these data suggest that ROS-JNK-caspase-3 pathway may be one of the regulatory mechanisms in CHOP-mediated liver damage during ALF, demonstrating that CHOP is critical for the development of ALF.

The molecular mechanisms of CHOP-mediated liver damage during ALF are complex and may be involved in the activation of multiple intercellular signalling pathways. We found that GaIN/LPS treatment in WT mice significantly increased ERO1α expression. However, the expression of ERO1α was not changed in the CHOP-deficient mice. Moreover, disruption of ERO1α diminished caspase-3 activity and TM-induced cytotoxicity, whereas overexpression of ERO1α resulted in increased cell death, suggesting that ERO1α may be a key regulator in CHOP-mediated hepatocyte death. Indeed, recent studies have shown that ERO1α plays an important role in ER stress-induced apoptosis in macrophages [29]. ERO1α oxidizes PDI to generate oxidative stress from molecular oxygen that forms H2O2, which results in a hyperoxidizing environment within the ER leading to increased cell death [22,30]. Consistent with these results, our in vitro and in vivo data demonstrate that ERO1α is essential for CHOP-mediated cell death induced by oxidative stress.

In conclusion, our studies demonstrate that UPR signalling pathways are activated during ALF, especially for the CHOP/ERO1α pathway, which is a key signalling pathway underlying ALF-related oxidative stress and hepatocellular apoptosis. Disruption of CHOP inhibited ERO1α activation, which in turn alleviated ROS production and enhanced cytoprotection against apoptosis induced by the ROS-JNK-caspase-3 pathway. By identifying the molecular mechanisms of CHOP-mediated ERO1α signalling in hepatocellular apoptosis, our study provides a novel therapeutic approach to management of ALF.

Abbreviations

     
  • ATF

    activating transcription factor

  •  
  • ALF

    acute liver failure

  •  
  • BHA

    butylated hydroxyanisole

  •  
  • CHOP

    C/EBP homologous protein

  •  
  • CREB-2

    cAMP response element binding protein 2

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • eIF2α

    eukaryotic initiation factor α

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERO1α

    endoplasmic reticulum oxidoreductin 1-α

  •  
  • GaIN

    galactosamine

  •  
  • GPX-1

    glutathione peroxidase 1

  •  
  • H&E

    haematoxylin and eosin

  •  
  • HPRT

    hypoxanthine phosphoribosyltransferase

  •  
  • IL-6

    interleukin-6

  •  
  • IRE1

    inositol-requiring enzyme-1

  •  
  • JNK

    Jun N-terminal kinase

  •  
  • LDH

    lactate dehydrogenase

  •  
  • LPS

    lipopolysaccharide

  •  
  • MDA

    malondialdehyde

  •  
  • MPO

    myeloperoxidase

  •  
  • NS

    siRNA, non-specific siRNA

  •  
  • PDI

    protein disulfide isomerase

  •  
  • PERK

    PKR-like ER kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • RNS

    reactive nitrogen species

  •  
  • sALT

    serum alanine aminotransferase

  •  
  • sAST

    serum aspartate aminotransferase

  •  
  • SOD

    superoxide dismutase

  •  
  • TM

    tunicamycin

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • TUNEL

    terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling

  •  
  • UPR

    unfolded protein response

  •  
  • WT

    wild-type

AUTHOR CONTRIBUTION

Jianhua Rao, Chuangyong Zhang and Feng Zhang conceptualized and designed the experiment. Jianhua Rao and Ping Wang analysed and interpreted the data. Jianhua Rao, Xiongxiong Pan and Guoqiang Li performed data collection. Jianhua Rao, Chuangyong Zhang and Ping Wang wrote the manuscript. Ling Lu, Xiaofeng Qian, Jianjie Qin, Xuehao Wang and Feng Zhang critically revised the article. Jianhua Rao and Feng Zhang performed funding related tasks.

We are grateful to Bibo Ke at UCLA for revising the English text.

FUNDING

The present study was supported by the International Collaboration Foundation of Jiangsu Province [grant numbers BZ2011041, BK2009439], Basic research program-Youth Fund Project of Jiangsu Province [grant number BK20140092] and the National Nature Science Foundation of China [grant numbers 81400650, 81470901, 81310108001, 81210108017, 81273261, 81270583).

