Endoplasmic Reticulum Stress In Hepatocytes Research Articles

Phosphorylation of eIF2α mitigates endoplasmic reticulum stress and hepatocyte necroptosis in acute liver injury

Introduction and objectivesNecroptosis and endoplasmic reticulum (ER) stress has been implicated in acute and chronic liver injury. Activated eukaryotic initiation factor 2 alpha (eIF2α) attenuates protein synthesis and relieves the load of protein folding in the ER. In this study, we aimed to analyze the impact of eIF2α phosphorylation on hepatocyte necroptosis in acute liver injury. Materials and methodsMale BALB/c mice were injected with tunicamycin or d-galactosamine, and LO2 cells were incubated with tunicamycin to induce acute liver injury. 4-Phenylbutyric acid (PBA) and salubrinal were used to inhibit ER stress and eIF2α dephosphorylation, respectively. We analyzed the eIF2α phosphorylation, ER stress, and hepatocyte necroptosis in mice and cells model. ResultsTunicamycin or d-galactosamine significantly induced ER stress and necroptosis, as well as eIF2α phosphorylation, in mice and LO2 cells (p<0.05). ER stress aggravated tunicamycin-induced hepatocyte necroptosis in mice and LO2 cells (p<0.05). Elevated eIF2α phosphorylation significantly mitigated hepatocyte ER stress (p<0.05) and hepatocyte necroptosis in mice (34.37±3.39% vs 22.53±2.18%; p<0.05) and LO2 cells (1±0.11 vs 0.33±0.05; p<0.05). Interestingly, tumor necrosis factor receptor (TNFR) 1 protein levels were not completely synchronized with necroptosis. TNFR1 expression was reduced in d-galactosamine-treated mice (p<0.05) and cells incubated with tunicamycin for 12 and 24h (p<0.05). ER stress partially restored TNFR1 expression and increased necroptosis in tunicamycin-incubated cells (p<0.05). ConclusionsThese results imply that ER stress can mediate hepatocyte necroptosis independent of TNFR1 signaling and elevated eIF2α phosphorylation can mitigate ER stress during acute liver injury.

Corrigendum to “Zhou LX, Yang AN, Chen JK, Zhao L, Wang YH, Liu XM, Cai X, Zhang MH, Jiang YD, Cao J. Endoplasmic reticulum oxidoreductin 1α mediates homocysteine-induced hepatocyte endoplasmic reticulum stress” [Shijie Huaren Xiaohua Zazhi 2014; 22(34): 5228-5234

Corrigendum to “Zhou LX, Yang AN, Chen JK, Zhao L, Wang YH, Liu XM, Cai X, Zhang MH, Jiang YD, Cao J. Endoplasmic reticulum oxidoreductin 1α mediates homocysteine-induced hepatocyte endoplasmic reticulum stress” [Shijie Huaren Xiaohua Zazhi 2014; 22(34): 5228-5234

Abstract 2659: IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death

Abstract Hepatocellular carcinoma (HCC) belongs to the most deadly types of cancer since it has a poor prognosis due to its aggressive metastatic phenotype. Endoplasmic reticulum (ER) stress is closely associated with the incidences of a variety of liver diseases including liver injury, regeneration, fibrosis, cirrhosis and often HCC. G protein-coupled receptor (GPCR) signals lead to activation of their downstream pathways through G proteins. Previously, we reported that Gα12 overexpression enhances HCC malignancy, promoting cancer migration and microvascular invasion. However, the role of Gα12 in ER stress-associated pathophysiology in the liver was unclear. Given the link between ER stress and HCC malignancy, this study investigated the effect of ER stress on Gα12, the role of Gα12 in ER stress-mediated hepatocyte death, and the underlying pathway. In the analyses of RNA-sequencing data from mouse liver or primary mouse embryonic fibroblasts challenged with ER stress, Gα12 and C/EBP homologous protein (CHOP) levels correlated with each other. Increases in Gα12 level by unfolded protein response (UPR) under ER stress condition was verified using primary hepatocytes or hepatocyte-derived cell line, confirmative of the close association between Gα12 and UPR. Of note, enforced Gα12 expression under ER stress condition augmented IRE1α level in a RhoA/Rock-dependent pathway. Among the canonical pathways, IRE1α gene silencing distinctly inhibited Gα12 induction elicited by ER stress in hepatocytes. Consistently, overexpression of IRE1α enhanced Gα12 level upon tunicamycin treatment. Similar outcomes were obtained in cells or mice treated with acetaminophen, a representative liver toxicant inducing ER stress response. Overall, our results provide evidence that IRE1α-dependent increase of Gα12 potentiates ER stress response in the liver as mediated by the RhoA-ROCK pathway, enhancing catastrophic death of hepatocytes. Citation Format: Jihoon Tak, Tae Hyun Kim, Sang Geon Kim. IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2659.

