A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Abstract This study assessed the molecular mechanism of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) protection against IPEC-1 cell damage induced by deoxynivalenol (DON). The cells were divided into six groups, including the CON group, the EPA group, the DHA group, the DON group, the EPA+DON group, and the DHA+DON group. RNA sequencing was used to investigate the potential mechanism, and qRT-PCR was employed to verify the expression of selected genes. Changes in ultrastructure were used to estimate pathological changes and endoplasmic reticulum (ER) injury in IPEC-1 cells. Transferrin receptor 1 (TFR1) was tested by ELISA. Fe2+ and malondialdehyde (MDA) contents were estimated by spectrophotometry, and reactive oxygen species (ROS) was assayed by fluorospectrophotometry. RNA sequencing analysis showed that EPA and DHA had a significant effect on the expression of genes involved in ER stress and iron balance during DON-induced cell injury. The results showed that DON increased ER damage, the content of MDA and ROS, the ratio of X-box binding protein 1s (XBP-1s)/X-box binding protein 1u (XBP-1u), the concentration of Fe2+, and the activity of TFR1. However, the results also showed that EPA and DHA decreased the ratio of XBP-1s/XBP-1u to relieve DON-induced ER damage of IPEC-1 cells. Moreover, EPA and DHA (especially DHA) reversed the factors related to iron balance. It can be concluded that EPA and DHA reversed IPEC-1 cell damage induced by DON. DHA has the potential to protect IPEC-1 cells from DON-induced iron imbalance by inhibiting ER stress.

Download Full-text

Transient Cerebral Ischemia Activates Processing of xbp1 Messenger RNA Indicative of Endoplasmic Reticulum Stress

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000054216.21675.ac ◽

2003 ◽

Vol 23 (4) ◽

pp. 449-461 ◽

Cited By ~ 46

Author(s):

Wulf Paschen ◽

Christoph Aufenberg ◽

Svenja Hotop ◽

Thorsten Mengesdorf

Keyword(s):

Endoplasmic Reticulum ◽

Cerebral Ischemia ◽

Er Stress ◽

Messenger Rna ◽

Stress Proteins ◽

Focal Cerebral Ischemia ◽

Signal Transduction Pathway ◽

Transient Cerebral Ischemia ◽

Mrna Levels ◽

Xbp1 Mrna

Cells respond to conditions associated with endoplasmic reticulum (ER) dysfunction with activation of the unfolded protein response, characterized by a shutdown of translation and induction of the expression of genes coding for ER stress proteins. The genetic response is based on IRE1-induced processing of xbp1 messenger RNA (mRNA), resulting in synthesis of new XBP1proc protein that functions as a potent transcription factor for ER stress genes. xbp1 processing in models of transient global and focal cerebral ischemia was studied. A marked increase in processed xbp1 mRNA levels during reperfusion was observed, most pronounced (about 35-fold) after 1-h occlusion of the right middle cerebral artery. The rise in processed xbp1 mRNA was not paralleled by a similar increase in XBP1proc protein levels because transient ischemia induces severe suppression of translation. As a result, mRNA levels of genes coding for ER stress proteins were only slightly increased, whereas mRNA levels of heat-shock protein 70 rose about 550-fold. Under conditions associated with ER dysfunction, cells require activation of the entire ER stress-induced signal transduction pathway, to cope with this severe form of stress. After transient cerebral ischemia, however, the block of translation may prevent synthesis of new XBP1proc protein and thus hinder recovery from ischemia-induced ER dysfunction.

Download Full-text

Obesity-Linked Variants of Melanocortin-4 Receptor Are Misfolded in the Endoplasmic Reticulum and Can Be Rescued to the Cell Surface by a Chemical Chaperone

Endocrine Reviews ◽

10.1210/edrv.31.4.9985 ◽

2010 ◽

Vol 31 (4) ◽

pp. 605-605

Author(s):

Susana Granell ◽

Sameer Mohammad ◽

Ramanagouda Ramanagoudr-Bhojappa ◽

Giulia Baldini

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Er Stress ◽

Binding Protein ◽

Intracellular Localization ◽

Specific Inhibitor ◽

Surface Expression ◽

Cell Surface Expression ◽

Chemical Chaperone ◽

Melanocortin 4 Receptor

Abstract Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein. Expression of the X-box-binding-protein-1 with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.

Download Full-text

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00483.2010 ◽

2011 ◽

Vol 301 (5) ◽

pp. G773-G782 ◽

Cited By ~ 50

Author(s):

Ersin Seyhun ◽

Antje Malo ◽

Claus Schäfer ◽

Christopher A. Moskaluk ◽

Ralf-Thorsten Hoffmann ◽

...

Keyword(s):

Acute Pancreatitis ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Stress Responses ◽

Cell Damage ◽

Binding Protein ◽

Edema Formation ◽

Tauroursodeoxycholic Acid ◽

Caspase 12

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH2-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.

