Increased ER Stress Research Articles

In silico analysis reveals PRDX4 as a prognostic and oncogenic marker in renal papillary cell carcinoma

BackgroundAlterations in the tumor microenvironment leads to the accumulation of reactive oxygen species (ROS). When in low levels, ROS act as a signaling molecule and contribute to tumor cell proliferation whereas its elevation results in oxidative stress and eventually cell death. It is known that antioxidant systems regulate the ROS levels and thus cell fate. Among these systems, peroxiredoxins (PRDXs) were found to be upregulated in various cancers. However their exact contribution to carcinogenesis is not yet clear. AimHerein, the expression pattern and prognostic value of PRDXs were explored in cancer setting by using in silico analysis tools and publicly available datasets. ResultsPan-cancer analysis revealed that PRDXs are differentially expressed in normal and tumor tissues. Further analysis showed that higher PRDX4 levels was associated with poor prognosis and clinicopathological and histological features associated with a more aggressive renal papillary cell carcinoma (KIRP) profile. Hypoxia, ER stress and protein folding were shown to be pathways positively correlated with PRDX4 levels. Furthermore, PRDX4 was found to be strong regulator of protein homeostasis. Kaplan-Meier analysis revealed that PRDX4 is a potent prognostic marker in Type 2 KIRP and this might be due to increased ER stress and oxidative stress levels in this subtype. ConclusionsThe data suggest that PRDX4 can be used as a prognostic marker for KIRP patients. Its association with more aggressive tumor characteristics also underlines that it might be used for targeted therapy.

Overexpression of Pdx1, reduction of p53, or deletion of CHOP attenuates pancreas hypoplasia in mice with pancreas-specific O-GlcNAc transferase deletion

Deletion of O-GlcNAc transferase (Ogt) in pancreatic epithelial progenitor cells results in pancreatic hypoplasia at birth, partly due to increased apoptosis during embryonic development. Constitutive loss of Ogt in β-cells results in increased ER stress and apoptosis, and in the Ogt-deficient pancreas, transcriptomic data previously revealed both tumor suppressor protein p53 and pancreatic duodenal homeobox 1 (Pdx1), key cell survival proteins in the developing pancreas, as upstream regulators of differentially expressed genes. However, the specific roles of these genes in pancreatic hypoplasia are unclear. In this study, we explored the independent roles of p53, ER stress protein CHOP, and Pdx1 in pancreas development and their use in the functional rescue of pancreatic hypoplasia in the context of Ogt loss. Using invivo genetic manipulation and morphometric analysis, we show that Ogt plays a key regulatory role in pancreas development. Heterozygous, but not homozygous, loss of pancreatic p53 afforded a partial rescue of β-cell, α-cell, and exocrine cell masses, while whole body loss of CHOP afforded a partial rescue in pancreas weight and a full rescue in exocrine cell mass. However, neitherwas sufficient to fully mitigate pancreatic hypoplasia at birth in the Ogt-deficient pancreas. Furthermore, overexpression of Pdx1 in the pancreatic epithelium resulted in partial rescues in pancreas weight and β-cell mass in the Ogt loss background. These findings highlight the requirement of Ogt in pancreas development by targeting multiple proteins such as transcription factor Pdx1 and p53 in the developing pancreas.

Copa-1 mutants experience heightened endoplasmic reticulum stress sensitivity in a C. elegans COPA Syndrome model.

COPA Syndrome is a rare, autosomal dominant autoimmune/autoinflammatory disease caused by mutations in COPA , which codes for the alpha subunit of the Coat Protein Complex I (COPI). COPI coated vesicles move proteins in retrograde from the Golgi Apparatus to the Endoplasmic Reticulum. At the cellular level, COPA mutations cause ER stress, though the downstream genetic mechanisms of COPA Syndrome remain undefined. Here, we model COPA Syndrome in Caenorhabditis elegans , using CRISPR/Cas9 to generate patient alleles in copa-1 , the C. elegans COPA ortholog. The two alleles made thus far are superficially wild type under normal growth conditions. However, these animals demonstrate an increased ER stress sensitivity.

Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine β-coronavirus host cell response

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine β-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where β-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

Polystyrene microplastic particles induce autophagic cell death in BEAS‐2B human bronchial epithelial cells

The detection of high levels of microplastics in indoor and outdoor air has increased concerns regarding its toxic effects on the respiratory system. They are not easily degradable and can be deposited deep in the lungs. Although several studies have reported inhalation toxicities of microplastics, they are still controversial due to a lack of evidence. Herein, we evaluated the inhalation toxicities of three differently charged polystyrene microplastics (PS-MPs), the most abundant microplastics in the air. Cytotoxicity and ROS generation were evaluated using WST-1 and DCF-DA assays, respectively. To evaluate the toxic effects on the lung, inflammatory responses were analyzed after repeated exposure to the PS-MPs through intratracheal instillation. To explore the mechanism of toxicity, autophagy and ER stress-associated proteins were analyzed. Only the positively charged PS-MPs (NH2 -PS-MPs) showed cytotoxicity and increased ROS generation in BEAS-2B cells. Similarly, only NH2 -PS-MPs significantly increased the expression and secretion of the pro-inflammatory cytokine IL-β in the animal experiments. The expression of ER stress proteins indicated that NH2 -PS-MPs increased ER stress via PERK-EIF2α and ATF4-CHOP pathways. Moreover, accumulation of NH2 -PS-MPs in lysosomes and deformity of the nucleus were observed in BEAS-2B cells with autophagy induction. Taken together, our results demonstrated that NH2 -PS-MPs induced autophagic cell death in bronchial epithelial cells, leading to inflammatory responses in the lungs. These results suggest that repeated inhalation of microplastics can result in inflammatory responses in the lung through cellular damage of lung epithelial cells, and that inhalation microplastics should be monitored to reduce inhalation health risks.

PERK/EIF2AK3 integrates endoplasmic reticulum stress-induced apoptosis, oxidative stress and autophagy responses in immortalised retinal pigment epithelial cells

Retinal pigment epithelium (RPE) performs essential functions for ensuring retinal homeostasis and is a key site for pathogenic changes leading to age-related macular degeneration (AMD). Compromised proteostasis in RPE results in ER stress and ER stress-dependent antioxidant, apoptosis and autophagic responses. ER stress induces the unfolded protein response (UPR) in which EIF2AK3, encoding the protein kinase RNA-like ER kinase (PERK), acts as a key regulator. Downregulated EIF2AK3 gene expression has recently been identified in AMD using human donor RPE, however the molecular mechanisms that integrate the various ER-mediated cellular pathways underpinning progressive RPE dysfunction in AMD have not been fully characterised. This study investigated the downstream effects of PERK downregulation in response to Brefeldin A (BFA)-induced ER stress in ARPE-19 cells. PERK downregulation resulted in increased ER stress and impaired apoptosis induction, antioxidant responses and autophagic flux. ARPE-19 cells were unable to efficiently induce autophagy following PERK downregulation and PERK presented a role in regulating the rate of autophagy induction. The findings support PERK downregulation as an integrative event facilitating dysregulation of RPE processes critical to cell survival known to contribute to AMD development and highlight PERK as a potential future therapeutic target for AMD.

Apoptosis Induction Associated with Enhanced ER Stress Response and Up-Regulation of c-Jun/p38 MAPK Proteins in Human Cervical Cancer Cells by Colocasia esculenta var. aquatilis Hassk Extract

Colocasia esculenta var. Aquatilis Hassk, elephant ear (CF-EE) has been widely used as traditional food and medicine. It also shows other therapeutic properties, such as antimicrobial and anti-cancer activity. In this study, we aim to investigate the effect of CF-EE extract on apoptosis induction associated with ER stress in cervical cancer HeLa cells. Cell viability was determined by MTT assay. Assessments of nuclear morphological changes, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) production were conducted by hoeshst33342, JC-1, and DCFH-DA fluorescence staining, respectively. Sub-G1 DNA content was analyzed by flow cytometry, and protein expression was determined by Western blotting. The results demonstrate that CF-EE extract suppressed HeLa cell growth and induced nuclear condensation and apoptotic bodies. There was also a loss of mitochondrial membrane potential and increased apoptosis marker protein expression, including Bax, cleaved-caspase-7, and cleaved-PARP. In addition, the results show that CF-EE extract induced ROS, increased ER stress proteins (GRP78 and CHOP), enhanced p38 and c-Jun phosphorylation, and inhibited Akt expression in HeLa cells. In summary, CF-EE extract induced apoptotic cell death-associated ROS-induced ER stress and the MAPK/AKT signaling pathway. Therefore, CF-EE extract has anticancer therapeutic potential for cervical cancer treatment in the future.