Zinc Attenuates the Cytotoxicity of Some Stimuli by Reducing Endoplasmic Reticulum Stress in Hepatocytes.

Zinc is an essential trace element and plays critical roles in cellular integrity and biological functions. Excess copper induced both oxidative stress and endoplasmic reticulum (ER) stress in liver-derived cultured cells. Excess copper also induced impairment of autophagic flux at the step of autophagosome–lysosome fusion, as well as Mallory–Denk body (MDB)-like inclusion body formation. Zinc ameliorated excess copper-induced impairment of autophagic flux and MDB-like inclusion body formation via the maintenance of ER homeostasis. Furthermore, zinc also ameliorated free fatty acid-induced impairment of autophagic flux. These results indicate that zinc may be able to protect hepatocytes from various ER stress-related conditions.

From the Editor’s Desk…: October 2018

From the Editor’s Desk…: October 2018

Impact of Hepatic Mig6 on Glucose Homeostasis and Oxidative Stress

Hepatic epidermal growth factor receptor (EGFR) expression and activation are decreased during steatosis in humans and several animal models of obesity. Restoring EGFR activation in obesity-induced endoplasmic reticulum (ER) stress and diabetes is a potential therapy to improve liver function in type 2 diabetes (T2D). Mitogen-inducible gene 6 (Mig6) is an inducible feedback inhibitor of EGFR activity and, therefore, a target for enhancing EGFR signaling during diet-induced obesity and T2D. We previously observed that liver Mig6 expression was increased during obesity-induced insulin resistance in C57Bl/6J mice fed a high fat diet and by both pharmacological- and fatty acid-driven ER stress in hepatocytes, leading to decreased EGF-mediated EGFR activation. To test the hypothesis of Mig6 as a target to treat insulin resistance in T2D, we examined liver-specific Mig6 knockout mice (LKO) and their littermate controls (CON) during diet-induced obesity. We identified enhanced whole-body glucose tolerance and hepatic insulin action in LKO compared to CON mice. Moreover, liver ballooning, an oxidative stress marker, was also improved in LKO mice. Using whole transcriptome sequencing, we sought to determine the molecular mechanisms leading to liver function improvement in obesity-induced insulin resistance in LKO mice. We identified that gene expression and protein accumulation of key enzymes of both peroxisomal (e.g., Acox1, Ehhadh) and mitochondrial (e.g., Cpt2, Acadl) beta-oxidation were decreased by Mig6 ablation. This observation correlated with a compromised gene expression of liver gluconeogenic enzymes. In conclusion, we posit that hepatic Mig6 deficiency partially reverts the effects of obesity-induced diabetes by decreasing hepatic glucose output and reducing cellular oxidative stress, thereby establishing Mig6 as a therapeutic candidate for treating insulin resistance in obesity and T2D. Disclosure J.M. Irimia-Dominguez: None. A.J. Lutkewitte: None. P.T. Fueger: None.

Comparative Proteomic Identification of Protein Disulphide Isomerase A6 Associated with Tert-Butylhydroperoxide-Induced Liver Injury in Rat Hepatocytes

