Endoplasmic Reticulum Stress Response Protein Research Articles

Duality of jacalin-related lectin: Master regulator and chaperone

Lectins have chaperone-like activity with carbohydrate-binding sites, key in cellular homeostasis. However, the specific interactions of lectin with endoplasmic-reticulum (ER) proteins during stress are unclear. Recently, Das et al. (2023) study reveals lectins' critical role in root cap stress response, through regulating ER stress-responsive proteins, suggesting pathways for enhancing plant resilience.

Abstract C049: Loss of RNF43 creates an OXPHOS dependency in early pancreatic neoplasia

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival of 12%. RNF43 is frequently mutated in pancreatic cystic neoplasms, including intraductal papillary mucinous neoplasms (IPMNs), which are precursor lesions of PDAC. RNF43 encodes for an E3 ubiquitin ligase and was initially identified as a negative Wnt regulator. To elucidate the role of RNF43-mutated neoplasms in an organ specific context, we have generated a genetically engineered mouse model which has pancreas specific loss of RNF43 (Hosein, Gastroenterology 2022). In this study we examine some of the metabolic dependencies created by Rnf43 loss on the background of mutant Kras, and how the Kras;Rnf43 model differs from the commonly used Kras;p53 model of PDAC. We have generated cell lines harboring mutant Kras and bi-allelic loss of Rnf43 from the resulting lesions in these mice (henceforth referred to as Kras;Rnf43 lines).Isogenic Kras;Rnf43 cell lines with re-expression of full-length Rnf43 (Kras;Rnf43-RNF43OE) were generated to confirm if observed phenotypes were Rnf43 dependent. Single-cell RNA sequencing (scRNA-seq) was performed for comparison between murine PDAC arising in Kras;Rnf43 versus Kras;p53 mice. We further compared metabolic parameters in Kras;Rnf43 and Kras;Rnf43-RNF43OE cells using the Seahorse platform. Mitochondrial ultrastructure by transmission electron microscopy (TEM) was analyzed in Kras;Rnf43 compared to Kras;p53 cells. To visualize endoplasmic reticulum (ER)-mitochondria contact sites (ERMCS), a split green fluorescent protein-based contact site sensor (SPLICS) was transfected and analyzed. Compared to Kras;p53 PDAC, Kras;Rnf43 tumors demonstrated upregulated mitochondrial transcripts and gene signatures of ER stress. Further, the ER stress response protein IRE1 was markedly elevated in Kras;Rnf43 cells versus Kras;p53 cells. The Kras;Rnf43 lines had enhanced mitochondrial oxidative phosphorylation (OXPHOS) activity and demonstrated evidence of enhanced mitochondrial quality control. ERMCS is a dynamic contact region between ER and mitochondria, which regulates biological processes including calcium homeostasis, ER stress and mitochondrial dynamics. The Kras;Rnf43 cells had significantly increased ERMCS compared to Kras;p53 cells. Finally, using IACS-010759, a small molecule inhibitor of OXPHOS, we demonstrate that Kras;Rnf43 cells had both increased in vitro sensitivity as well as in vivo susceptibility to tumor growth inhibition in orthotopic models, which translated into increased overall survival of mice. In conclusion, we provide evidence showing that bi-allelic loss of RNF43 is associated with increased ER stress and enhanced IRE1 dependent stress response. ER stress is mitigated via increased mitochondrial OXPHOS and ERMCS. Importantly, our results suggest that this creates a metabolic synthetic essentiality in the context of RNF43 loss, providing an avenue to suppress the progression of RNF43-mutated IPMNs, and potentially other solid cancers by targeting mitochondrial OXPHOS. Citation Format: Akiko Sagara, Brandon Chen, Nathaniel G. Yee, Megan J. Siemann, Costas A. Lyssiotis, Yatrik M. Shah, Sonja M. Woermann, Anirban Maitra. Loss of RNF43 creates an OXPHOS dependency in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C049.

