Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.     

A transgenic mouse model for monitoring endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER lumen, and is associated with vascular and neurodegenerative diseases. Although the connection between ER stress and some disease-related proteins has been studied using animal models of these diseases, no in vivo data concerning ER stress are available. Here we report a new method for monitoring ER stress in vivo, based on XBP-1 mRNA splicing by inositol requiring-1 (IRE-1) during ER stress. The stress indicator was constructed by fusing XBP-1 and venus, a variant of green fluorescent protein. During stress, the spliced indicator mRNA is translated into an XBP-1-venus fusion protein, which can be detected by its fluorescence. We used transgenic animals expressing the ER stress indicator to show that it can be used to monitor physiological and pathological ER stress in vivo.     

Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase expression by endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is induced in infectious and inflammatory conditions, but its role in inflammatory responses still remains elusive. In this study we found tunicamycin (TM) and brefeldin A (BFA), two ER stressors, could attenuate lipopolysaccharide (LPS)-elicited inducible nitric oxide synthase (iNOS) gene expression in murine RAW264.7 macrophages, and this effect was not resulting from the effects on IKK or MAPKs activation. However, ER stressors could block NF-κB binding to the iNOS promoter in late-phase signaling evoked by LPS. Results indicated that inhibition of RelB nuclear translocation and p300 expression are involved in the anti-inflammatory actions of ER stressors. We also found that ER stressors could block LPS- and IFN (α, β, and γ)-mediated STAT1 phosphorylation. Our results suggest that activation of MKP-1 via a Ca/calmodulin/calcineurin pathway accounts for the inhibitory effect of ER stressors on IFN signaling. MKP-1 was downregulated by IFN-γ and is a newly identified protein phosphatase targeting STAT1. Taken together, these results indicate that multiple mechanisms are involved in the inhibition of LPS-induced iNOS gene expression by ER stressors. These include downregulation of RelB and p300, upregulation of MKP-1, and inhibition of the JAK/STAT signaling pathway.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress

Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5′-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery.     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Aberrant expression profiles of isoproterenol-induced endoplasmic reticulum stress response genes in mouse myocardium

In this study, we identified the aberrant expression profiles of isoproterenol- (ISO; synthetic catecholamine)induced endoplasmic reticulum (ER) stress response genes in mouse myocardium. Mouse models of acute catecholamine cardiotoxicity were induced by ISO for 6, 12, and 24 h. We performed whole genome oligo microarrays of damaged mouse cardiac tissues, and we found 26 ER stress-related genes whose expression changed significantly for at least one time point. The functional analysis of those genes indicated that myocardial cells were protected by increasing folding capacity, inhibiting general protein translation, and promoting the degradation of misfolded proteins; however, some of them underwent apoptosis in the early stage of ER stress after ISO induced.     

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.     

GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress

Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin biosynthesis, beta cell survival, and glucose homeostasis. We show that a murine model of diabetes is associated with the development of ER stress in beta cells and that treatment with the GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in vivo. Exendin-4 attenuated translational downregulation of insulin and improved cell survival in purified rat beta cells and in INS-1 cells following induction of ER stress in vitro. GLP-1R agonists significantly potentiated the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in INS-1 beta cells in a PKA-dependent manner. The effects of exendin-4 on the induction of ATF-4 were mediated via enhancement of ER stress-stimulated ATF-4 translation. Moreover, exendin-4 reduced ER stress-associated beta cell death in a PKA-dependent manner. These findings demonstrate that GLP-1R signaling directly modulates the ER stress response leading to promotion of beta cell adaptation and survival.     

Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury

Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.     

Oxidative stress on mouse embryo development in vitro.

Oxygen radicals are involved in the in vitro block phenomenon of embryo development, because a low oxygen tension and superoxide dismutase (SOD) have been shown to promote the in vitro development of mouse embryos. One of the target molecules damaged by oxygen radicals may be the thiol (SH) group of proteins because it is readily oxidized. In this study, we evaluated the effects of thioredoxin, which is a powerful protein disulfide reductase, on mouse (Institute of Cancer Research, ICR) preimplantation embryo development. Culture of mouse pronuclear embryos recovered 17 h after human chorionic gonadotrophin (hCG) administration in the presence of thioredoxin (200 micrograms/mL) significantly increased the blastulation rate (75.3%) when compared to the control culture system (8.9%). The effects of thioredoxin were observed only from the pronuclear stage to the two-cell stage (17-48 h after hCG administration). An additive effect of thioredoxin and SOD, or thioredoxin and a low oxygen tension, was observed. These results suggest that the oxidation of the SH group of proteins is one of the causes of developmental blockage of embryos in vitro. The target protein for reduction by thioredoxin has not been identified yet, but thioredoxin will be a new clue for clarifying the mechanism of blocking development in vitro.     

Recombinant Clostridium difficile toxin B induces endoplasmic reticulum stress in mouse colonal carcinoma cells

Clostridium difficile is the main cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans and animals. Its pathogenicity is primarily linked to the secretion of two exotoxins （TcdA and TcdB）. Although great progress in the toxic mechanism of TcdA and TcdB has been achieved, there are many conflicting reports about the apop- totic mechanism. More importantly, apoptotic endoplasmic reticulum （ER） stress has been reported in cells treated with Shiga toxins---another kind of cytotoxins that can cause diar- rhea and colitis. Herein we checked whether TcdB can induce ER stress. The results showed that recombinant TcdB （rTcdB） activated molecular markers of unfolded protein re- sponse, suggesting that rTcdB induced ER stress in CT26 cells. However, rTcdB did not induce the up-regulation of C/EBP homologous protein （CHOP）, a classic mediator of apoptotic ER stress, but it activated the precursor of cysteine aspartic acid-specific protease 12 （caspase-12）, a controver- sial mediator of apoptotic ER stress. Besides, glucosyltrans- ferase activity-deficient mutant recombinant TcdB induced ER stress, though it has no cytotoxic or cytopathic effect on CT26 cells. Altogether, these data demonstrated that ER stress induced by rTcdB is glucosyltransferase-independent, indicating that ER stress induced by rTcdB is non-apoptotic. This work also offers us a new insight into the molecular mechanism of CHOP protein expression regulation and the role of CHOP expression in ER stress.