Background/Aims: Oxidants are important human toxicants. They have been implicated in the occurrence and development of liver diseases. Increased intracellular tert-butylhydroperoxide (t-BHP) may be critical for oxidant toxicity, and is commonly used for evaluating mechanisms involving oxidative stress, but the method remains controversial. Methods: Primary cultures of hepatocytes as well as human Hep G2 and mouse FL83B liver cells were obtained. Cell viability was measured by annexin V–FITC/propidium iodide and DAPI staining to determine the effects of t-BHP treatment on acute liver injury. A proteomic assay provided information that was used to identify the differentially expressed proteins following t-BHP treatment; immunohistochemistry and western blotting were performed to detect the expression of PDIA6 activity in apoptotic and endoplasmic reticulum (ER) stress pathways. Results: Our results demonstrate that t-BHP treatment of liver cells increased cell cytotoxicity and the generation of reactive oxygen species. This treatment also increased the level of PDIA6; this was validated in vitro and in vivo based on a comparison of t-BHP-treated and -untreated groups. Treatment of mouse liver FL83B cells with t-BHP activated caspase 3, increased the expression of apoptotic molecules, caused cytochrome c release, and induced Bcl-2, Bax and IRE1α/TRAF2 complex formation. t-BHP-dependent induction of apoptosis was accompanied by sustained phosphorylation of the IRE1α/ASK1/JNK1/2/p38 pathways and PDIA6 expression. Furthermore, t-BHP induced liver FL83B cell viability and apoptosis by upregulating the levels of PDIA6; this process could be involved in the activation of the IRE1α/ASK1/JNK1/2/p38 signalling pathways. Conclusions: We conclude that t-BHP induced an apoptosis cascade and ER stress in hepatocytes by upregulation of PDIA6, providing a new mechanism underlying the effects of t-BHP on liver injury.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Overexpression of apolipoprotein A-I alleviates endoplasmic reticulum stress in hepatocytes

BackgroundAbnormal lipid metabolism may contribute to an increase in endoplasmic reticulum (ER) stress, resulting in the pathogenesis of non-alcoholic steatohepatitis. Apolipoprotein A-I (apoA-I) accepts cellular free cholesterol and phospholipids transported by ATP-binding cassette transporter A1 to generate nascent high density lipoprotein particles. Previous studies have revealed that the overexpression of apoA-I alleviated hepatic lipid levels by modifying lipid transport. Here, we examined the effects of apoA-I overexpression on ER stress and genes involved in lipogenesis in both HepG2 cells and mouse hepatocytes.MethodsHuman apoA-I was overexpressed in HepG2 hepatocytes, which were then treated with 2 μg/mL tunicamycin or 500 μM palmitic acid. Eight-week-old male apoA-I transgenic or C57BL/6 wild-type mice were intraperitoneally injected with 1 mg/kg body weight tunicamycin or with saline. At 48 h after injecting, blood and liver samples were collected.ResultsThe overexpression of apoA-I in the models above resulted in decreased protein levels of ER stress makers and lipogenic gene products, including sterol regulatory element binding protein 1, fatty acid synthase, and acetyl-CoA carboxylase 1. In addition, the cellular levels of triglycerides and free cholesterol also decreased. Some of gene products which are related to ER stress-associated apoptosis were also affected by apoA-I overexpression. These results suggested that apoA-I overexpression could reduce steatosis by decreasing lipid levels and by suppressing ER stress and lipogenesis in hepatocytes.ConclusionApoA-I expression could significantly reduce hepatic ER stress and lipogenesis in hepatocytes.

Modeling Acute ER Stress in Vivo and in Vitro.

The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.

Therapeutic efficacy and mechanism of action of ginsenoside Rg1 in treating acute hepatic failure in mice

