INS1-EGFP-L10a stable cell lines treated with Thapsigargin
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ABSTRACT:
ORGANISM(S): Mus musculus Rattus norvegicus
PROVIDER: GSE39212 | GEO | 2012/09/01
SECONDARY ACCESSION(S): PRJNA170233
REPOSITORIES: GEO
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Project description:INS1-EGFP-L10a stable cell lines were induced with Doxycycline (Dox) for 24 hours, then either untreated or treated for 30 minutes with 1uM Thapsigargin to induce ER stress. Total RNA was purified with Trizol, and RIP RNA samples were extracted by immunoaffinity purification using an EGFP antibody We aimed to determine which genes are enriched under ER stress at the polyribosomal level. To do this we compared expression profiles of Total RNA with and without ER stress, and Immunoaffinity purified RNA (RIP) with and without ER stress. Microarray results of INS-1 EGFP-L10a cells either untreated or treated with 1uM Thapsigargin (Tg) in triplicate. Both Total RNA and RNA isolated from ribosomal Immunoprecipitation (RIP) When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this M-bM-^@M-^\Terminal UPRM-bM-^@M-^]. TXNIP becomes rapidly induced by hyperactivated IRE1a, an ER bifunctional kinase/endoribonuclease (RNase). IRE1a controls TXNIP mRNA stability by reducing levels of a TXNIP destabilizing micro-RNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing Caspase-1 cleavage and interleukin 1b (IL-1b) secretion. Txnip gene deletion reduces pancreatic b-cell death during ER stress, and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1a RNase inhibitors suppress TXNIP production to block IL-1b secretion. In summary, the IRE1a-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death, and may be targeted to develop new treatments for degenerative diseases driven by ER stress. There are 12 samples total all in INS-1 EGFP L10a cells- 3 untreated total RNA (reference sample), 3 treated with 1uM Tg 30 min total RNA, 3 untreated RIP RNA (reference sample), 3 treated with 1uM for 30 min RIP RNA
2012-09-01 | E-GEOD-39212 | biostudies-arrayexpress
Project description:During mammalian cell growth and differentiation, the unfolded protein response (UPR) homeostatically adjusts endoplasmic reticulum (ER) protein-folding capacity to match changing cellular secretory demands. However, under high/chronic ER stress conditions the UPR triggers apoptosis. This dichotomy is promoted in part by differential activation levels of the ER transmembrane kinase/endoribonuclease (RNase) IRE1a. IRE1a kinase auto-phosphorylation operates as a rheostat to control downstream RNase-induced outputs that either sustain adaptive ER protein-folding or cause apoptosis. We have previously shown that IRE1a’s RNase activity can be activated or fully inactivated by ATP-competitive kinase inhibitors. Here we developed a new class of ATP-competitive kinase inhibitors that partially antagonize the RNase of IRE1a at full occupancy. An atomic level resolution co-crystal structure shows that these small molecule partial antagonists—which we named ‘PAIR’s for (Partial Antagonists of IRE1a RNase)—occupy the ATP-binding site of IRE1a and partially displace a helix (helix aC) in the kinase domain that forms part of a dimeric interface. In insulin-producing beta cells, PAIRs permit adaptive XBP1 mRNA splicing, while quelling destructive/terminal outputs from extra-XBP1 mRNA endonucleolytic decay, thus preventing apoptosis. Preservation of XBP1 splicing by PAIRs permits B lymphocytes to differentiate into immunoglobulin-producing plasma cells. In summary, we propose that an intermediate RNase-inhibitory “sweet spot,” achieved by PAIR-bound IRE1a, may capture a structural conformation naturally available to IRE1a that could represent a desirable therapeutic state for drugging this master UPR sensor/effector.
2021-06-01 | GSE164496 | GEO
Project description:Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide b cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in b cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1a) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, b cell specific EIF2A viral overexpression reduced ER stress, improved insulin signaling, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for b cell protection and the circumvention of EIF2S1-mediated translational repression.
