XBP1 mRNA Splicing Research Articles

Mir-331 negatively regulates thyroglobulin secretion via ERp29

We investigated the effect of endoplasmic reticulum ER protein 29 ERp29 on thyroglobulin Tg secretion and its negative regulation by microRNAs miRNAs . ERp29 overexpression promoted Tg secretion from thyrocytes of PCCL3 cells via activation of ER stress sensors, including activation of transcription factor 6 fragmentation, XBP1 mRNA splicing by inositol-requiring enzyme 1, and phosphorylation of eukaryotic initiation factor 2 alpha as downstream actions of RNA-dependent protein kinase-like ER kinase. Thyroid hormone receptor beta is a target gene of mir-331, one of the most abundant miRNAs observed in ERp29-overexpressing cells. mir-331 negatively regulated Tg expression and secretion by ~70% compared with the control. To overcome hypothyroidism, Tg secretion at the molecular level is required to counteract negative regulation of intracellular mir-331. Our findings may provide insight into the treatment of diseases caused by poor ER protein secretion, including ER storage diseases.

IRE1α Promotes Zika Virus Infection via XBP1.

Zika virus (ZIKV) is an emergent member of the Flaviviridae family which causes severe congenital defects and other major sequelae, but the cellular processes that support ZIKV replication are incompletely understood. Related flaviviruses use the endoplasmic reticulum (ER) as a membranous platform for viral replication and induce ER stress during infection. Our data suggest that ZIKV activates IRE1α, a component of the cellular response to ER stress. IRE1α is an ER-resident transmembrane protein that possesses a cytosolic RNase domain. Upon activation, IRE1α initiates nonconventional cytoplasmic splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor, which upregulates ER-related targets. We find that ZIKV infection induces XBP1 mRNA splicing and induction of XBP1 target genes. Small molecule inhibitors of IRE1α, including those specific for the nuclease function, prevent ZIKV-induced cytotoxicity, as does genetic disruption of IRE1α. Optimal ZIKV RNA replication requires both IRE1α and XBP1. Spliced XBP1 has been described to cause ER expansion and remodeling and we find that ER redistribution during ZIKV infection requires IRE1α nuclease activity. Finally, we demonstrate that inducible genetic disruption of IRE1α and XBP1 impairs ZIKV replication in a mouse model of infection. Together, our data indicate that the ER stress response component IRE1α promotes ZIKV infection via XBP1 and may represent a potential therapeutic target.

The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

A Role for the Ribosome-Associated Complex in Activation of the IRE1 Branch of UPR

The ubiquitous ribosome-associated complex (RAC) is a chaperone that spans ribosomes, making contact near both the polypeptide exit tunnel and the decoding center, a position prime for coordination of translation and folding. Loss of RAC results in growth defects and sensitization to translational stress. The mechanism of RAC in responding to specific stresses remains obscure. Data presented here uncover an essential function of mammalian RAC in the unfolded protein response (UPR), coupling the inositol-requiring protein 1α (IRE1α) branch to translation. Knockdown of RAC sensitizes mammalian cells to ER stress by selectively interfering with IRE1α activation. Higher-order oligomerization of IRE1α kinase/endoribonuclease and accurate ribosome stalling of Xbp1 mRNA, as required for IRE1α mediated Xbp1 mRNA splicing activity, depends upon RAC. These results reveal a surveillance function for RAC on the ribosomes: modulating IRE1α clustering and the fitness of Xbp1 mRNA splicing in coordination with translation.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Diphtheria toxoid nanoparticles improve learning and memory impairment in animal model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder involving memory. The present study aimed at evaluating the effects of encapsulated diphtheria toxoid (DT) on behavioral learning impairment, and XBP1 mRNA splicing in AD. A DT-loaded nanoparticle (NP) carrier was prepared using the ionic gelation method. Sixty-three rats were divided into nine groups: (1) healthy, (2-4) sham, and (5-9) AD models: (5) AD was induced by intracerebroventricular injection of amyloid beta (Aβ) 1-42. (6) The rats received a subcutaneous diphtheria vaccine only 28 days before Aβ injection. (7) The rats received an intranasal diphtheria vaccine, in group 8, induced by administering empty chitosan NPs. 9) it was induced by administering chitosan NPs carrying DT. Morris water maze (MWM) test was used to examine the animals' learning and memory. Also, X-box binding protein 1 (XBP-1) mRNA gene splicing was studied in the hippocampus by reverse-transcription polymerase chain reaction (RT-PCR). For the first time, chitosan NPs were prepared with an average diameter size of 40 nm and the effectiveness of approximately 70% during DT encapsulation. In comparison with the healthy group, the AD models exhibited significant impairment of learning and memory (P < 0.05), while DT-administrated animals showed significant improvements in learning and memory impairment (P < 0.05). XBP-1 mRNA gene splicing was only detected in an untreated AD group, while encapsulated DT completely inhibited splicing. The therapeutic effects of DT chitosan NPs against learning and memory impairment were observed in this study, and XBP1 mRNA splicing was reported in the animal models.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