Objective: To examine the regulatory effect of ginsenoside Rg1 (G-Rg1) on endoplasmic reticulum stress and its effect on hepatocellular apoptosis in carbon tetrachloride (CCl(4))-induced acute liver failure (ALF). Methods: Forty healthy, adult male C57/BL mice were randomly divided into normal saline control (NS) group, G-Rg1 blank control (G-Rg1) group, CCl(4) model (CCl(4)) group, and G-Rg1 preventive treatment (CCl(4)+G-Rg1) group, and an ALF mouse model was established by CCl(4) induction. Blood and liver specimens were collected from all mice upon sacrifice at 12 hours post-intraperitoneal injection. Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and total bilirubin (TBil) levels were determined using commercial test kits. The mRNA expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was measured using real-time PCR. The protein expression of GRP78, CHOP, caspase12, and caspase3 were measured by Western blot. Histological changes in the liver were assessed by hematoxylin-eosin staining, and the expression of GRP78 and caspase3 was detected by immunohistochemistry. Hepatocyte apoptosis was determined using terminal transferase dUTP nick end labeling. Quantitative data were analyzed using one-way ANOVA, and subsequent pairwise comparisons were performed using the LSD-t method. Results: Serum ALT, AST, and TBil levels in the CCl(4)+G-Rg1 group were significantly reduced compared with those in the CCl(4) group (ALT: 691.30 ± 108.06 U/L vs 980.66 ± 110.29 U/L, F = 365.07, P < 0.05; AST: 195.40 ± 15.41 U/L vs 319.44 ± 89.32 U/L, F = 115.64, P < 0.05; TBil: 1.09 ± 0.11 mg/dl vs 1.56 ± 0.12 mg/dl, F = 211.29, P < 0.05). The relative mRNA expression of GRP78 and CHOP was significantly lower in the CCl(4) + G-Rg1 group than in the CCl(4) group (P < 0.05). The relative protein expression of caspase3, GRP78, caspase12, and CHOP was significantly reduced to different extents in the CCl(4)+G-Rg1 group compared with those in the CCl4 group (P < 0.05). The CCl(4) + G-Rg1 group showed reduced liver tissue degeneration and necrosis compared with the CCl(4) group. Furthermore, the CCl(4)+G-Rg1 group showed significantly fewer brown granules in the liver than the CCl4 group (P < 0.05), indicating that G-Rg1 preventive treatment reduced CCl(4)-induced hepatocyte apoptosis. Conclusion: G-Rg1 prophylaxis can inhibit inflammation and reduce hepatocyte necrosis and apoptosis during CCl(4)-induced ALF. Its mechanism may involve the suppression of endoplasmic reticulum stress-related signaling molecules to alleviate hepatocyte endoplasmic reticulum stress and apoptosis. The results of this study suggest that G-Rg1 may inhibit liver inflammation and hepatocyte apoptosis through multiple targets to protect liver function.

Combination of tauroursodeoxycholic acid and N-acetylcysteine exceeds standard treatment for acetaminophen intoxication.

Acetaminophen overdose in mice is characterized by hepatocyte endoplasmic reticulum stress, which activates the unfolded protein response, and centrilobular hepatocyte death. We aimed at investigating the therapeutic potential of tauroursodeoxycholic acid, a hydrophilic bile acid known to have anti-apoptotic and endoplasmic reticulum stress-reducing capacities, in experimental acute liver injury induced by acetaminophen overdose. Mice were injected with 300mg/kg acetaminophen, 2hours prior to receiving tauroursodeoxycholic acid, N-acetylcysteine or a combination therapy, and were euthanized 24hours later. Liver damage was assessed by serum transaminases, liver histology, terminal deoxynucleotidyl transferase dUTP nick end labelling staining, expression profiling of inflammatory, oxidative stress, unfolded protein response, apoptotic and pyroptotic markers. Acetaminophen overdose resulted in a significant increase in serum transaminases, hepatocyte cell death, unfolded protein response activation, oxidative stress, NLRP3 inflammasome activation, caspase 1 and pro-inflammatory cytokine expressions. Standard of care, N-acetylcysteine and, to a lesser extent, tauroursodeoxycholic treatment were associated with significantly lower transaminase levels, hepatocyte death, unfolded protein response activation, oxidative stress markers, caspase 1 expression and NLRP3 levels. Importantly, the combination of N-acetylcysteine and tauroursodeoxycholic acid improved serum transaminase levels, reduced histopathological liver damage, UPR-activated CHOP, oxidative stress, caspase 1 expression, NLRP3 levels, IL-1β levels and the expression of pro-inflammatory cytokines and this to a greater extend than N-acetylcysteine alone. These findings indicate that a combination strategy of N-acetylcysteine and tauroursodeoxycholic acid surpasses the standard of care in acetaminophen-induced liver injury in mice and might represent an attractive therapeutic opportunity for acetaminophen-intoxicated patients.

Association between betatrophin/ANGPTL8 and non-alcoholic fatty liver disease: animal and human studies.