Gypenoside XVII attenuates renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-triggered pyroptosis

BackgroundRenal ischemia-reperfusion (I/R) is one of the main causes of acute kidney injury (AKI), for which there is currently no effective treatment. Recently, the interaction between endoplasmic reticulum (ER) stress and pyroptosis during AKI has been investigated. AimThe purpose of this study was to investigate the protective effects of Gypenoside XVII (GP-17) against I/R-induced renal injury. MethodsIn this study, mice were divided into 6 groups, sham group, I/R group, GP-17 low-, medium-, high-dose group, and positive control 4-PBA group. The renal I/R was performed in mice by clamping the bilateral renal pedicles for 40 min, and then reperfusing for 24 h. Blood and kidney samples were collected for analysis. ResultsThe results showed that GP-17 improved renal function and alleviated renal histopathological abnormalities caused by I/R. In addition, GP-17 pretreatment significantly decreased the expression or phosphorylation of ER stress response proteins including BIP, p-PERK, and CHOP. Besides, GP-17 inhibited the expression of pyroptosis proteins including caspase-1, GSDMD, and apoptotic protein BAX. The inflammatory factor IL-1β in these GP-17 pretreatment groups was also significantly reduced. ConclusionGP-17 blocked NLRP3 inflammasome activation by inhibiting ERS, thereby inhibiting renal tubular cell pyroptosis and apoptosis, and prevented renal I/R injury.

Upregulation of mesencephalic astrocyte-derived neurotrophic factor (MANF) expression offers protection against alcohol neurotoxicity.

Alcohol exposure has detrimental effects on both the developing and mature brain. Endoplasmic reticulum (ER) stress is one of the mechanisms that contributes to alcohol-induced neuronal damages. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-responsive protein and is neuroprotective in multiple neuronal injury and neurodegenerative disease models. MANF deficiency has been shown to exacerbate alcohol-induced ER stress and neurodegeneration. However, it is unknown whether MANF supplement is sufficient to protect against alcohol neurotoxicity. Alcohol alters MANF expression in the brain, but the mechanisms underlying alcohol modulation of MANF expression remain unclear. This study was designed to determine how alcohol alters MANF expression in neuronal cells and whether exogeneous MANF can alleviate alcohol neurotoxicity. We showed that alcohol increased MANF transcription and secretion without affecting MANF mRNA stability and protein degradation. ER stress was necessary for alcohol-induced MANF upregulation, as pharmacological inhibition of ER stress by 4-PBA diminished alcohol-induced MANF expression. In addition, the presence of ER stress response element II (ERSE-II) was required for alcohol-stimulated MANF transcription. Mutations or deletion of this sequence abolished alcohol-regulated transcriptional activity. We generated MANF knockout (KO) neuronal cells using CRISPR/Cas9. MANF KO cells exhibited increased unfolded protein response (UPR) and were more susceptible to alcohol-induced cell death. On the other hand, MANF upregulation by the addition of recombinant MANF protein or adenovirus gene transduction protected neuronal cells against alcohol-induced cell death. Further studies using early postnatal mouse pups demonstrated that enhanced MANF expression in the brain by intracerebroventricular (ICV) injection of MANF adeno-associated viruses ameliorated alcohol-induced cell death. Thus, alcohol increased MANF expression through inducing ER stress, which could be a protective response. Exogenous MANF was able to protect against alcohol-induced neurodegeneration.

Ubiquitin-like protein 5 is a novel player in the UPR–PERK arm and ER stress–induced cell death

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells invivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

CARD6: Warm Ischemic Time, Cold Static Storage and Impairment of Cardiac Endoplasmic Reticular Stress in Human Hearts Donated After Circulatory Death and Brain Death

