Endoplasmic Reticulum Stress Stress Research Articles

Glycated LDL increase VCAM-1 expression and secretion in endothelial cells and promote monocyte adhesion through mechanisms involving endoplasmic reticulum stress.

Type 2 Diabetes Mellitus is a worldwide epidemic, and its atherosclerotic complications produce morbidity and mortality in affected patients. It is known that the vascular cell adhesion molecule-1 (VCAM-1) levels are increased in the sera of diabetic patients. Our aim was to investigate the impact of the endoplasmic reticulum stress (ERS) in VCAM-1 expression and secretion in human endothelial cells (HEC) exposed to glycated low-density lipoproteins (gLDL). The results showed that 24h incubation of HEC with gLDL induces (i) stimulation of VCAM-1 expression and secretion, determining increased monocyte adhesion to HEC; (ii) RAGE up-regulation and free cholesterol loading; (iii) ERS activation (increased eIF2α phosphorylation and CHOP mRNA levels, and decreased GRP78 protein expression); and (iv) oxidative stress [increased levels of reactive oxygen species (ROS) and glutamate cysteine ligase catalytic unit gene expression]. Treatment of gLDL-exposed HEC with ERS inhibitors, salubrinal (Sal) and sodium phenylbutyrate (PBA), decreased intracellular ROS. Incubation of gLDL-exposed cells with the anti-oxidant N-acetyl-cysteine (NAC)reduced ERS, revealed by decreased eIF2α phosphorylation and CHOP gene expression and increased GRP78 expression, thus validating the interconnection between ERS and oxidative stress. Sal, PBA, NAC and inhibitors of p38MAP kinase and NF-kB induced the decrease of VCAM-1 expression and of the ensuing monocyte adhesion induced by gLDL. In conclusion, in HEC, gLDL stimulate the expression of cellular VCAM-1, the secretion of soluble VCAM-1, and the adhesion of monocytes through mechanisms involving p38MAP kinase and NF-kB signalling pathways activated by RAGE, ERS and oxidative stress, thus contributing to diabetic atherosclerosis.