Betatrophin/angiopoietin-like protein 8 (ANGPTL8) is a liver-secreted protein recently identified as a potent stimulator of beta cell proliferation in mice. However, it is unclear how betatrophin is regulated in humans with non-alcoholic fatty liver disease (NAFLD). We investigated the role of betatrophin in mice and in humans with and without NAFLD. Serum betatrophin levels were examined by ELISA in 164 subjects, including 96 patients with NAFLD. Levels were significantly elevated in subjects with NAFLD compared with controls (1.301 ± 0.617 vs. 0.900 ± 0.574 μg/L, P < 0.001), even after stratification by diabetic or obesity status. Circulating betatrophin positively correlated with obesity or glycemic indices, liver enzyme profiles, and NAFLD status, and was confirmed by multivariate regression analyses (β = 0.195, P = 0.040). However, when including insulin resistance index in the model, the significant association between betatrophin level and NAFLD was diminished due to a mediation effect of insulin resistance on this relationship. Palmitate or tunicamycin increased betatrophin expression in HepG2 cells, while a chemical chaperone blocked its induction. Hepatic expression of betatrophin was elevated in mice with NAFLD including db/db or ob/ob mice and mice with a high-fat or methionine-choline deficient diet. In conclusion, circulating betatrophin was increased in mice and humans with NAFLD and its expression was induced by endoplasmic reticulum stress in hepatocytes (Clinical trial no. NCT02285218).

Cystatin C attenuates insulin signaling transduction by promoting endoplasmic reticulum stress in hepatocytes

It has been reported that cystatin c (Cys C) closely correlates with metabolic disorders such as obesity and diabetes. However, it is still unknown whether Cys C plays a role for these disorders. Our results showed that the insulin signal transduction was largely impaired by Cys C in hepatocytes. In myotubes, however, the insulin signal transduction was not affected. Following experiments revealed that Cys C could induce endoplasmic reticulum stress (ER stress) in hepatocytes, whereas Cys C had no such an effect in myotubes. The alleviation of ER stress by 4-Phenyl butyric acid (4-PBA) restored the impaired insulin signal transduction in Cys C-treated hepatocytes. These results provided direct evidence that, by inducing ER stress, Cys C impairs insulin signal transduction in hepatocytes.

ER Stress Cooperates with Hypernutrition to Trigger TNF-Dependent Spontaneous HCC Development

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of viral hepatitis, insulin resistance, hepatosteatosis, and nonalcoholic steatohepatitis (NASH), disorders that increase risk of hepatocellular carcinoma (HCC). To determine whether and how ER stress contributes to obesity-driven hepatic tumorigenesis we fed wild-type (WT) and MUP-uPA mice, in which hepatocyte ER stress is induced by plasminogen activator expression, with high-fat diet. Although both strains were equally insulin resistant, the MUP-uPA mice exhibited more liver damage, more immune infiltration, and increased lipogenesis and, as a result, displayed classical NASH signs and developed typical steatohepatitic HCC. Both NASH and HCC development were dependent on TNF produced by inflammatory macrophages that accumulate in the MUP-uPA liver in response to hepatocyte ER stress.

Exendin-4 attenuates endoplasmic reticulum stress through a SIRT1-dependent mechanism

Accumulation of excess hepatic lipids contributes to insulin resistance and liver disease associated with endoplasmic reticulum (ER) stress. Exendin-4 is an agonist of the glucagon-like peptide 1 receptor and plays a role in improving insulin resistance and liver disease by increasing silent mating type information regulation 2 homolog (SIRT) 1. However, the effects and mechanism of action of exendin-4 on responses to palmitic acid (PA)-induced ER stress in hepatocytes have not been clearly defined. We investigated whether exendin-4 attenuates PA-induced ER stress via SIRT1 in HepG2 cells. PA treatment induced increased expression of PRKR-like endoplasmic reticulum kinase, inositol-requiring kinase 1α (IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) mRNA. Exendin-4 decreased the expression of P-IRE1α, ATF6, X-box binding protein-1 and CHOP, and increased the expression of SERCA2b. A significant decrease in the hepatic expression of PUMA, BAX, cytochrome c, and cleaved caspase-3 were observed in hepatocytes treated with exendin-4. The TUNEL assay consistently showed that exendin-4 reversed hepatocyte apoptosis induced by treatment with PA. Inhibition of SIRT1 by nicotinamide and siRNA significantly increased the expression of ER stress marker genes in cells treated with both PA and exendin-4. In conclusion, increased SIRT1 by exendin-4 attenuates PA-induced ER stress and mitochondrial dysfunction in hepatocytes.