Background: Endoplasmic reticulum (ER) plays an important role in maintaining normal myocardial function at the cellular level. Overexpression of ER stress-associated proteins during ischemia impact cardiac function. Initial result of heart transplantation using donation after circulatory death (DCD) heart with short warm ischemic time (WIT, less than 30 minutes) was encouraging. However, little is known about the impact of various WIT on donor heart and with subsequent cold storage as used in normothermic regional perfusion(NRP) on the DCD hearts. In this study we investigated expression of myocardial ER stress response proteins in the DCD hearts to evaluate the impact of warm ischemic time (WIT) and subsequent cold storage. We also studied expression of myocardial ER stress response proteins in the brain death donor (DBD) hearts as a control group. Methods: A total of 23 human hearts, DCD group, n=17, with 3 WIT sub-groups [WIT 20 (n=6), 40 (n=6), ≥60 (n=5) minutes] and DBD group, n=6, no warm ischemia (control: WIT:0) were studied. All the DCD hearts received del Nido cardioplegia and all DBD hearts received Celsior solution before they were placed at cold storage for 6 hours. Left ventricular biopsies were performed at hour 0 (T0), 2 (T2), 4 (T4) and 6 (T6). Myocardial ER stress response proteins ATF6 (Activating Transcription Factor 6) and GRP78 (78-kDa glucose-regulated protein) were measured through immunoblotting after normalizing to GAPDH loading control in all cardiac biopsies. Results: Representative western blots showing the protein expressions patterns among the DBD and three different groups of DCD hearts under increasing time points (T0:0hrs, T2:2hrs, T4:4hrs, T6:6hrs) of cold static storage conditions (Fig.1A). At baseline, the proteins expression found to be comparable between DBD, DCD:20 and 40 groups, while DCD:≥60 group remained significantly higher (Fig.1B,C). We noticed differential temporal increase of ER stress proteins after 2 hours of cold storage and became prominent at hour 6 in DCD groups when compared to DBD group; while the DCD:60 group always remained highly significant throughout the cold storage compared to all other groups (Fig.1D,E). Conclusions: Our study concluded that duration of warm ischemia and subsequent cold storage condition in DCD hearts were associated with elevated ER stress response in myocardium. The length of cold storage was also found to be linked with elevated ER stress in DCD hearts. The DCD hearts with more than 60 min warm ischemic has the highest ES stress response suggesting profound alteration in ER homeostasis which may promote cardiac damage. Such findings are important in guiding clinical decision making on selecting DCD donor hearts. On the other hand the DBD hearts with up to 6 hour cold storage exerted minimal ER stress, supporting current DBD heart transplant donor preservation practice.

Altered MANF Expression in Pancreatic Acinar and Ductal Cells in Chronic Alcoholic Pancreatitis: A Cross-Sectional Study

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress response protein that plays an important role in pancreatic functions. As both alcohol and ER stress response proteins are involved in the pathogenesis of pancreatitis, we sought to investigate the expression of MANF in chronic alcoholic pancreatitis (CAP) and chronic non-alcoholic pancreatitis (CNP). A cohort of chronic pancreatitis tissues was gathered from routine surgical pathology (n = 77) and autopsy (n = 10) cases and tissue microarrays were created. Sampled tissues were reviewed and designated as representing CAP (n = 15), CNP (n = 58), or normal pancreatic tissue (NPT) (n = 27). MANF immunohistochemistry (IHC) and digital image analysis were performed to obtain an estimation of tissue fibrosis and an optical density (OD) of MANF IHC in ducts and acini for each case. The averaged values for these variables among histologic designations were compared. The amount of fibrous tissue of the combined CAP and CNP group (chronic alcoholic and non-alcoholic pancreatitis, CANP) exceeded that of the NPT group (70% vs. 34%, p < 0.0001). The MANF OD in ducts of CANP was significantly higher than that of NPT (0.19 vs. 0.10, p < 0.05). The MANF OD in ducts of CAP was significantly higher than that of CNP (0.27 vs. 0.17, p < 0.05). The MANF OD in acini of CAP was significantly lower than that in CNP (0.81 vs. 1.05, p < 0.05). Finally, there was a statistically significant positive relationship between the amount of fibrosis and MANF OD in ducts (p < 0.001). MANF expression was higher in ducts of CAP than CNP. In contrast, MANF expression in acini was lower in CAP than CNP and NPT. There was a positive correlation between fibrosis and MANF levels in the ducts.