MP84-10 ERG INDUCES ENDOPLASMIC RETICULUM STRESS (ER) AND UNFOLDED PROTEIN RESPONSE (UPR) TO INITIATE PROSTATE CANCER CARCINOGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-10 ERG INDUCES ENDOPLASMIC RETICULUM STRESS (ER) AND UNFOLDED PROTEIN RESPONSE (UPR) TO INITIATE PROSTATE CANCER CARCINOGENESIS Taduru L. Sreenath, Shiela S. Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Parameet Kumar, Denise Young, Rishita Gupta, Shilpa Katta, Ahmed Mohamed, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Charles J. Bieberich, Peter Nelson, David G. McLeod, Valeri Vasioukhin, and Shiv Srivastava Taduru L. SreenathTaduru L. Sreenath More articles by this author , Shiela S. MacalindongShiela S. Macalindong More articles by this author , Natallia MikhalkevichNatallia Mikhalkevich More articles by this author , Shashwat SharadShashwat Sharad More articles by this author , Parameet KumarParameet Kumar More articles by this author , Denise YoungDenise Young More articles by this author , Rishita GuptaRishita Gupta More articles by this author , Shilpa KattaShilpa Katta More articles by this author , Ahmed MohamedAhmed Mohamed More articles by this author , Shyh-Han TanShyh-Han Tan More articles by this author , Albert DobiAlbert Dobi More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , Isabell A. SesterhennIsabell A. Sesterhenn More articles by this author , Charles J. BieberichCharles J. Bieberich More articles by this author , Peter NelsonPeter Nelson More articles by this author , David G. McLeodDavid G. McLeod More articles by this author , Valeri VasioukhinValeri Vasioukhin More articles by this author , and Shiv SrivastavaShiv Srivastava More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2234AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High levels of the ERG expression due to gene fusions (predominantly TMPRSS2-ERG) is frequently detected in prostate cancer (CaP) in Western countries. Better understanding of the roles of the ERG oncogenic functions in CaP initiation and/or progression will enhance its utility as the therapeutic target. Transgenic mice generated to understand ERG’s role in the development of prostate adenocarcinoma have shown focal proliferative and dysplastic PIN lesions and later developed adenocarcinoma in older mice. Mice with ERG and PTEN alterations exhibit more accelerated malignant progression in prostate. The present study was aimed towards understanding the biological functions of ERG in early stages oncogenic transformation of prostate epithelium. METHODS Morphological analyses of mouse prostate glands were analyzed by H&E staining and by electron microscopy. Luminal cell surface markers were analyzed by FACS analysis. Luminal cells were analyzed for their potential to proliferate and form spheres by prostate sphere formation assay. ER stress and UPR marker proteins were analyzed by Western blot analysis. Analysis of representative ER stress pathway in ERG-positive human prostate cancer was analyzed in CPDR-80 gene-chip array data set representing well- and poor differentiated tumors. RESULTS Histological examination of ERG-Tg mouse prostates revealed increased luminal cell death due to apoptosis. Subsequently, TEM analysis revealed significant morphological differences such as increased numbers of lysosomes, autophagosomes, concentric membrane bodies with ribosomes, and lipid droplets in the cytoplasm of transgenic secretory luminal epithelial cells. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to induced cell death. Western blots of ERG-Tg mouse and LNCaP-ERG transfectants showed increased expression of ER stress sensors and UPR proteins. A strong positive correlation was also observed between ERG and P4HB/PDI, one of the ER stress response proteins in human prostate tumors. CONCLUSIONS Cumulative data from experimental models of ERG and human CaP suggests that ERG mediates ER stress resulting into the activation of UPR, cell death with eventual biological selection for cell survival. Taken together these data indicate a critical biological function for ERG in prostate cancer initiation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1093 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Taduru L. Sreenath More articles by this author Shiela S. Macalindong More articles by this author Natallia Mikhalkevich More articles by this author Shashwat Sharad More articles by this author Parameet Kumar More articles by this author Denise Young More articles by this author Rishita Gupta More articles by this author Shilpa Katta More articles by this author Ahmed Mohamed More articles by this author Shyh-Han Tan More articles by this author Albert Dobi More articles by this author Gyorgy Petrovics More articles by this author Isabell A. Sesterhenn More articles by this author Charles J. Bieberich More articles by this author Peter Nelson More articles by this author David G. McLeod More articles by this author Valeri Vasioukhin More articles by this author Shiv Srivastava More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The molecular mechanisms of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathways in oral squamous cell carcinoma under endoplasmic reticulum stress.

Proteasome inhibitor Carbobenzoxy-Leu-Leu-leucinal (MG132) induces the unfolded protein response (UPR) in oral squamous cell carcinoma (OSCC). X-box binding protein 1 (XBP1) is a key UPR component that regulates endoplasmic reticulum stress (ER) homeostasis. This study was aimed to investigate the activation of IRE1α-TRAF2-ASK1-JNK pathway by silencing the XBP1 expression in an OSCC cell line. The XBP1 specific short hairpin RNA (shRNA) plasmid vector was constructed and then transfected into the Tca-8113 cells. The effect of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathway under MG132 induced endoplasmic reticulum stress in Tca-8113 were investigated by real-time RT-PCR or western blot. Cell apoptosis was detected by flow cytometry. XBP1 expression was reduced in transfected groups and MG132 groups. shRNA-XBP1 induces IRE1α-TRAF2-ASK1 signaling activation to activate pro-apoptotic ASK1-JNK signaling. Moreover, combined shRNA-XBP1 with MG132 further enhanced downregulated XBP1 expression and upregulated activation of ASK1-JNK signaling. Silencing XBP1 expression under MG132 induced ER stress block the XBP1 survival pathway and synergism with MG132 to promote Tca8113 cell apoptosis. These findings provide a therapeutic option in oral squamous cell carcinoma by inhibition of proteasome and XBP1 splicing.