1455-P: The Loss of Pancreatic ß-Cell Complement 1q-Like-3 Secreted Protein Increases Insulin Secretion in Lean and Obese Mice

Previously, we identified complement-1q like-3 (C1ql3) secreted protein as a hub gene affecting pancreatic islet function in obese mice using network biology. We demonstrated that the recombinant C1ql3 protein inhibits insulin secretion stimulated by exendin-4 (an agonist for GLP-1 receptor) from human and mouse islets. Our goal is to investigate the role of β-cell C1ql3 in pancreatic β-cell function in physiological and pathophysiological states. We generated mice with β-cell-specific deletion of C1ql3 (βKO) by crossing C1ql3fl/fl and RIP-Cre+ mice. C1ql3 βKO mice showed significantly improved glucose clearance after oral glucose challenge than control mice, whereas no difference in insulin action was observed. Moreover, the plasma insulin levels were significantly increased at 15 min after the glucose challenge in the C1ql3 βKO vs. control mice. C1ql3 βKO islets significantly increased insulin secretion stimulated by cAMP and Exendin-4 than control islets. These results suggest that the loss of C1ql3 in β-cells increases insulin secretion to improve glucose tolerance. Obesity increases the expression-coupled secretion of C1ql3 from mouse islets. Adenovirus mediated overexpression of C1ql3 in islets showed increased ER stress - a key trigger for β-cell dysfunction in obesity- as well as decreased expression of genes involving β-cell function and maintenance. Interestingly, reduction in C1ql3 secretion is inversely correlated with increased glucose stimulated insulin secretion from islets of C1ql3 βKO Leptinob/ob compared to Leptinob/ob control mice. These findings identify that the loss of β-cell C1ql3 maintains ER stress to compensate β-cell workload during obesity and increases insulin secretion to maintain glucose homeostasis. Our work shows that an autocrine regulation of the C1ql3-signaling pathway contributes to β-cell dysfunction in obesity and type 2 diabetes. Disclosure M.Rahman: None. H.Alsharif: n/a. S.Bhatnagar: None. Funding American Diabetes Association (1-18-PDF-103) ; 1R01DK120684-01

Rhinacanthin-C but Not -D Extracted from Rhinacanthus nasutus (L.) Kurz Offers Neuroprotection via ERK, CHOP, and LC3B Pathways.

Neurodegenerative diseases present an increasing problem as the world’s population ages; thus, the discovery of new drugs that prevent diseases such as Alzheimer’s, and Parkinson’s diseases are vital. In this study, Rhinacanthin-C and -D were isolated from Rhinacanthus nasustus, using ethyl acetate, followed by chromatography to isolate Rhinacanthin-C and -D. Both compounds were confirmed using NMR and ultra-performance-LCMS. Using glutamate toxicity in HT-22 cells, we measured cell viability and apoptosis, ROS build-up, and investigated signaling pathways. We show that Rhinacanthin-C and 2-hydroxy-1,4-naphthoquinone have neuroprotective effects against glutamate-induced apoptosis in HT-22 cells. Furthermore, we see that Rhinacanthin-C resulted in autophagy inhibition and increased ER stress. In contrast, low concentrations of Rhinacanthin-C and 2-hydroxy-1,4-naphthoquinone prevented ER stress and CHOP expression. All concentrations of Rhinacanthin-C prevented ROS production and ERK1/2 phosphorylation. We conclude that, while autophagy is present in HT-22 cells subjected to glutamate toxicity, its inhibition is not necessary for cryoprotection.