Electroacupuncture stimulation of HT7 alleviates sleep disruption following acute caffeine exposure by regulating BDNF-mediated endoplasmic reticulum stress in the rat medial septum

Acupuncture stimulation can protect the brain against caffeine-induced sleep disruption. This study investigated whether electroacupuncture stimulation acupuncture point HT7 alleviates sleep disruption by regulating mBDNF and ER stress in the medial septum. Acute exposure to caffeine (15 mg/kg, i.p.) increased the wake time and decreased REM sleep, which HT7 stimulation alleviated. HT7 stimulation ameliorated the acute caffeine exposure-induced increase in the expression of BiP, an endoplasmic reticulum stress response protein, in the rat medial septum. Interestingly, HT7 stimulation induced the expression of mBDNF and pTrkB in the medial septum. The next experiment investigated whether TrkB phosphorylated by HT7 stimulation induced BiP expression in the rat medial septum. Before electroacupuncture stimulation at HT7, ANA-12 was administered to caffeine-treated rats. In rats administered ANA-12 in the medial septum, HT7 stimulation did not reduce BiP expression. These findings suggest that HT7 stimulation improves wake time and REM sleep dysfunction by regulating the BDNF-mediated endoplasmic reticulum stress response in the medial septum. These results indicate that the alleviation of endoplasmic reticulum stress in the medial septum by HT7 stimulation and the subsequent amelioration of insomnia may depend on phosphorylated TrkB activation.

Chrysin protects cardiac H9c2 cells against H2O2-induced endoplasmic reticulum stress by up-regulating the Nrf2/PERK pathway.

Oxidative and endoplasmic reticulum (ER) stress-mediated cardiac apoptosis is an essential pathological process in cardiovascular diseases (CVDs). Chrysin (Chy) is a natural flavonoid that exerts several health benefits, particularly anti-oxidative and anti-apoptotic effects. However, its protective effect against CVDs and its mechanism of action at a molecular level remains unclear. Therefore, the present study aimed to investigate the interaction of ER stress response protein with Chy by computational analysis and molecular action in H2O2-induced oxidative and ER stress in cardiomyoblast cells. H9c2 cells were pre-treated with 50μM of Chy for 24h and exposed to H2O2 for 1h. Explore the Chy-mediated Nrf2 signalling on ER stress reduction, H9c2 cell lines were transfected with Nrf2 siRNA for 48h and further treated with Chy for 24h and subjected to H2O2 for 1h. Chy pre-treatment increased the Nrf2-regulated gene expression, reduced the ER stress signalling genes such as CHOP and GRP78, and increased the PERK and AFT6 expression compared to H2O2-treated cells. Chy preincubation down-regulated the expression of PI3K, NF-κB, and caspase-3. Fluorescence staining revealed that Chy reduced intracellular ROS generation, ER stress, apoptosis, and increased MMP. This beneficial effect of Chy was abolished when silencing Nrf2 in H9c2 cells. Overall, the present study confirmed that Chy showed the cardioprotective effect by attenuating ER stress via the activation of Nrf2 signalling. Therefore, the study concluded that improving Nrf2 signalling by Chy supplementation could provide a promising therapeutic target in oxidative and ER stress-mediated CVDs complications.

Panaxnotoginseng extract and total saponin suppress diet-induced obesity and endoplasmic reticulum stress in epididymal white adipose tissue in mice