2022-08-31 | GSE166829 | GEO
Project description:The unfolded protein response (UPR) couples cellular translation rates and gene expression to the protein folding status of the endoplasmic reticulum (ER). Upon activation, the UPR machinery elicits a general suppression of protein synthesis and activation of stress gene expression, which act coordinately to restore protein folding homeostasis. We report here that UPR activation promotes the release of signal sequence-encoding mRNAs from the ER to the cytosol as a mechanism to decrease protein influx into the ER. This release of mRNA begins rapidly, then gradually recovers with ongoing stress. Upon release into the cytosol, these mRNAs have divergent fates: some synthesize full-length proteins, while others are translationally inactive and retain nascent protein chains. Together, these findings identify the dynamic subcellular localization of mRNAs and translation as a regulatory feature of the cellular response to protein folding stress. Cells were treated with a timecourse of Thapsigargin or DTT, then fractionated and analyzed by mRNA-seq or ribosome profiling
2014-08-28 | E-GEOD-53743 | biostudies-arrayexpress
Project description:Objective: Induction of ER stress has been shown to promote NP cell apoptosis and disc degeneration. However, little is known about its regulation by hypoxia and its contribution to extracellular matrix homeostasis. Methods: NP cells were culture under hypoxia (1% O2) and normoxia (21% O2) to assess ER stress status. Tunicamycin and thapsigargin were used to induce canonical ER stress pathways and effects on cell secretome were assessed by proteomic analysis using mass spectrometry. The relevance of these findings to aging and degeneration was investigated by analyzing microarray data of NP tissues from aged mice (GSE134955) and degenerated human discs (GSE70362). Results: NP cells exhibited lower ER stress levels under hypoxia. ER stress inducers tunicamycin and thapsigargin promoted a canonical unfolded protein response. UPR further induced HIF-1 activity under hypoxia. NP cell secretome under ER stress showed an increased secretory pathway with a concomitant decrease in collagens, HAPLN1, and cell adhesion related proteins. Similar to our cell culture studies, NP tissues from aged mice and degenerated human discs presented higher levels of UPR markers and decreased levels of matrix components.
2020-12-03 | PXD019151 | Pride
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the master UPR sensor IRE1a kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1a-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1a as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1a through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1a was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1a, perhaps as a strategy to eliminate detection by the host immune system.
2022-07-20 | GSE193169 | GEO
Project description:Erguler2013 - Unfolded protein stress response
The model investigates the mechanism by which UPR (unfolded protein response) outcome switches between survival and death.
This model is described in the article:
A mathematical model of the unfolded protein stress response reveals the decision mechanism for recovery, adaptation and apoptosis.
Erguler K, Pieri M, Deltas C.
BMC Syst Biol. 2013 Feb 21;7(1):16.
Abstract:
BACKGROUND: The unfolded protein response (UPR) is a major signalling cascade acting in the quality control ofprotein folding in the endoplasmic reticulum (ER). The cascade is known to play an accessory rolein a range of genetic and environmental disorders including neurodegenerative and cardiovasculardiseases, diabetes and kidney diseases. The three major receptors of the ER stress involved withthe UPR, i.e. IRE1a, PERK and ATF6, signal through a complex web of pathways to convey anappropriate response. The emerging behaviour ranges from adaptive to maladaptive depending on theseverity of unfolded protein accumulation in the ER; however, the decision mechanism for the switchand its timing have so far been poorly understood.
RESULTS:
Here, we propose a mechanism by which the UPR outcome switches between survival and death.We compose a mathematical model integrating the three signalling branches, and perform a comprehensivebifurcation analysis to investigate possible responses to stimuli. The analysis reveals threedistinct states of behaviour, low, high and intermediate activity, associated with stress adaptation, tolerance,and the initiation of apoptosis. The decision to adapt or destruct can, therefore, be understoodas a dynamic process where the balance between the stress and the folding capacity of the ER playsa pivotal role in managing the delivery of the most appropriate response. The model demonstratesfor the first time that the UPR is capable of generating oscillations in translation attenuation and theapoptotic signals, and this is supplemented with a Bayesian sensitivity analysis identifying a set ofparameters controlling this behaviour.