Expression of XBP1s in peritoneal mesothelial cells is critical for inflammation-induced peritoneal fibrosis

Intraperitoneal inflammation is the most important determinant of peritoneal fibrosis in patients with long-term peritoneal dialysis (PD). Spliced x-box binding protein-1 (XBP1s), a major proximal effector of unfolded protein response (UPR) signaling, plays an indispensable role in inflammation. Our study demonstrated that the inflammatory factor interleukin-1β (IL-1β) dose- and time-dependently induced XBP1s upregulation and interleukin-6 (IL-6) secretion, as well as the expression of the fibrotic marker fibronectin. However, these effects were prevented by the IRE1 endonuclease inhibitor STF083010 since it time-dependently reduced IL-1β-induced Xbp1 mRNA splicing, XBP1s protein expression, inflammatory factor IL-6 secretion and the expression of the fibrotic marker fibronectin in human peritoneal mesothelial cells (HPMCs). The overexpression and knockdown of XBP1s in HPMCs had a similar effect on fibronectin expression. In a rat model of peritoneal inflammation, STF083010 significantly attenuated chlorhexidine digluconate-induced XBP1s and α-smooth muscle actin expression, as well as fibrotic tissue proliferation, in the peritoneum. Our results suggest that XBP1s is a strong pathogenic factor that mediates inflammation-induced peritoneal fibrosis in peritoneal dialysis.

EIF2\u03b1-CHOP-BCl-2/JNK and IRE1\u03b1-XBP1/JNK signaling promote apoptosis and inflammation and support the proliferation of Newcastle disease virus

Newcastle disease virus (NDV) causes severe infectious disease in poultry and selectively kills tumor cells, by inducing apoptosis and cytokines secretion. In this report, we study the mechanisms underlying NDV-induced apoptosis by investigating the unfolded protein response (UPR). We found that NDV infection activated all three branches of the UPR signaling (PERK-eIF2α, ATF6, and IRE1α) and triggered apoptosis, in avian cells (DF-1 and CEF) and in various human cancer cell types (HeLa, Cal27, HN13, A549, H1299, Huh7, and HepG2). Interestingly, the suppression of either apoptosis or UPR led to impaired NDV proliferation. Meanwhile, the inhibition of UPR by 4-PBA protected cells from NDV-induced apoptosis. Further study revealed that activation of PERK-eIF2α induced the expression of transcription factor CHOP, which subsequently promoted apoptosis by downregulating BCL-2/MCL-1, promoting JNK signaling and suppressing AKT signaling. In parallel, IRE1α mediated the splicing of XBP1 mRNA and resulted in the translation and nuclear translocation of XBP1s, thereby promoting the transcription of ER chaperones and components of ER-associated degradation (ERAD). Furthermore, IRE1α promoted apoptosis and cytokines secretion via the activation of JNK signaling. Knock down and overexpression studies showed that CHOP, IRE1α, XBP1, and JNK supported efficient virus proliferation. Our study demonstrates that the induction of eIF2α-CHOP-BCL-2/JNK and IRE1α-XBP1/JNK signaling cascades promote apoptosis and cytokines secretion, and these signaling cascades support NDV proliferation.