Abstract 19375: Rapamycin Treatment Regulates miRNAs Associated With Mitochodrial and Endoplasmic Reticulum Functions in a Pig Model of Miocardial Ischemia-reperfusion Injury

Introduction: Previously(Lassaletta et al,PMC3943541)we showed that Rapamycin treatment of normal pigs subjected to an acute myocardial ischemia-reperfusion injury (IRI) was associated with increase myocardial necrosis and refractory left ventricle (LV) defibrillations. In an effort to better understand rapamycin effects we applied a non-bias genomic analysis to determine if microRNAs were modified by rapamycin treatment in this pre-clinical animal model of IRI. Hypothesis: We hypothesized that microRNA expression were modified by rapamycin-treated and would identify novel and meaningful pharmacological mechanisms related to its detrimental effects on reperfusion injury. Methods: Young male Yorkshire swine (16-19 kg) received either no drug (controls, n=5) or were given 4mg of oral rapamycin daily (n=5). After two weeks of treatment, all animals underwent median sternotomy and the mid-LAD was occluded for 60 minutes followed by 120 minutes of reperfusion. RNA was extracted from LV non-ischemic and ischemic areas from each group and processed (according to the manufacture′s specifications at Brown University Genomic Core facility) for microRNA (miRNA) expression analysis using a Affymetrix GeneChip miRNA3.0 Array containing over 25,000 probe sets of which 257 miRNA probes of domestic pigs. Data were processed using MetaCore version 5.0 (GeneGo, St Joseph, MI) for both statistical (FDR: p<0.05) and pathway analysis. Results: miRNA regulated in 5 animals per group were annotated for human orthologs. A total of 23 miRNAs were differentially expressed. Interestingly, all miRNAs were down-regulated in the rapamycin-treated animals. Of those, nine had human orthologs: miR 181c-3p was highly represented followed by miR323a-5p, miR1245b-3p, miR4324, miR331-5p, miR491-5p and miR490-3p. Conclusions: Recent studies show that miRNA 181c regulates myocardial mitochondrial function through cytochrome c oxidase and complex IV gene expressions (PMCID: PMC4014556)and regulates Glucose-regulated protein (GRP78)/BiP [an endoplasmic reticulum stress (ER) chaperone](PMID: 24696166). Hence It is likely that rapamycin effects (detrimental or not) are associated with mitochondrial function and ER stress response.

Intermedin protects against renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress.

BackgroundIntermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. Endoplasmic reticulum stress (ERS) has been implicated in the pathology of renal ischemia/reperfusion (IRI). In the present study, we investigated whether IMD could reduce ERS damage after renal ischemia.MethodsThe kidneys of SD rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. The hypoxia/reoxygenation(H/R) model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. The serum creatinine concentration and lactate dehydrogenase (LDH) activity in the plasma were determined. Histologic examinations of renal tissues were performed with PAS staining. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Additionally, ER staining was used to detect the ERS response.ResultsIn the rat renal IRI model, we found that IMD gene transfer markedly improved renal function and pathology and decreased LDH activity and cell apoptosis compared with the kidneys that were transfected with the control plasmid. IMD significantly attenuated the ERS stress parameters compared with IRI group. Indeed, IMD down-regulated glucose-regulated protein 78 (GRP78), C/EBP homologous protein(CHOP), and caspase 12 protein and mRNA levels. Moreover, in the NRK-52E cell H/R model, IMD overexpression prevented the apoptosis induced by H/R. Furthermore, IMD ameliorated the ER structural changes and concomitantly decreased the levels of GRP78, CHOP and caspase-12.ConclusionThis study revealed that IMD protects against renal IRI by suppressing ERS and ERS-related apoptosis.

697 Novel Approach Reveals That Genetic Variation in/Near FXR/RXR Pathway Genes Predisposes to Nonalcoholic Fatty Liver Disease in Humans

Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte lipoapoptosis (p<0.05) and caspase 3/7 activity (p<0.05) in dose dependent manner. Interestingly, Toyocamycin also attenuated free fatty acid-induced steatosis. In addition, Toyocamycin attenuated expression of Bim protein andmRNA (p<0.05) following palmitate treatment. Furthermore, PAmediated-transcriptional activation of DR5was reduced by Toyocamycin (p<0.05). In contrast, palmitate-induced JNK phosphorylation, which also occurs downstream of IRE-1 arm of ER stress, was not inhibited by Toyocamycin. Conclusions: These data imply that Toyocamycin attenuates hepatocyte lipotoxicity as well as steatosis, likely through inhibition of XBP-1 splicing. Toyocamycin might be beneficial for NASH treatment.