References

References
1
Malhi
H.
Kaufman
R.J.
Endoplasmic reticulum stress in liver disease
J. Hepatol.
2011
, vol. 
54
 (pg. 
795
-
809
)
[PubMed]
2
Kaplowitz
N.
Than
T.A.
Shinohara
M.
Ji
C.
Endoplasmic reticulum stress and liver injury
Semin. Liver Dis.
2007
, vol. 
27
 (pg. 
367
-
377
)
[PubMed]
3
Duvigneau
J.C.
Kozlov
A.V.
Zifko
C.
Postl
A.
Hartl
R.T.
Miller
I.
Gille
L.
Staniek
K.
Moldzio
R.
Gregor
W.
, et al. 
Reperfusion does not induce oxidative stress but sustained endoplasmic reticulum stress in livers of rats subjected to traumatic-hemorrhagic shock
Shock.
2010
, vol. 
33
 (pg. 
289
-
298
)
[PubMed]
4
Xu
C.
Bailly-Maitre
B.
Reed
J.C.
Endoplasmic reticulum stress: cell life and death decisions
J. Clin. Invest.
2005
, vol. 
115
 (pg. 
2656
-
2664
)
[PubMed]
5
Fawcett
T.W.
Martindale
J.L.
Guyton
K.Z.
Hai
T.
Holbrook
N.J.
Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response
Biochem. J.
1999
, vol. 
339
 (pg. 
135
-
141
)
[PubMed]
6
Zinszner
H.
Kuroda
M.
Wang
X.
Batchvarova
N.
Lightfoot
R.T.
Remotti
H.
Stevens
J.L.
Ron
D.
CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum
Genes Dev.
1998
, vol. 
12
 (pg. 
982
-
995
)
[PubMed]
7
Tabas
I.
Ron
D.
Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress
Nat. Cell Biol.
2011
, vol. 
13
 (pg. 
184
-
190
)
[PubMed]
8
Back
S.H.
Scheuner
D.
Han
J.
Song
B.
Ribick
M.
Wang
J.
Gildersleeve
R.D.
Pennathur
S.
Kaufman
R.J.
Translation attenuation through eIF2alpha phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells
Cell Metab.
2009
, vol. 
10
 (pg. 
13
-
26
)
[PubMed]
9
Song
B.
Scheuner
D.
Ron
D.
Pennathur
S.
Kaufman
R.J.
Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes
J. Clin. Invest.
2008
, vol. 
118
 (pg. 
3378
-
3389
)
[PubMed]
10
Ferret
P.J.
Hammoud
R.
Tulliez
M.
Tran
A.
Trébéden
H.
Jaffray
P.
Malassagne
B.
Calmus
Y.
Weill
B.
Batteux
F.
Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse
Hepatology
2001
, vol. 
33
 (pg. 
1173
-
1180
)
[PubMed]
11
Aram
G.
Potter
J.J.
Liu
X.
Wang
L.
Torbenson
M.S.
Mezey
E.
Deficiency of nicotinamide adenine dinucleotide phosphate, reduced form oxidase enhances hepatocellular injury but attenuates fibrosis after chronic carbon tetrachloride administration
Hepatology
2009
, vol. 
49
 (pg. 
911
-
919
)
[PubMed]
12
Morgan
M.J.
Liu
Z.G.
Reactive oxygen species in TNFalpha-induced signaling and cell death
Mol. Cells
2010
, vol. 
30
 (pg. 
1
-
12
)
[PubMed]
13
Storz
P.
Forkhead homeobox type O transcription factors in the responses to oxidative stress
Antioxid. Redox. Signal.
2011
, vol. 
14
 (pg. 
593
-
605
)
[PubMed]
14
Fubini
B.
Hubbard
A.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis
Free Radic. Biol. Med.
2003
, vol. 
34
 (pg. 
1507
-
1516
)
[PubMed]
15
Sun
H.
Chen
L.
Zhou
W.
Hu
L.
Li
L.
Tu
Q.
Chang
Y.
Liu
Q.
Sun
X.
Wu
M.
, et al. 
The protective role of hydrogen-rich saline in experimental liver injury in mice
J. Hepatol.
2011
, vol. 
54
 (pg. 
471
-
480
)
[PubMed]
16
Rao
J.
Qin
J.
Qian
X.
Lu
L.
Wang
P.
Wu
Z.
Zhai
Y.
Zhang
F.
Li
G.
Wang
X.
Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI) through Inhibiting ATF4-CHOP pathway in mice
PLoS One
2013
, vol. 
8
 pg. 
e65568
 