ERAP1 is a critical regulator of inflammasome-mediated proinflammatory and ER stress responses

BackgroundIn addition to its role in antigen presentation, recent reports establish a new role for endoplasmic reticulum aminopeptidase 1 (ERAP1) in innate immunity; however, the mechanisms underlying these functions are not fully defined. We previously confirmed that loss of ERAP1 functions resulted in exaggerated innate immune responses in a murine in vivo model. Here, we investigated the role of ERAP1 in suppressing inflammasome pathways and their dependence on ER stress responses.ResultsUsing bone marrow-derived macrophages (BMDMs), we found that loss of ERAP1 in macrophages resulted in exaggerated production of IL-1β and IL-18 and augmented caspase-1 activity, relative to wild type macrophages. Moreover, an in vivo colitis model utilizing dextran sodium sulfate (DSS) confirmed increased levels of proinflammatory cytokines and chemokines in the colon of DSS treated ERAP1−/− mice as compared to identically stimulated WT mice. Interestingly, stimulated ERAP1−/− BMDMs and CD4+ T cells simultaneously demonstrated exaggerated ER stress, assessed by increased expression of ER stress-associated genes, a state that could be reverted to WT levels with use of the ER stress inhibitor Tauroursodeoxycholic acid (TUDCA).ConclusionsTogether, these results not only suggest that ERAP1 is important for regulating inflammasome dependent innate immune response pathways in vivo, but also propose a mechanism that underlies these changes, that may be associated with increased ER stress due to lack of normal ERAP1 functions.

Targeting histone deacetylase-3 blocked epithelial-mesenchymal plasticity and metastatic dissemination in gastric cancer

Background and purposeHistone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression.Experimental approachClinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/β-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography.Key resultsHDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPβ signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPβ. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPβ activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/β-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo.Conclusions and implicationsHonokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked.Graphical abstractConceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress–activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination.• Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPβ signaling.• HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol.• Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

Targeting ER protein TXNDC5 in hepatic stellate cell mitigates liver fibrosis by repressing non-canonical TGFβ signalling

Background and objectivesLiver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide...

Multiomic analysis on human cell model of wolfram syndrome reveals changes in mitochondrial morphology and function

BackgroundWolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress.MethodsWe performed transcriptomic and proteomic analysis on WFS human cell model—skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency.ResultsProteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated.ConclusionsOur results show the functional and morphological secondary mitochondrial damage in patients with WFS.Graphical EQy9i2UA_HE4ksLyXi_ZKvVideo

Investigating the contribution of the unfolded protein response to prostate cancer bone metastasis

Zeynep Kaya, John C. Christianson, Ian G. Mills, Srinivasa R. Rao, Claire M. Edwards
 
 The majority of deaths from PCa arise following metastasis, particularly to the skeleton. ER stress and the unfolded protein response (UPR) promote primary PCa, however the contribution of the UPR to PCa bone metastasis remains unknown. The aim of this work was to determine the role of the UPR in PCa bone metastasis, focusing on the osteogenic potential of PCa cells, EMT and migration, and PCa-induced bone disease. Using paired cell lines ARCaPE and ARCaPM which differ in their epithelial (E) and mesenchymal (M) characteristics, we found that components of IRE1 and ATF6 pathways are higher in ARCaPE cells than in ARCaPM and decreased upon osteogenic differentiation of ARCaPM cells. Inhibition of the IRE1 or PERK pathway increased ALP activity in ARCaPM cells. Inhibition of specific arms of the UPR produced a varied response in EMT markers with no effect on migration of ARCaPM cells. Increasing ER stress using tunicamycin significantly reduced migration of ARCaPM cells. The bone disease associated with PCa bone metastases is driven by alterations in a complex signaling network, including the RANKL/OPG pathway and Wnt signaling. ER stress, induced by tunicamycin, decreased RANKL and Dkk1 expression and increased OPG expression in ARCaPM cells. This osteolytic response to ER stress was blocked by PERK inhibition. Taken together, my research demonstrates that the UPR has multiple effects in bone metastatic PCa cells, including a reduction in migration and in osteolytic factors following UPR activation, suggesting a novel mechanism by which the UPR may modulate PCa-induced bone disease.

Activated PI3Kδ signals compromise plasma cell survival via limiting autophagy and increasing ER stress.