696 XBP-1 Inhibitor Toyocamycin Attenuates Palmitate-Induced Hepatocyte Apoptosis and Steatosis

Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte lipoapoptosis (p<0.05) and caspase 3/7 activity (p<0.05) in dose dependent manner. Interestingly, Toyocamycin also attenuated free fatty acid-induced steatosis. In addition, Toyocamycin attenuated expression of Bim protein andmRNA (p<0.05) following palmitate treatment. Furthermore, PAmediated-transcriptional activation of DR5was reduced by Toyocamycin (p<0.05). In contrast, palmitate-induced JNK phosphorylation, which also occurs downstream of IRE-1 arm of ER stress, was not inhibited by Toyocamycin. Conclusions: These data imply that Toyocamycin attenuates hepatocyte lipotoxicity as well as steatosis, likely through inhibition of XBP-1 splicing. Toyocamycin might be beneficial for NASH treatment.

GW24-e1859 Shenyuan, an extract of American ginseng and corydalis tuber formula, attenuates cardiomyocyte apoptosis via inhibition of endoplasmic reticulum stress and oxidative stress in a porcine model of myocardial infarction

ObjectivesTo evaluate the anti-apoptotic effect of Shenyuan, which is composed of the bioactive components extracted from the mixture of American Ginseng and Corydalis Tuber, and to explore the inhibition of...

Abstract SY28-03: Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies.

Abstract SY28-03: Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies.

MCP-1 Induced Protein Promotes Adipogenesis via Oxidative Stress, Endoplasmic Reticulum Stress and Autophagy

Obesity involves inflammation. MCP-1, an inflammatory chemokine, and MCP-1-induced protein (MCPIP) are known to induce adipogenesis that causes increase in the number of adipocytes. Here we elucidate the intermediate processes through which MCPIP induces adipogenesis. Forced expression of MCPIP in 3T3-L1 preadipocytes caused increased reactive oxygen/nitrogen species (ROS/RNS) production and inducible-nitric oxide synthase (iNOS) expression, endoplasmic reticulum stress (ER), as indicated by expression of ER chaperones and protein disulfide isomerase, and autophagy as indicated by expression of beclin-1 and cleavage of LC3. Treatment of ROS inhibitor, apocynin attenuated MCPIP induction of adipogenesis as measured by the induction of transcription factors involved in adipogenesis, adipocyte markers and lipid droplet accumulation. Inhibition of ER stress with taurursodeoxycholate or knockdown of inositol requiring enzyme 1 (IRE1) inhibited MCPIP induced autophagy and adipogenesis. Preadipocytes in adipogenesis-inducing cocktail manifested ER stress and autophagy. Knockdown of MCPIP attenuated these effects. MCPIP induced p38 activation and p38 inhibitor, SB203580, attenuated MCPIP-induced adipogenesis.

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

BackgroundCells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.ResultsExpression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival.ConclusionsThe inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in β-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.

Functional polymorphisms of HSPA5: Possible association with bipolar disorder

Altered endoplasmic reticulum stress (ER) response signaling is suggested in bipolar disorder. Previously, we preliminarily reported the genetic association of HSPA5 ( GRP78/ BiP) with bipolar disorder. Here, we extended our analysis by increasing the number of Japanese case-control samples and NIMH Genetics Initiative bipolar trio samples (NIMH trios), and also analyzed schizophrenia samples. In Japanese, nominally significant association of one haplotype was observed in extended samples of bipolar disorder but not in schizophrenia. In NIMH trios, no association was found in total samples. However, an exploratory analysis suggested that the other haplotype was significantly over-transmitted to probands only from the paternal side. The associated haplotype in Japanese or NIMH pedigrees shared three common polymorphisms in the promotor, which was found to alter promotor activity. These findings suggested promotor polymorphisms of HSPA5 may affect the interindividual variability of ER stress response and may confer a genetic risk factor for bipolar disorder.