Download Full-text

Protection of pancreatic β-cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies

Journal of Endocrinology ◽

10.1677/joe-06-0148 ◽

2007 ◽

Vol 193 (1) ◽

pp. 65-74 ◽

Cited By ~ 70

Author(s):

Shin Tsunekawa ◽

Naoki Yamamoto ◽

Katsura Tsukamoto ◽

Yuji Itoh ◽

Yukiko Kaneko ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Islet Cells ◽

Binding Protein ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic β-cells against their cell death. In in vivo experiments, we used β-cell-specific calmodulin-overexpressing mice where massive apoptosis takes place in their β-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1α, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in β-cell-specific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic β-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

Download Full-text

Reducing endoplasmic reticulum stress does not improve steatohepatitis in mice fed a methionine- and choline-deficient diet

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00052.2012 ◽

2012 ◽

Vol 303 (1) ◽

pp. G54-G59 ◽

Cited By ~ 22

Author(s):

Anne S. Henkel ◽

Amanda M. Dewey ◽

Kristy A. Anderson ◽

Shantel Olivares ◽

Richard M. Green

Keyword(s):

Endoplasmic Reticulum ◽

Stress Response ◽

Er Stress ◽

Hepatic Steatosis ◽

Binding Protein ◽

Mrna Levels ◽

Primary Role ◽

Chemical Chaperones ◽

Er Stress Response ◽

Markers Of Inflammation

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of nonalcoholic steatohepatitis. The ER stress response is activated in the livers of mice fed a methionine- and choline-deficient (MCD) diet, yet the role of ER stress in the pathogenesis of MCD diet-induced steatohepatitis is unknown. Using chemical chaperones on hepatic steatosis and markers of inflammation and fibrosis in mice fed a MCD diet, we aim to determine the effects of reducing ER stress. C57BL/6J mice were fed a MCD diet with or without the ER chemical chaperones 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) for 2 wk. TUDCA and PBA effectively attenuated the ER stress response in MCD diet-fed mice, as evidenced by reduced protein levels of phosphorylated eukaryotic initiation factor 2α and phosphorylated JNK and suppression of mRNA levels of CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78 kDa, and X-box binding protein 1. However, PBA and TUDCA did not decrease MCD diet-induced hepatic steatosis. MCD diet-induced hepatic inflammation, as evidenced by increased plasma alanine aminotransferase and induction of hepatic TNFα expression, was also not reduced by PBA or TUDCA. PBA and TUDCA did not attenuate MCD diet-induced upregulation of the fibrosis-associated genes tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9. ER chemical chaperones reduce MCD diet-induced ER stress, yet they do not improve MCD diet-induced hepatic steatosis, inflammation, or activation of genes associated with fibrosis. These data suggest that although the ER stress response is activated by the MCD diet, it does not have a primary role in the pathogenesis of MCD diet-induced steatohepatitis.

Download Full-text

Downregulation of miR-384-5p attenuates rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through inhibiting endoplasmic reticulum stress

AJP Cell Physiology ◽

10.1152/ajpcell.00226.2015 ◽

2016 ◽

Vol 310 (9) ◽

pp. C755-C763 ◽

Cited By ~ 17

Author(s):

Mingfang Jiang ◽

Qiang Yun ◽

Feng Shi ◽

Guangming Niu ◽

Yang Gao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Target Genes ◽

Binding Protein ◽

Quantitative Polymerase Chain Reaction ◽

Inhibitory Effect ◽

Luciferase Reporter ◽

Stress Signaling

Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of Parkinson's disease (PD). However, the role of microRNAs (miRNAs) in this process involved in PD remains poorly understood. Recent studies indicate that miR-384-5p plays an important role for cell survival in response to different insults, but the role of miR-384-5p in PD-associated neurotoxicity remains unknown. In this study, we investigated the role of miR-384-5p in an in vitro model of PD using dopaminergic SH-SY5Y cells treated with rotenone. We found that miR-384-5p was persistently induced by rotenone in neurons. Also, the inhibition of miR-384-5p significantly suppressed rotenone-induced neurotoxicity, while overexpression of miR-384-5p aggravated rotenone-induced neurotoxicity. Through bioinformatics and dual-luciferase reporter assay, miR-384-5p was found to directly target the 3′-untranslated region of glucose-regulated protein 78 (GRP78), the master regulator of ER stress sensors. Quantitative polymerase chain reaction and Western blotting analysis showed that miR-384-5p negatively regulated the expression of GRP78. Inhibition of miR-384-5p remarkably suppressed rotenone-evoked ER stress, which was evident by a reduction in the phosphorylation of activating transcription factor 4 (ATF4) and inositol-requiring enzyme 1 (IRE1α). The downstream target genes of ER stress including CCAAT/enhancer-binding protein-homologous protein (CHOP) and X box-binding protein-1 (XBP-1) were also decreased by the miR-384-5p inhibitor. In contrast, overexpression of miR-384-5p enhanced ER stress signaling. In addition, knockdown of GRP78 significantly abrogated the inhibitory effect of miR-384-5p inhibitors on cell apoptosis and ER stress signaling. Moreover, we observed a significant increase of miR-384-5p expression in primary neurons induced by rotenone. Taken together, our results suggest that miR-384-5p mediated ER stress by negatively regulating GRP78 and that miR-384-5p inhibition might be a novel and promising approach for the treatment of PD.