[PubMed]
17
Rao
J.
Yue
S.
Fu
Y.
Zhu
J.
Wang
X.
Busuttil
R.W.
Kupiec-Weglinski
J.W.
Lu
L.
Zhai
Y.
ATF6 mediates a pro-inflammatory synergy between ER stress and TLR activation in the pathogenesis of liver ischemia-reperfusion injury
Am. J. Transplant.
2014
, vol. 
14
 (pg. 
1552
-
1561
)
[PubMed]
18
Ho
Y.
Samarasinghe
R.
Knoch
M.E.
Lewis
M.
Aizenman
E.
DeFranco
D.B.
Selective inhibition of mitogen-activated protein kinase phosphatases by zinc accounts for extracellular signal-regulated kinase 1/2-dependent oxidative neuronal cell death
Mol. Pharmacol.
2008
, vol. 
74
 (pg. 
1141
-
1151
)
[PubMed]
19
Ozcan
L.
Tabas
I.
Role of endoplasmic reticulum stress in metabolic disease and other disorders
Annu. Rev. Med.
2012
, vol. 
63
 (pg. 
317
-
328
)
[PubMed]
20
Novoa
I.
Zeng
H.
Harding
H.P.
Ron
D.
Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha
J. Cell Biol.
2001
, vol. 
153
 (pg. 
1011
-
1022
)
[PubMed]
21
Ogasawara
J.
Watanabe-Fukunaga
R.
Adachi
M.
Matsuzawa
A.
Kasugai
T.
Kitamura
Y.
Itoh
N.
Suda
T.
Nagata
S.
Lethal effect of the anti-Fas antibody in mice
Nature
1993
, vol. 
364
 (pg. 
806
-
809
)
[PubMed]
22
Tiegs
G.
Wolter
M.
Wendel
A.
Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice
Biochem. Pharmacol.
1989
, vol. 
38
 (pg. 
627
-
631
)
[PubMed]
23
Marciniak
S.J.
Yun
C.Y.
Oyadomari
S.
Novoa
I.
Zhang
Y.
Jungreis
R.
Nagata
K.
Harding
H.P.
Ron
D.
CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum
Genes Dev.
2004
, vol. 
18
 (pg. 
3066
-
3077
)
[PubMed]
24
Harding
H.P.
Zhang
Y.
Zeng
H.
Novoa
I.
Lu
P.D.
Calfon
M.
Sadri
N.
Yun
C.
Popko
B.
Paules
R.
, et al. 
An integrated stress response regulates amino acid metabolism and resistance to oxidative stress
Mol. Cell
2003
, vol. 
11
 (pg. 
619
-
633
)
[PubMed]
25
Han
J.
Back
S.H.
Hur
J.
Lin
Y.H.
Gildersleeve
R.
Shan
J.
Yuan
C.L.
Krokowski
D.
Wang
S.
Hatzoglou
M.
, et al. 
ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death
Nat. Cell Biol.
2013
, vol. 
15
 (pg. 
481
-
490
)
[PubMed]
26
Chen
L.
Ren
F.
Zhang
H.
Wen
T.
Piao
Z.
Zhou
L.
Zheng
S.
Zhang
J.
Chen
Y.
Han
Y.
, et al. 
Inhibition of glycogen synthase kinase 3β ameliorates D-GalN/LPS-induced liver injury by reducing endoplasmic reticulum stress-triggered apoptosis
PLoS One
2012
, vol. 
7
 pg. 
e45202
 
[PubMed]
27
Ji
C.
Mehrian-Shai
R.
Chan
C.
Hsu
Y.H.
Kaplowitz
N.
Role of CHOP in hepatic apoptosis in the murine model of intragastric ethanol feeding
Alcohol. Clin. Exp. Res.
2005
, vol. 
29
 (pg. 
1496
-
1503
)
[PubMed]
28
Uzi
D.
Barda
L.
Scaiewicz
V.
Mills
M.
Mueller
T.
Gonzalez-Rodriguez
A.
Valverde
A.M.
Iwawaki
T.
Nahmias
Y.
Xavier
R.
, et al. 
CHOP is a critical regulator of acetaminophen-induced hepatotoxicity
J. Hepatol.
2013
, vol. 
59
 (pg. 
495
-
503
)
[PubMed]
29
Li
G.
Mongillo
M.
Chin
K.T.
Harding
H.
Ron
D.
Marks
A.R.
Tabas
I.
Role of ERO1-alpha-mediated stimulation of inositol 1, 4, 5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis
J. Cell Biol.
2009
, vol. 
186
 (pg. 
783
-
792
)
[PubMed]
30
Haynes
C.M.
Titus
E.A.
Cooper
A.A.
Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death
Mol. Cell
2004
, vol. 
15
 (pg. 
767
-
776
)
[PubMed]

Author notes

1

These authors contributed equally to the present work.

Supplementary data