Epigallocatechin-3-gallate protects against cisplatin-induced nephrotoxicity by inhibiting endoplasmic reticulum stress-induced apoptosis

Cisplatin (CP)-induced nephrotoxicity hampers its application in clinic. Green tea, particularly its predominant polyphenolic constituent epigallocatechin-3-gallate (EGCG), possesses anti-inflammatory, antioxidant, and anti-apoptotic properties. The present study was designed to investigate the protective effects of EGCG against CP-induced nephrotoxicity in mice. Male C57/BL6 mice in different groups received single injection of CP (20 mg/kg) and EGCG (100 mg/kg) in various sets and kidney tissues and blood were collected after killing. Then, samples were used for biochemical and immunohistochemical assay. Our results showed EGCG decreased biochemical factors and immunohistochemical damage induced by CP. Besides, expression of phosphorylated-extracellular signal-regulated kinase (p-ERK), glucose-regulated protein 78 (GRP78), caspase-12, and apoptosis of kidney were decreased by EGCG via inhibition of endoplasmic reticulum (ER) stress-induced apoptosis.     

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or,a novel flavoheme reductase

NADH cytochrome b₅ oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.     

Over-expression of methionine sulfoxide reductase A in the endoplasmic reticulum increases resistance to oxidative and ER stresses

MsrA and MsrB catalyze the reduction of methionine-S- suifoxide and methionine-R-sulfoxide, respectively, to methionine in different cellular compartments of mammalian cells. One of the three MsrBs, MsrB3, is an endoplasmic reticulum （ER）-type enzyme critical for stress resistance including oxidative and ER stresses. However, there is no evidence for the presence of an ER-type MsrA or the ER local- ization of MsrA. In this work, we developed an ER-targeted recombinant MsrA construct and investigated the potential effects of methionine-S-sulfoxide reduction in the ER on stress resistance. The ER-targeted MsrA construct contained the N-terminal ER-targeting signal peptide of human MsrB3A （MSPRRSLPRPLSLCLSLCLCLCLAAALGSAQ） and the C-terminal ER-retention signal sequence （KAEL）. The over-expression of ER-targeted MsrA significantly increased cellular resistance to H202-induced oxidative stress. The ER-targeted MsrA over-expression also significantly enhanced resistance to dithiothreitol-induced ER stress; however, it had no positive effects on the resistance to ER stresses induced by tunicamycin and thapsigargin. Collectively, our data suggest that methionine-S-sulfoxide reduction in the ER compartment plays a protective role against oxidative and ER stresses.     

Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers

Herp is a stress-response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER-folding, decrease ER protein load, and participate in ER-associated degradation (ERAD). Intra-muscle-fiber ubiquitinated multiprotein-aggregates containing, among other proteins, either amyloid-beta (Abeta) or phosphorylated tau are characteristic of sporadic inclusion-body myositis (s-IBM). ER stress and proteasome inhibition appear to play a role in s-IBM pathogenesis. We have now studied Herp in s-IBM muscle fibers and in ER-stress-induced or proteasome-inhibited cultured human muscle fibers. In s-IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co-localized with Abeta, GRP78, and beta2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER-stress-induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome-inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s-IBM muscle fibers: (i) increase of Herp might be due to both ER-stress and proteasome inhibition; (ii) co-localization of Herp with Abeta, proteasome, and ER-chaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.     

Neuregulin‐1 protects myocardial cells against H2O2‐induced apoptosis by regulating endoplasmic reticulum stress

Neuregulin‐1 (NRG‐1) is a stress‐mediated growth factor secreted by cardiovascular endothelial cells and provides the protection to myocardial cells, but the underlying mechanisms are not fully understood. This study aimed to demonstrate that NRG‐1 protects myocardial cells exposed to oxidative damage by regulating endoplasmic reticulum (ER) stress. Neonatal rat cardiac myocytes (NRCMs) were isolated and treated with H₂O₂ as a cellular model of ER stress. NRCMs were pretreated with different concentrations of NRG‐1. We found that NRG‐1 increased the viability and reduced the apoptosis of NRCMs treated by H₂O₂. Moreover, NRG‐1 reduced lactate dehydrogenase level, increased superoxide dismutase activity and decreased malondialdehyde content in NRCMs treated by H₂O₂. Finally, we demonstrated that NRG‐1 alleviated ER stress and decreased CHOP and GRP78 protein levels in NRCMs treated by H₂O₂. Taken together, these data indicate that NRG‐1 relieves oxidative and ER stress in NRCMs and suggest that NRG‐1 is a promising agent for cardioprotection. Copyright © 2014 John Wiley & Sons, Ltd.     