BackgroundInvestigation on protective effects of Panax notoginseng against obesity and its related mechanisms is incomplete. Present study aimed to investigate the potential anti-obesity effect of the total saponins (PNS) and ethanolic extract of P. notoginseng (PNE).MethodsSix-week-old male C57BL/6J mice received 45% kcal fat diet for 12 weeks to induce obesity. Oral administration of PNS and PNE at 20 mg/kg/day was applied for the last 4 weeks in the obese mice. Lipid profile was determined by ELISA. Histological examination was performed in liver and fat tissues. Protein levels were measured by Western blot.ResultsPNS and PNE did not cause weight loss. PNE but not PNS decreased the mass of epididymal and retroperitoneal white adipose tissue, accompanied by a reduction in adipocyte hypertrophy. PNS and PNE improved lipid profile by reducing the concentrations of triglyceride, total cholesterol and low-density lipoprotein cholesterol in plasma or liver samples. PNS and PNE also relieved fatty liver in obese mice. PNS and PNE inhibited expression and phosphorylation of endoplasmic reticulum (ER) stress-responsive proteins in hypertrophic adipose tissue.ConclusionsPNS and PNE can regulate ER stress-mediated apoptosis and inflammation to alleviate obesity.

Ganoderic acid A ameliorates non-alcoholic streatohepatitis (NASH) induced by high-fat high-cholesterol diet in mice.

Non-alcoholic steatohepatitis (NASH) is becoming a huge global health problem. Previous studies have revealed that ganoderic acids have hepatoprotective and hypocholesterolemic effects. In the present study, to evaluate the anti-NASH activity of ganoderic acid A (GAA), male 6-week-old C57BL/6J mice were divided into the following four groups, which were administered different diets: Normal diet (ND group), high-fat high-cholesterol diet (HFHC group), HFHC diet supplemented with 25 mg/kg/day (GAAL group) or 50 mg/kg/day of GAA (GAAH group). After 12 weeks of GAA treatment, histopathological results revealed that compared with that of the HFHC group, GAA significantly inhibited fat accumulation, steatosis, inflammation and fibrosis in the liver. GAA effectively reduced serum aspartate transaminase and alanine transaminase levels compared with the HFHC model. Furthermore, the endoplasmic reticulum (ER) stress-responsive proteins, including glucose-regulated protein 78, phosphorylated (p)-eukaryotic initiation factor-2α and p-JNK, were significantly suppressed by GAA, while ERp57, p-MAPK and p-AKT were significantly increased after GAA treatment. Taken together, it was concluded that GAA could resist HFHC diet-induced NASH. In terms of its underlying mechanism, GAA could improve liver inflammation and fibrosis by inhibiting hepatic oxidative stress and the ER stress response induced by HFHC.

Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER\u2013mitochondria crosstalk in SiHa cells

BackgroundTrichomonas vaginalis causes lesions on the cervicovaginal mucosa in women; however, its pathogenesis remains unclear. We have investigated the involvement of the endoplasmic reticulum (ER) in the induction of apoptosis by T. vaginalis and its molecular mechanisms in human cervical cancer SiHa cells.MethodsApoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response and Bcl-2 family protein expression were evaluated using immunocytochemistry, flow cytometry, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide dye staining and western blotting.ResultsTrichomonas vaginalis induced mitochondrial ROS production, apoptosis, the ER stress response and mitochondrial dysfunction, such as MMP depolarization and an imbalance in Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA; ER stress inhibitor) significantly alleviated apoptosis, mitochondrial ROS production, mitochondrial dysfunction and ER stress response in a dose-dependent manner. In addition, T. vaginalis induced the phosphorylation of apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinases (JNK) in SiHa cells, whereas 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis and ER stress response in SiHa cells, in a dose-dependent manner. Furthermore, T. vaginalis excretory/secretory products also induced mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells, in a time-dependent manner.ConclusionsTrichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress responses, and also promotes ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells. These data suggest that T. vaginalis-induced apoptosis is affected by ROS and ER stress response via ER–mitochondria crosstalk.Graphical

Dysregulation of the miR-30a/BiP axis by cigarette smoking accelerates oral cancer progression