PiggyBac-mediated phenotypic correction of factor VIII deficiency

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

Endoplasmic reticulum oxidoreductin 1α mediates homocysteine-induced hepatocyte endoplasmic reticulum stress

Endoplasmic reticulum oxidoreductin 1α mediates homocysteine-induced hepatocyte endoplasmic reticulum stress

Effects of Radix Salvia Miltiorrhiza on expression of activating transcription factor-6α during the periods of hepatic ischemia-reperfusion in rats

Objective To investigate the expression of activating transcription factor-6α (ATF-6α) and cysteinylasparate specific proteinase-12 (Caspase-12) during the periods of hepatic ischemia-reperfusion iujury and the effect of radix Salvia mihiorrhiae (RSM).Methods A total of 80 Wistar rats were randomly divided into 4 groups:normal group (group A),sham-operated group (group B),ischemia-reperfusion group (group C) and RSM-pretreated group (group D).The model of hepatic ischemia-reperfusion injury was established by occlusion of hepatic pedicle.The expression levels of ATF-6α and Caspase-12 were detected by using Western blotting and immunohistochemistry at 45 min after ischemia and 0,3,12,24,72 h after reperfusion.Results There was no significant difference between group A and group B (P＞0.05).In groupC [0h (346.46±103.89),3 h (1190.70±134.88),12h (5456.18± 288.46),24 h (1772.60 ± 139.46),72 h (235.69 ± 68.15)],the expression of ATF-6α was higher than in group A (9.57±1.26) and groupB [0h (9.90±3.73),3 h (9.69±3.52),12 h (9.50± 2.31),24h (9.44 ±1.73),72 h (9.32 ±2.71)] (P＜0.01).In group C [0h (654.13 ±102.12),3 h (3004.09 ±505.77),12 h (6896.13 ±841.14),24 h (2364.03 ±276.68),72 h (674.96 ± 153.92)],the expression of Caspase-12 was higher than in group A (127.97 ±4.33) and group B [0 h (154.58 ±44.20),3 h (130.58 ±38.40),12 h (144.62 ±36.24),24 h (138.37 ±24.66),72 h (147.87 ±33.10)] (P ＜0.01).In group D,the expression levels of ATF-6α and Caspase-12 around the central veins and portal area were higher than in group A and group B (P ＜0.01),but lower than in group C (P ＜ 0.01).Conclusion The protective effect of RSM-pretreatment may regulate hepatocyte endoplasmic reticulum stress steady state and reduce the apoptosis during the periods of hepatic ischemia/reperfusion by maintaining the proper expression of ATF-6α and down-regulating the over-expression of Caspase-12 in rats. Key words: Radix salvia miltiorrhiza; Liver ischemia; Reperfusion injury; Endoplasmic reticulum stress ; Activating transcription factor-6α ; Caspase-12

The Transcription Factor c-Jun Protects Against Sustained Hepatic Endoplasmic Reticulum Stress Thereby Promoting Hepatocyte Survival

Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N-terminal kinase (JNK). The AP-1 (activating protein 1) transcription factor c-Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis. Because the functions of c-Jun during the ER stress response are poorly understood, we addressed this issue in primary hepatocytes and livers of hepatocyte-specific c-Jun knockout mice. ER stress was induced pharmacologically in vitro and in vivo and resulted in a rapid and robust induction of c-Jun protein expression. Interestingly, ER-stressed hepatocytes lacking c-Jun displayed massive cytoplasmic vacuolization due to ER distension. This phenotype correlated with exacerbated and sustained activation of canonical ER stress signaling pathways. Moreover, sustained ER stress in hepatocytes lacking c-Jun resulted in increased cell damage and apoptosis. ER stress is also a strong inducer of macroautophagy, a cell-protective mechanism of self-degradation of cytoplasmic components and organelles. Interestingly, autophagosome numbers in response to ER stress were reduced in hepatocytes lacking c-Jun. To further validate these findings, macroautophagy was inhibited chemically in ER-stressed wildtype hepatocytes, which resulted in cytoplasmic vacuolization and increased cell damage closely resembling the phenotypes observed in c-Jun-deficient cells. Our findings indicate that c-Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death and provide evidence that c-Jun functionally links ER stress responses and macroautophagy.