Exercise protects against diabetic cardiomyopathy by the inhibition of the endoplasmic reticulum stress pathway in rats

To explore the protective effect of exercise training on the injury of myocardium tissues induced by streptozotocin (STZ) in diabetic rats and the relationship with endoplasmic reticulum stress (ERS), the male sprague-dawley (SD) rats were fed with high-fat and high-sugar diet for 4 weeks, followed by intraperitoneal injection of STZ, 40 mg/kg, to establish a diabetes model, and then 10 rats were randomly selected as diabetes mellitus (DM) controls and 20 eligible diabetic rats were randomized into two groups: low-intensity exercise training (n = 10) and high-intensity exercise training (n = 10). After 12 weeks of exercise training, rats were killed and serum samples were used to determine cardiac troponin-I (cTn-I). Myocardial tissues were sampled for morphological analysis to detect myocardial cell apoptosis, and to analyze protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12. Different intensities (low and high) significantly reduced serum cTn-I levels compared with the DCM group (p < 0.01), and significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Hematoxylin and eosin and Masson staining indicated that exercise training could attenuate myocardial apoptosis. Additionally, exercise training significantly reduced GRP78, CHOP, and cleaved caspase-12 protein expression in an intensity-dependent manner. These findings suggest that exercise appeared to ameliorate diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in diabetic rats.     

Plin3 protects against alcoholic liver injury by facilitating lipid export from the endoplasmic reticulum

Hepatic lipid accumulation is the most common pathological characteristic of alcoholic liver disease (ALD). In mammalian cells, excess neutral lipids are stored in lipid droplets (LDs). As a member of perilipin family proteins, Plin3 was recently found to regulate the LD biogenesis. However, the roles and mechanism of Plin3 in ALD progression remain unclear. Herein, we found that alcohol stimulated Plin3 expression in both mouse livers and cultured AML12 mouse hepatic cells, which was accompanied by excess LD accumulation in hepatocytes. The elevations of Plin3 in alcohol-treated hepatocytes paralleled with the levels of both PPARα and γ, and the protein degradation of Plin3 was also reduced after alcohol exposure. Moreover, Plin3 knockdown increased cellular sensitivity to alcohol-induced apoptosis, endoplasmic reticulum (ER) stress, and inflammatory cytokines release, including TNF-α, IL-1, and IL-6β. Notably, alcohol exacerbated triglycerides (TG) accumulation in the ER and caused ER dilation in Plin3-knockdown AML12 cells. Finally, we observed that Plin3 interacted with dynein subunit Dync1i1 and mediated the colocalization of LDs and microtubules, while high concentration of alcohol disrupted microtubules and caused dispersion of excess small LDs in cytoplasm. Summarily, Plin3 promotes lipid export from the ER and reduces ER lipotoxic stress, thereby, protecting against alcoholic liver injury. Moreover, Plin3 could be an adapter protein mediating LD transport by microtubules. This study explored the roles of Plin3 in alcohol-induced hepatic injury, suggesting Plin3 as a potential target for the prevention of ALD progression.     

Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase

Methionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes.     

慢性低氧性肺动脉高压大鼠肺组织内质网应激介导的凋亡的变化

目的:探讨内质网应激介导的凋亡在低氧性肺动脉高压大鼠肺组织中的变化及意义.方法:清洁级雄性SD大鼠22只随机被均分成对照组和低氧组(n=11).采用常压低氧法复制慢性低氧高二氧化碳性肺动脉高压模型,4周后,测定肺动脉平均压(mPAP)、右心室游离壁(RV)和左心室加室间隔(LV+S)重量比、肺细小动脉管壁面积/管总面积...     