BackgroundCigarette smoking is the most significant cause of oral cancer progression. Cigarette smoke condensate (CSC) has been shown to induce endoplasmic reticulum (ER) stress. Binding immunoglobulin protein (BiP) being as an ER stress regulator, has been reported to be implicated in malignant behaviors. Therefore, the aim of this study was to investigate the role of the ER stress-responsive protein, BiP, in CSC-induced oral squamous cell carcinoma (OSCC) malignancy.MethodsThe biological role of BiP in CSC-induced tumor progression was investigated in OSCC cells (YD38 and SCC25) and in a tumor xenograft mouse model. The expressions of related genes were investigated using quantitative RT-PCR and Western blot analysis. Cell migration and invasion were assessed using scratch wound healing and Transwell invasion assays. The effects of conditioned media from OSCC cells on the angiogenic activities of endothelial cells were analyzed using a tube formation assay. The interaction between miR-30a and BiP mRNA was detected using a luciferase reporter assay.ResultsOur results demonstrated that CSC increased the expression of BiP in time- and dose-dependent manners in YD38 and SCC25 cells, and that silencing BiP abrogated CSC-induced cell invasion and tumor-associated angiogenesis. Notably, the putative miR-30a binding site was observed in the 3′untranslated region (UTR) of BiP mRNA, and miR-30a suppressed BiP expression by targeting 3′UTR of BiP transcript. In addition, CSC increased the expression of BiP in OSCC cells by downregulating miR-30a. We also showed that BiP promoted invasion and tumor-associated angiogenesis by increasing the production and secretion of vascular endothelial growth factor in CSC-exposed OSCC cells. Moreover, BiP inhibition suppressed OSCC growth and reduced tumor vessel density in tumor-bearing mice administered with CSC.ConclusionsThese observations suggest that epigenetic regulation of BiP via miR-30a downregulation is involved in CSC-induced OSCC progression.

Primary Osteoarthritis Early Joint Degeneration Induced by Endoplasmic Reticulum Stress Is Mitigated by Resveratrol

Increasing numbers of people are living with osteoarthritis (OA) due to aging and obesity, creating an urgent need for effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with endoplasmic reticulum (ER) stress. The I-ERS mouse, an ER stress-driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. In this study, ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block, and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP) largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation, and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1β, MMP-13, pS6, number of TUNEL-positive chondrocytes, and senescence marker p16 INK4a. The finding, that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA.

Downregulation of XBP1 protects kidney against ischemia-reperfusion injury via suppressing HRD1-mediated NRF2 ubiquitylation

Ischemia-reperfusion (IR) injury to the renal epithelia is associated with endoplasmic reticulum stress (ERS) and mitochondria dysfunction, which lead to oxidative stress-induced acute kidney injury (AKI). X-box binding protein 1 (XBP1), an ERS response protein, could play a prominent role in IR-induced AKI. In this study, we revealed that XBP1 and its downstream target HRD1 participated in the crosstalk between ERS and mitochondrial dysfunction via regulation of NRF2/HO-1-mediated reactive oxidative stress (ROS) signaling. Mice with reduced expression of XBP1 (heterozygous Xbp1±) were resistant to IR-induced AKI due to the enhanced expression of NRF2/HO-1 and diminished ROS in the kidney. Downregulation of XBP1 in renal epithelial cells resulted in reduced HRD1 expression and increased NRF2/HO-1 function, accompanied with enhanced antioxidant response. Furthermore, HRD1 served as an E3-ligase to facilitate the downregulation of NRF2 through ubiquitination-degradation pathway, and the QSLVPDI motif on NRF2 constituted an active site for its interaction with HRD1. Thus, our findings unveil an important physiological role for XBP1/HRD1 in modulating the antioxidant function of NRF2/HO-1 in the kidney under stress conditions. Molecular therapeutic approaches that target XBP1-HRD1-NRF2 pathway may represent potential effective means to treat renal IR injury.