While phosphatidylinositide 3-kinase delta (PI3Kδ) plays a critical role in humoral immunity, the requirement for PI3Kδ signaling in plasma cells remains poorly understood. Here, we used a conditional mouse model of activated PI3Kδ syndrome (APDS), to interrogate the function of PI3Kδ in plasma cell biology. Mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) in B cells generated increased numbers of memory B cells and mounted an enhanced secondary response but exhibited a rapid decay of antibody levels over time. Consistent with these findings, aPIK3CD expression markedly impaired plasma cell generation, and expression of aPIK3CD intrinsically in plasma cells was sufficient to diminish humoral responses. Mechanistically, aPIK3CD disrupted ER proteostasis and autophagy, which led to increased plasma cell death. Notably, this defect was driven primarily by elevated mTORC1 signaling and modulated by treatment with PI3Kδ-specific inhibitors. Our findings establish an essential role for PI3Kδ in plasma cell homeostasis and suggest that modulating PI3Kδ activity may be useful for promoting and/or thwarting specific immune responses.

Abstract P402: Caveolin-1 Upregulation Leads To Decreased Erg And Stress-induced Ventricular Tachycardia In Cystic Fibrosis Hearts

Background: Cardiac manifestations of cystic fibrosis are classically accepted as cor pulmonale, yet emerging evidence supports that cardiac abnormalities can develop early when respiratory function is preserved. Myocardial cystic fibrosis transmembrane regulator (CFTR) channels, inactive in normal conditions, are activated in response to catecholamine stress. Methods and Results: In multiple mouse models of homozygous CFTR mutations (S489X or F508d), we demonstrate increased susceptibility to ventricular tachycardia (VT) in response to epinephrine, and this is associated with prolongation of QTc intervals (repolarization). Despite having normal left ventricular ejection fraction and histology, CF S489X mut/mut hearts showed 460 proteins significantly altered compared with WT hearts by tandem mass tag (TMT) proteomics. The Ingenuity Pathway Analysis of proteomics data revealed mitochondrial dysfunction and increased caveolin-1 signaling among the top pathways affected in CF S489X mut/mut hearts. In mouse hearts and human induced pluripotent stem cells derived cardiomyocytes (iPS-CM) with CFTR mutations, we observed increased ER stress and caveolin-1 (cav-1). These changes are concomitant with reduced levels of the ERG channel, a K+ channel contributing to phase 3 repolarization, mutations of which cause long QT syndrome II and VT. Patch clamp analysis showed decreased phase 2-3 repolarization current (including hERG current) in CF F508d iPS-CM compared with control iPS-CM, consistent with the findings of decreased membranous hERG and elevated Cav-1. Suppression of Cav-1 successfully restores membranous hERG in two separate lines of mutant iPS-CM generated from CF patients with F508d mutation. In F508d mut/mut mice, suppression of cardiac Cav-1 upregulation by reducing ER stress using TUDCA or crossing with heterozygous Cav-1 +/- abolished the stress-induced VT. Our analysis of a large-scale real-world clinical database from TriNetX, including 4520 CF men, 3510 CF women and the same number of well-matched controls demonstrated novel findings of increased risk for QT prolongation and VT in CF patients. Retrospective analysis of 126 CF patients with available EKG in our hospital revealed significant QTc prolongation in CF patients compared with matched controls, for both males (CF 447.8±4.5 vs cont. 429.2±2.5) and females (CF 454.0±3.5 vs cont.437.5±2.4). The QTc interval is not affected by lung function (%predicted FEV1) and is an independent predictor of survival in CF patients. Conclusions: In mice and iPS-CM models of CF, we demonstrated impaired repolarization and increased susceptibility to stress-induced VT. This is mediated by ER-stress-induced upregulation of Cav-1, which leads to internalization of ERG and subsequent decrease in repolarization current.

Intracellular Accumulation of IFN-λ4 Induces ER Stress and Results in Anti-Cirrhotic but Pro-HCV Effects.