Application of a New Multiplexed Array for Rapid, Sensitive, Simultaneous and Quantitative Assessment of Spliced and Unspliced XBP1

BackgroundIRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them.MethodsA multiplexed isoform-specific XBP1 array utilising Biochip array technology (BAT™) was assessed for specificity and suitability when using cell protein lysates. The array was applied to RIPA protein lysates from several relevant pre-clinical models with an aim to quantify XBP1 isoforms in comparison with RT-PCR or immunoblot reference methods.ResultsA novel reliable, specific and sensitive XBP1 biochip was successfully utilised in pre-clinical research. Application of this biochip to detect XBP1 splicing at the protein level in relevant breast cancer models, under basal conditions as well as pharmacological inhibition and paclitaxel induction, confirmed the findings of previous studies. The biochip was also applied to non-adherent cells and used to quantify changes in the XBP1 isoforms upon activation of the NLRP3 inflammasome.ConclusionsThe XBP1 biochip enables isoform specific quantification of protein level changes upon activation and inhibition of IRE1α RNase activity, using a routine clinical methodology. As such it provides a research tool and potential clinical tool with a quantified, simultaneous, rapid output that is not available from any other published method.

Dysregulation of the unfolded protein response (UPR) in the dorsolateral prefrontal cortex in elderly patients with schizophrenia

Abnormalities in protein localization, function, and posttranslational modifications are targets of schizophrenia (SCZ) research. As a major contributor to the synthesis, folding, trafficking, and modification of proteins, the endoplasmic reticulum (ER) is well-positioned to sense cellular stress. The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction to environmental and pathological perturbation in ER function. The UPR is a highly orchestrated and complex cellular response, which is mediated through the ER chaperone protein, BiP, three known ER transmembrane stress sensors, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), inositol requiring enzyme 1α (IRE1α), and their downstream effectors. In this study, we measured protein expression and phosphorylation states of UPR sensor pathway proteins in the dorsolateral prefrontal cortex (DLPFC) of 22 matched pairs of elderly SCZ and comparison subjects. We observed increased protein expression of BiP, decreased PERK, and decreased phosphorylation of IRE1α. We also observed decreased p-JNK2 and increased sXBP1, downstream targets of the IRE1α arm of the UPR. The disconnect between decreased p-IRE1α and increased sXBP1 protein expression led us to measure sXbp1 mRNA. We observed increased expression of the ratio of sXbp1/uXbp1 transcripts, suggesting that splicing of Xbp1 mRNA by IRE1α is increased and drives upregulation of sXBP1 protein expression. These findings suggest an abnormal pattern of UPR activity in SCZ, with specific dysregulation of the IRE1α arm. Dysfunction of this system may lead to abnormal responses to cellular stressors and contribute to protein processing abnormalities previously observed in SCZ.

P2B.17: A model to study ischemia-reperfusion injury in human intestinal organoids

Introduction: Ischemia-reperfusion (IR) injury is inevitable during intestinal transplantation. IR damages the intestinal epithelium, which functions as a physical and immunological barrier and is therefore crucial in maintaining intestinal homeostasis. In order to investigate potential therapeutic targets to protect the epithelium during intestinal IR and promote a regenerative response, we aim to validate a model to study IR in human intestinal organoids. Intestinal organoids have been shown to closely resemble self-renewal kinetics, 3D architecture, and cell-type composition of the intestinal epithelium in vivo. Methods: A well-established human experimental model to study IR was used for temporal expression profiling of the in vivo intestinal response to IR. The top perturbed pathway was further validated using qPCR. Intestinal epithelial organoids were cultured from crypts isolated from surgical specimens of normal human small intestine. To simulate IR, organoids were subjected to 12 hours of hypoxia (1% O2) followed by 30 and 120 minutes of reoxygenation. Activation of the UPR response was assessed by qPCR for CHOP, GADD34, BiP and XBP1 splicing, and, in addition, signs of endoplasmic reticulum (ER) stress were evaluated using electron microscopy (EM). Results: The unfolded protein response (UPR) was the top perturbed pathway during reperfusion of the ischemically injured human small intestine in vivo. Subjecting small intestinal organoids to 12 hours of hypoxia followed by 30 minutes of reoxygenation significantly increased expression of UPR-related genes CHOP and GADD34 and splicing of XBP1 mRNA compared to organoids not subjected to hypoxia. In addition, EM showed dilated ER after reoxygenation which is indicative of ER stress. Conclusion: In line with findings in the in vivo human IR model, revealing the response to unfolded protein as a highly regulated process during reperfusion, hypoxia-reoxygenation in intestinal organoids induces significant activation of the UPR. Intestinal organoids can be used to improve insight in the pathophysiology of epithelial IR injury and regeneration, which could lead to new therapeutic targets.