Glucose-Regulated Protein 94 Mediates the Proliferation and Metastasis through the Regulation of ETV1 and MAPK Pathway in Colorectal Cancer

Colorectal cancer (CRC) is a worldwide health problem. Glucose-regulated protein 94 (GRP94) is known as an important endoplasmic reticulum-stress response protein that shows correlation with aggressive cancer behavior. However, the role of GRP94 in CRC is still unclear. Our results showed that silencing GRP94 (GRP94-KD) reduced cell proliferation, invasion and migration of CRC cells and suppressed tumorigenesis in the xenograft mouse model. Rescue assay showed that ETV1 overexpression reversed the effect of GRP94 on cell proliferation and migration. In the molecular mechanism, we found that knockdown of GRP94 inhibited the level of MAPK pathway, including ERK/p-ERK, JNK/p-JNK, and p38/p-p38 signals. Cyclooxygenase-2 and epithelial-mesenchymal transformation biomarkers, such as N-cadherin, vimentin, and β-catenin were suppressed in GRP94 knockdown cells. Treatment of specific inhibitors of MAPK pathway showed that ERK/p-ERK, and p38/p-p38 inhibitors significantly influenced ETV1 expression as compared to JNK/p-JNK inhibitor. Our results indicated that silencing GRP94 repressed the ability of EMT process, cancer cell proliferation, metastasis, and CRC tumorigenesis. Therefore, GRP94 may play an important role in CRC by regulating ETV1 and MAPK pathway.

Acute high-intensity aerobic exercise increases gene expression of calcium-related proteins and activates endoplasmic reticulum stress responses in diabetic hearts

Regular aerobic exercise training has a wide range of beneficial cardiac effects, but recent data also show that acute very strenuous aerobic exercise may impose a transient cardiac exhaustion. The aim of this study was to assess the response to acute high-intensity aerobic exercise on properties of mitochondrial respiration, cardiomyocyte contractile function, Ca2+ handling and transcriptional changes for key proteins facilitating Ca2+ handling and endoplasmic reticulum (ER) stress responses in type 2 diabetic mice. Diabetic mice were assigned to either sedentary control or an acute bout of exercise, consisting of a 10×4 minutes high-intensity interval treadmill run. Mitochondrial respiration, contractile and Ca2+ handling properties of cardiomyocytes were analysed 1 hour after completion of exercise. Gene expression levels of key Ca2+ handling and ER stress response proteins were measured in cardiac tissue samples harvested 1 hour and 24 hours after exercise. We found no significant changes in mitochondrial respiration, cardiomyocyte contractile function or Ca2+ handling 1 hour after the acute exercise. However, gene expression of Atp2a2, Slc8a1 and Ryr2, encoding proteins involved in cardiomyocyte Ca2+ handling, were all significantly upregulated 24 hours after the acute exercise bout. Acute exercise also altered gene expression of several key proteins in ER stress response and unfolded protein response, including Grp94, total Xbp1, Gadd34, and Atf6. The present results show that despite no significant alterations in functional properties of cardiomyocyte function, Ca2+ handling or mitochondrial respiration following one bout of high intensity aerobic exercise training, the expression of genes involved in Ca2+ handling and key components in ER stress and the unfolded protein response were changed. These transcriptional changes may constitute important steps in initiating adaptive remodelling to exercise training in type 2 diabetes.

Exposure to etoxazole induces mitochondria-mediated apoptosis in porcine trophectoderm and uterine luminal epithelial cells

Etoxazole is an organofluorine insecticide widely used in agriculture. Exposure to insecticides is a serious environmental problem owing to their cytotoxic effects in humans and animals. Reproductive toxicity of various organofluorine insecticides have been shown in previous studies. However, few studies have evaluated the toxicity of etoxazole in mammals. We aimed to examine the toxic effects of etoxazole in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. To estimate the effects of etoxazole, we conducted assays after treatment with multiple concentration of etoxazole (0, 2, 4, 6 and 9 μM) to pTr and pLE cells for 0–72 h. Etoxazole decreased the cell proliferation, viability, and migration of pTr and pLE cells. Further, etoxazole induced apoptosis via cell cycle arrest and disruption of mitochondrial membrane potential. We also found that pro-apoptotic proteins and endoplasmic reticulum (ER) stress-response proteins were activated in response to etoxazole. Finally, we observed that etoxazole altered the PI3K/AKT and MAPK signaling pathways and the mRNA expression of genes associated with implantation. Collectively, these results suggest that etoxazole disrupts normal cellular physiology and might cause early implantation failure.