CONCLUSIONS:
This work contributes largely to the understanding of one of the most ubiquitous signalling pathwaysinvolved in protein folding quality control in the metazoan ER. The insights gained have direct consequenceson the management of many UPR-related diseases, revealing, in addition, an extended listof candidate disease modifiers. Demonstration of stress adaptation sheds light to how preconditioningmight be beneficial in manifesting the UPR outcome to prevent untimely apoptosis, and paves the wayto novel approaches for the treatment of many UPR-related conditions.
In the paper, PERKA refers to the amount of phosphorylated PERK monomer. However, it refers to the active complex in the model. The complex with the model parameterization is formed of 4 monomers (n=4). So, the value of PERKA should be multiplied by 4, in order to generate the figures in the paper (eg. Figure 12).
An additional parameter (tmr=10)) is used in the model. This parameter is not mentioned in the paper. The model values of kf(=10) and kr(=1) are not consistent with that of the paper (kf=100, kr=10, in the paper). However, this is corrected by the introduction of "tmr" in the model, which is multiplied with kf and kr to get the resulting values.
The term "tmr" was missing in the kinetic laws of the reactions reu7 and reu8, in the original model. This has been corrected as per the author's request.
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2024-09-02 | BIOMD0000000446 | BioModels
Project description:The unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum (ER) stress, is induced by a range of environmental factors. Here we describe the identification and characterization of a synthetic small molecule, erstressin, which activates the UPR. Erstressin induced rapid phosphorylation of PERK and eIF2a and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activated the transcription of multiple genes involved in the UPR. Coincident with these effects, erstressin also downregulated the transcription of the inflammation-associated enzyme inducible nitric oxide synthase (iNOS) in cytokine-activated cells. A close analog of erstressin that failed to induce the UPR did not attenuate expression of iNOS, suggesting that both biological effects of erstressin were mediated by a common mechanism. Further, the structurally-distinct ER stressor thapsigargin also inhibited iNOS expression. Together these chemical genetic studies reveal an unanticipated anti-inflammatory role for the UPR. Experiment Overall Design: We utilized microarrays to understand the global effect of a novel compund, erstressin, on cytokine stimulated cells over time.
2008-06-15 | E-GEOD-7806 | biostudies-arrayexpress
Project description:We are currently studying how these information-carrying oligosaccharides are involved in cellular stress responses, particularly in human genetic diseases called Congenital Disorders Of Glycosylation in which oligosaccharide assembly is defective. We are interested in endoplasmic stress responses/unfolded protein responses. ER glycans and ER lectins are intimately involved in the folding of ER proteins. Therefore, ER lectins and ER glycosyltransferases may be controlled by these stress responses, to produce and/or recognize key glycans in the stress response. Our model system is the human dermal fibroblast. We have successfully calibrated the ER stress responses in these cells by determining the magnitudes of various stress effects (such as chaperone transcription) with different forms of ER stress. We are also completing a study in which activation the signaling molecules Ire1 and ATF6 are also calibrated. We prepared multiple identical aliquots of RNA from cells stressed under several of these calibrated conditions. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR): Study of the role of ER stress in the expression of genes for ER transferases and lectins that participate in ER folding and quality control using human skin fibroblasts. The UPR response in these cells has been calibrated using various readouts in response to different stresses: 1) 40 nM L-azetidine-2-carboxylate (AZC) 1 hour, 2) 0.2 mg/ml castanospermine, 24 hours, 3) 2 mM Dithiothreitol (DTT), 20 minutes, 4) 100nm Thapsigargin (TG), 30 minutes, 5) 5 ug/ml tunicamycin, 5 hours, 6) untreated control cells
2011-02-17 | E-GEOD-27349 | biostudies-arrayexpress
Project description:Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-time PCR analyses confirmed that four of these factors, VEGF, FGF2, angiogenin and IL-8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGF regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGF promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGF expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGF gene, although its contribution to VEGF transcription appeared to be fairly modest. We also found that VEGF mRNA stability is increased in response to UPR activation, via activation of the AMP and p38MAP kinases, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGF protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGF expression at multiple levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer. We used microarrays to perform genome wide expression analysis in Daoy medulloblastoma cells to identify gene profiles that change in response to thapsigargin treatment which causes induction of the unfolded protein response.
2011-05-13 | E-GEOD-21979 | biostudies-arrayexpress