Upregulation of miR-34a by Inhibition of IRE1α Has Protective Effect against Aβ-Induced Injury in SH-SY5Y Cells by Targeting Caspase-2.

Background Neurotoxicity induced by the amyloid-β (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Based on accumulating evidence in AD research, both endoplasmic reticulum stress (ER stress) and alterations in the microRNA (miRNA) network contribute to the pathogenesis of the disease, making them potential therapeutic targets for AD. The present study was performed to investigate whether miR-34a and the inositol-requiring enzyme 1 (IRE1) are involved in the regulation of Aβ-induced cytotoxicity. Methods Human neuroblastoma SH-SY5Y cells were treated with Aβ1-40. Cell viability was assessed by the MTT assay. The integrity of the plasma membrane was assessed by LDH release. The expression levels of XBP1s, IRE1α, p-IRE1α, and Caspase-2 were detected by Western blot analysis. Spliced-XBP1 mRNA and miR-34a were detected by reverse transcription- (RT-) PCR and quantitative real-time PCR, respectively. Caspase-2 activity was measured using the Caspase-2 cellular activity assay kit. The IRE1 inhibitor (STF-083010) was used to determine the role of IRE1α on miR-34a expression. SH-SY5Y cells were transfected with miR-34a mimics to assess the role of miR-34a on the activation of Caspase-2 and the viability of Aβ-exposed SH-SY5Y cells. Results We showed that Aβ caused concentration- and duration-dependent death of SH-SY5Y cells. The expression levels of XBP1s, p-IRE1α, and Caspase-2 were increased, along with a corresponding decrease in the miR-34a levels in Aβ-exposed SH-SY5Y cells. The IRE1 inhibitor (STF-083010) upregulated the expression of miR-34a and suppressed the activation of Caspase-2, effectively alleviating the Aβ-induced death of SH-SY5Y cells. Transfection studies show that miR-34a mimics inhibit the expression of Caspase-2 and restore the viability of Aβ-exposed SH-SY5Y cells. Conclusion Aβ peptide induced downregulation of miR-34a through the activation of IRE1α, which may induce cytotoxicity by targeting Caspase-2. Upregulation of miR-34a by inhibition of IRE1α has protective effects against Aβ-induced injury in SH-SY5Y cells.

2121-P: nAChR Signaling Protects ß Cells from Apoptosis Induced by ER Stress and Specific Activation of IRE1α in INS1 Cells