Download Full-text

Diesel Exhaust Particulates Induce Neutrophilic Lung Inflammation by Modulating Endoplasmic Reticulum Stress-Mediated CXCL1/KC Expression in Alveolar Macrophages

Molecules ◽

10.3390/molecules25246046 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6046 ◽

Cited By ~ 1

Author(s):

Dong Im Kim ◽

Mi-Kyung Song ◽

Hye-In Kim ◽

Kang Min Han ◽

Kyuhong Lee

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Alveolar Macrophages ◽

Diesel Exhaust ◽

Lung Inflammation ◽

Binding Protein ◽

In Vitro Study ◽

Vitro Study

Diesel exhaust particulates (DEP) have adverse effects on the respiratory system. Endoplasmic reticulum (ER) abnormalities contribute to lung inflammation. However, the relationship between DEP exposure and ER stress in the respiratory immune system and especially the alveolar macrophages (AM) is poorly understood. Here, we examined ER stress and inflammatory responses using both in vivo and in vitro study. For in vivo study, mice were intratracheally instilled with 25, 50, and 100 μg DEP and in vitro AM were stimulated with DEP at 1, 2, and 3 mg/mL. DEP increased lung weight and the number of inflammatory cells, especially neutrophils, and inflammatory cytokines in bronchoalveolar lavage fluid of mice. DEP also increased the number of DEP-pigmented AM and ER stress markers including bound immunoglobulin protein (BiP) and CCAAT/enhancer binding protein-homologous protein (CHOP) were upregulated in the lungs of DEP-treated mice. In an in vitro study, DEP caused cell damage, increased intracellular reactive oxygen species, and upregulated inflammatory genes and ER stress-related BiP, CHOP, splicing X-box binding protein 1, and activating transcription factor 4 expressions in AM. Furthermore, DEP released the C-X-C Motif Chemokine Ligand 1 (CXCL1/KC) in AM. In conclusion, DEP may contribute to neutrophilic lung inflammation pathogenesis by modulating ER stress-mediated CXCL1/KC expression in AM.

Download Full-text

A Molecular Mechanism for Turning off IRE1α Signaling During Endoplasmic Reticulum Stress

10.1101/2020.04.03.024356 ◽

2020 ◽

Author(s):

Xia Li ◽

Sha Sun ◽

Suhila Appathurai ◽

Arunkumar Sundaram ◽

Rachel Plumb ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Mammalian Cells ◽

Mrna Splicing ◽

Xbp1 Mrna ◽

Long Time ◽

A Subunit ◽

Active Transcription ◽

J Domain ◽

Sustained Activation

SummaryMisfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress and thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63 deficient cells, IRE1α remains activated for a long time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.

Download Full-text

Pseudorabies Virus Infection Induces Endoplasmic Reticulum Stress Through PERK and IRE1 Pathways of Unfolded Protein Response

10.21203/rs.3.rs-1087342/v1 ◽

2021 ◽

Author(s):

Li Chen ◽

Minshu Ni ◽

Waqas Ahmed ◽

Yue Xu ◽

Xi Bao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein Response ◽

Pseudorabies Virus ◽

Viral Infections ◽

Host Cells ◽

Unfolded Protein ◽

Xbp1 Mrna ◽

Enhanced Expression ◽

Protein Response

Abstract Pseudorabies virus (PRV) is a pathogen of swine resulting in devastating disease. Some viral infections can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) to restore ER homeostasis. However, the mechanism of how PRV induces ER stress and UPR activation remains unclear. Here, levels of proteins or transcriptional factors of three UPR pathways were examined in suspension-cultured BHK-21 cells to investigate PRV-induced ER stress. Results showed that PRV triggered ER stress and UPR of the host cells with the upregulated expression of glucose-related protein 78 kD and 94 kD (GRP78 and GRP94). The protein kinase RNA-like ER kinase (PERK) pathway was activated to upregulate ATF4, CHOP, and GADD34 expression. Additionally, the inositol requiring kinase 1 (IRE1) pathway was triggered by splicing of X box-binding protein 1 (XBP1) mRNA and the enhanced expression of p58IPK and EDEM1. Furthermore, our data demonstrated that PRV took advantage of ER stress to accelerate its replication with the activation of the PERK and IRE1 pathways in suspension-cultured BHK-21 cells, and the glycoprotein B played a crucial role in ER stress.

Download Full-text