GPR177 promotes gastric cancer proliferation by suppressing endoplasmic reticulum stress-induced cell death

Gastric cancer is the fourth most common cancer worldwide. Despite the high incidence of gastric cancer, efficient chemotherapy treatments still need to be developed. In this study, we examined the anticancer effects of endoplasmic reticulum (ER) stress inducer tunicamycin in gastric cancer. Previously, we found that overexpression of WLS1/GPR177 correlated with poor prognosis in patients with gastric cancer. Furthermore, tunicamycin treatment downregulated GPR177 expression in a dose-dependent manner. GPR177 transports WNT ligand from ER to the plasma membrane, mediating its secretion to the extracellular matrix. In gastric cancer cells, GPR177 preferentially localizes to the ER. Small interfering RNA-mediated knockdown of GPR177 leads to sensitization to ER stress and induces apoptosis of cancer cells along with tunicamycin treatment. GPR177 suppression promoted the ER stress-mediated proapoptotic pathway, such as PERK-CHOP cascade. Furthermore, fluorouracil treatment combined with tunicamycin dramatically reduced cancer cell proliferation. Efficacy of tunicamycin chemotherapy treatments depended on GPR177 expression in gastric cancer cell lines. Together, our results indicate that ER stress can potentiate anticancer effects and suggest GPR177 as a potential gastric cancer therapeutic target.     

Effects of corticosteroid-hormones on the smooth endoplasmic reticulum of rat adrenocortical cells

Summary The ultrastructural changes occuring in adrenocortical cells of prednisolone-treated rats were evaluated by morphometric methods. They consist mostly in a conspicuous decrease in the smooth reticulum surface and in the lipid droplets. The diminution of the smooth reticulum accounts for about 60 % of the decrement in cellular volume and in adrenal weight. Since numerous enzymes of corticosteroid-synthesis are localized in the microsomal fraction of adrenocortical cells, it is reasonable to suggest that the decrement of these organelles is the morphological expression of the cellular deficit in hormone-synthesis.The possible mechanism of reduction of the reticulum membranes is discussed in relation to the probable regulation mechanism of the adrenocortical secretion. It is proposed that the reticulum decrement is the result of a reduced synthesis of new membranes, due to an inhibition of protein-synthesis in adrenocortical cells.The author wishes to express his sincere appreciation to Mr. G. Gottardo and to Dr. A. Gambino for their excellent technical assistance.     

内质网应激的细胞效应分子机制

内质网应激（endoplasmic reticulum stress,ERs）是内质网腔内错误折叠蛋白聚积的一种适应性反应,适度ERs通过激活未折叠蛋白反应起适应性的细胞保护作用,而过高和持久的ERs则通过诱导转录因子CHOP表达、激活caspase-12和c—Jun氨基末端激酶（JNK）等导致细胞凋亡。近年来,越来越多的研究提示内质网应激是神经退行性病变、2型糖尿病以及肥胖等疾病发生过程中的重要环节。对内质网应激的细胞效应分子机制进行综述。随着对ERs机制理解的深入,有可能会发现新的分子标志物或新的诊疗策略。     

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.     

Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases, including Parkinson's disease. The present study attempted to investigate the effect of hydrogen sulfide (H(2)S) on 6-hydroxydopamine (6-OHDA)-induced ER stress in SH-SY5Y cells. We found in the present study that exogenous application of sodium hydrosulfide (NaHS; an H(2)S donor, 100 μM) significantly attenuated 6-OHDA (50 μM)-induced cell death. NaHS also reversed the upregulation of cleaved poly(ADP-ribose) polymerase and caspase 9 in 6-OHDA-treated cells. Consistent with its cytoprotective effects, NaHS markedly reduced 6-OHDA induced-ER stress responses, including the upregulated levels of eukaryotic initiation factor-2α phosphorylation, glucose-regulated protein 78, and C/EBP homologous protein expression. The protective effect of H(2)S on ER stress was attenuated by blockade of Akt activity with an Akt inhibitor or inhibition of heat shock protein (Hsp)90 with geldanamycin but not by suppression of ERK1/2 with PD-98059. Blockade of Akt also significantly decreased the protein abundance of Hsp90 in SH-SY5Y cells. Moreover, overexpression of cystathionine β-synthase (a main H(2)S-synthesizing enzyme in the brain) elevated the Hsp90 protein level and suppressed 6-OHDA-induced ER stress. In conclusion, the protective effect of H(2)S against 6-OHDA-induced ER stress injury in SH-SY5Y cells involves the Akt-Hsp90 pathway.