Protective effects of UFL1 against endoplasmic reticulum stress-induced autophagy in bovine mammary epithelial cells.

Ubiquitin-fold modifier 1 (UFM1)-specific ligase 1 (UFL1) is an important component of the UFM1 conjugation system, which is required for various cellular processes including protein translation, apoptosis, autophagy, and signal transduction. However, both, the expression of UFL1 in mammary cells and its role in endoplasmic reticulum (ER) stress in bovine mammary epithelial cells (BMECs) remain to be fully elucidated. Here, we characterized the potential roles of UFL1 in BMECs. Amino acid sequence comparison indicated that bovine UFL1 shares a high level of sequence identity with the UFL1 of other ruminant species. Notably, UFL1 expression in BMECs was increased by endoplasmic reticulum (ER) stress induced by treatment with tunicamycin (TM). ER stress-related gene expression was further increased in UFL1 knockdown cells upon TM treatment. Moreover, UFL1 overexpression inhibited TM-stimulated ER stress and alleviated ER stress-induced autophagy. Together, our results indicated that UFL1 is a novel ER stress-responsive protein in BMECs. Thus, our study provides a basis for further research into ER stress-related processes in bovine mammary tissues and potential targets for alleviating ER stress in these cells.

Cross-talk between ER stress and mitochondrial pathway mediated adriamycin-induced testicular toxicity and DA-9401 modulate adriamycin-induced apoptosis in Sprague\u2013Dawley rats

BackgroundDA-9401 was prepared as a mixture of Chinese medicinal herb extracts from roots of Morinda officinalis How (Rubiaceae), outer scales of Allium cepa L. (Liliceae) and seeds of Cuscuta chinensis Lamark (Convolvulaceae). The present study was designed to investigate the possible protective role of DA-9401 in adriamycin (ADR)-induced testicular toxicity associated with oxidative stress, endoplasmic reticulum (ER) stress, and apoptosis.MethodsFifty healthy 8-week-old male Sprague–Dawley rats were equally divided into five groups. The first CTR group was treated with normal saline 2 ml/day by gavage. The second was treated with DA-100 (DA-9401 100 mg/kg/day). The third (ADR) group received ADR (2 mg/kg/once a week) intraperitoneally, while the combination of ADR and DA-9401 was given to the fourth ADR + DA-100 (100 mg/kg/day p.o) group and fifth ADR + DA-200 (200 mg/kg/day p.o) group. At the end of the 8-week treatment period, body weight, reproductive organ weights, fertility rate, pups per female were recorded, and serum were assayed for hormone concentrations. Tissues were subjected to semen analysis, histopathological changes, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), oxidative stress markers and expression levels of endoplasmic reticulum (ER) stress markers, apoptosis markers, tight junction protein markers, steroidogenic acute regulatory protein (StAR), cation channel of sperm (CatSper) and glycogen synthase kinase-3 (GSK-3) by western blot.ResultsDA-9401 administration to ADR-treated rats significantly decreased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, interleukin-6, TNF-α, MDA level, ROS/RNS level, ER stress response protein levels, tunnel positive cells, cleaved caspase-3, and Bax/Bcl2 ratio. Moreover, pretreatment with DA-9401 significantly increased body weight, reproductive organ weights, fertility rate, pups per female, Johnsen’s score, spermatogenic cell density, sperm count and sperm motility, serum testosterone concentration, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), tight junction protein markers, star protein level, CatSper, and GSK-3 level.ConclusionsADR treatment can markedly impair testicular function and induce testicular cell death presumably by causing significant changes in oxidative stress, ER stress, and mitochondrial pathway. DA-9401 exerts beneficial effects against oxidative stress, ER stress, and mitochondria-mediated cell death pathway in testis tissue by up-regulating expression levels of tight junction protein markers, steroidogenic acute regulatory protein, GSK-3 alpha, and cation channels of sperm.