IFNL3/IFNL4 polymorphisms are inversely associated with the risk of chronic hepatitis C virus (HCV) infection and cirrhosis, two major risk factors for developing hepatocellular carcinoma (HCC). To further explore these inverse associations and their molecular underpinnings, we analyzed IFNL3/IFNL4 polymorphisms represented by the IFNL4 genotype (presence of rs368234815-dG or rs12979860-T alleles) in HCV patients: 2969 from Japan and 2931 from Taiwan. IFNL4 genotype was associated with an increased risk of HCV-related HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058) in the general population of Japanese patients, but not in Taiwanese patients who achieved treatment-induced viral clearance. IFNL4 genotype was also associated with a decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwanese patients). We then engineered HepG2 cells to inducibly express IFN-λ4 in the presence or absence of interferon lambda receptor 1 (IFNLR1). Induction of IFN-λ4 resulted in its intracellular accumulation, mainly in lysosomes and late endosomes, and increased ER stress, leading to apoptosis and reduced proliferation. We identified the very-low-density lipoprotein receptor (VLDLR), which facilitates HCV entry into hepatocytes, as a transcript induced by IFN-λ4 but not IFN-λ3. Our results suggest that the molecular mechanisms underlying the anti-cirrhotic but pro-HCV associations observed for IFNL3/IFNL4 polymorphisms are, at least in part, contributed by intracellular accumulation of IFN-λ4 causing ER stress in hepatic cells.

Ouabain and chloroquine trigger senolysis of BRAF-V600E-induced senescent cells by targeting autophagy.

The expression of BRAF‐V600E triggers oncogene‐induced senescence in normal cells and is implicated in the development of several cancers including melanoma. Here, we report that cardioglycosides such as ouabain are potent senolytics in BRAF senescence. Sensitization by ATP1A1 knockdown and protection by supplemental potassium showed that senolysis by ouabain was mediated by the Na,K‐ATPase pump. Both ion transport inhibition and signal transduction result from cardioglycosides binding to Na,K‐ATPase. An inhibitor of the pump that does not trigger signaling was not senolytic despite blocking ion transport, demonstrating that signal transduction is required for senolysis. Ouabain triggered the activation of Src, p38, Akt, and Erk in BRAF‐senescent cells, and signaling inhibitors prevented cell death. The expression of BRAF‐V600E increased ER stress and autophagy in BRAF‐senescent cells and sensitized the cell to senolysis by ouabain. Ouabain inhibited autophagy flux, which was restored by signaling inhibitors. Consequently, we identified autophagy inhibitor chloroquine as a novel senolytic in BRAF senescence based on the mode of action of cardioglycosides. Our work underlies the interest of characterizing the mechanisms of senolytics to discover novel compounds and identifies the endoplasmic reticulum stress‐autophagy tandem as a new vulnerability in BRAF senescence that can be exploited for the development of further senolytic strategies.

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p < 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p < 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

High-fat diet associated sensitization to metabolic stress in Wfs1 heterozygous mice

Wolfram syndrome is a rare autosomal recessive disorder caused by mutations in the wolframin ER transmembrane glycoprotein (WFS1) gene and characterized by diabetes mellitus, diabetes insipidus, optic atrophy and deafness. In experimental models the homozygous Wfs1 mutant mice have a full penetrance and clearly expressed phenotype, whereas heterozygous mutants have a less-pronounced phenotype between the wild-type and homozygous mutant mice. Heterozygous WFS1 mutations have been shown to be significant risk factors for diabetes and metabolic disorders in humans. In the present study we analyzed the response of heterozygous Wfs1 mice to high fat diet (HFD) by exploring potential outcomes and molecular changes induced by this challenge. The HFD treatment increased the body weight (BW) similarly both in Wfs1 wild-type (WT) and heterozygous (HZ) mice, and therefore HFD also prevented the impaired BW gain found in Wfs1 mutant mice. In Wfs1HZ mutant mice, HFD impaired the normalized insulin secretion and the expression of ER stress genes in isolated pancreatic islets. These results suggest that Wfs1HZ mice have a decreased insulin response and pronounced cellular stress response due to a higher sensitivity to HFD as hypothesized. In Wfs1HZ mice, HFD increased the expression of Ire1α and Chop in pancreas and decreased that of Ire1α and Atf4 in liver. The present study shows that HFD can disturb insulin function with an increased ER stress in Wfs1HZ mice and only one functional Wfs1 gene copy is not sufficient to compensate this challenge. In conclusion, our study indicates that quantitative Wfs1 gene deficiency is sufficient to predispose the carriers of single functional Wfs1 copy to diabetes and metabolic syndrome and makes them susceptible to the environmental challenges such as HFD.