Background: We previously showed that nAChR signaling regulates activation of IRE1α and markers of terminal-unfolded protein response (T-UPR) under endoplasmic reticulum stress (ERS) in β-cells. However, it is still unknown if nAChR signaling affects the apoptosis under ERS or specific activation of IRE1α in β-cells. We also previously reported that IRE1α specific inhibitor, KIRA-Kinase Inhibiting RNase Attenuator-, suppresses apoptosis induced by ERS in INS1 cells and β-cells in Akita mice, which suggest that IRE1α plays a critical role in ERS-induced apoptosis. The aim of this study is to determine if and how nAChR signaling regulates the apoptosis induced by ERS or specific activation of IRE1α in β-cells. Methods: We investigated the role of nAChR signaling on IRE1α signaling under ERS inducers or doxycycline-inducible IRE1α-overexpression (OE) in INS-1 cells. Cells were treated with Nicotine as an agonist of nAChR, and Tunicamycin or Thapsigargin as ERS inducers. Activation of IRE1α was confirmed by XBP1 mRNA splicing and phosphorylation of IRE1α. That of T-UPR was determined by Insulin1/proinsulin and TXNIP mRNA and protein expression. Translocation of phosphatidylserine to the external cell surface was detected by flow cytometry with Annexin V-FITC staining. Activation of caspase-3 and -7, markers of activation of caspase cascade, was detected by the active caspase-3/-7 specific substrate. Results: Nicotine reduced XBP1 mRNA splicing and phosphorylation of IRE1α induced by ERS. Nicotine reversed increase of TXNIP and decrease of Insulin1/proinsulin expressions induced by both ERS and IRE1α OE. Increased Annexin positive cells and caspase-3/7 activation by ERS were reduced by Nicotine. Finally, increased caspase-3/7 activation by IRE1α OE was reversed by Nicotine as well as KIRA. Conclusion: nAChR signaling could regulate IRE1α signaling and protect β-cells from apoptosis induced by ERS and specific activation of IRE1α in INS1 cells. Disclosure T. Ishibashi: None. S. Morita: None. A. Doi: None. H. Iwakura: None. H. Ariyasu: None. M. Nishi: None. H. Furuta: None. T. Akamizu: None.

Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response Using Medaka Fish.

Three types of transmembrane protein, IRE1α/IRE1β, PERK, and ATF6α/ATF6β, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1β, ATF6α and ATF6β are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1β- and ATF6α/ATF6β-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.

Immobilization stress induces XBP1 splicing in the mouse brain

Cells activate the unfolded protein response (UPR) to cope with endoplasmic reticulum (ER) stress. In the present study, we investigated the possible involvement of psychological stress on UPR induction in the mouse brain. When mice were exposed to immobilization stress for 8 h, XBP1 mRNA splicing was significantly induced in the hippocampus, cortex, hypothalamus, cerebellum, and brain stem. On the other hand, we did not observe any increase in XBP1 splicing in the liver, suggesting that this effect is specific to the brain. Stress-induced XBP1 splicing was attenuated 2 days after immobilization stress. We did not observe increases in any other UPR genes, such as CHOP or GRP78, in mouse brains after immobilization stress. These findings indicate an important specific role of XBP1 in response to psychological stress in the mouse brain.

Cyclosporine A binding to COX-2 reveals a novel signaling pathway that activates the IRE1\u03b1 unfolded protein response sensor

Cyclosporine, a widely used immunosuppressant in organ transplantation and in treatment of various autoimmune diseases, activates the unfolded protein response (UPR), an ER stress coping response. In this study we discovered a new and unanticipated cyclosporine-dependent signaling pathway, with cyclosporine triggering direct activation of the UPR. COX-2 binds to and activates IRE1α, leading to IRE1α splicing of XBP1 mRNA. Molecular interaction and modeling analyses identified a novel interaction site for cyclosporine with COX-2 which caused enhancement of COX-2 enzymatic activity required for activation of the IRE1α branch of the UPR. Cyclosporine-dependent activation of COX-2 and IRE1α in mice indicated that cyclosporine-COX-2-IRE1α signaling pathway was functional in vivo. These findings identify COX-2 as a new IRE1α binding partner and regulator of the IRE1α branch of the UPR pathway, and establishes the mechanism underlying cytotoxicity associated with chronic cyclosporine exposure.

The Human Cytomegalovirus Endoplasmic Reticulum-Resident Glycoprotein UL148 Activates the Unfolded Protein Response.

Eukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER-resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol-requiring enzyme-1 (IRE1), as indicated by splicing of Xbp-1 mRNA, and the protein kinase R (PKR)-like ER kinase (PERK), as indicated by phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and accumulation of activating transcription factor 4 (ATF4). During wild-type HCMV infection, increases in Xbp-1 splicing, eIF2α phosphorylation, and accumulation of ATF4 accompany UL148 expression. UL148-null infections, however, show reduced levels of these UPR indicators and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. Small interfering RNA (siRNA) depletion of PERK dampened the extent of eIF2α phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2α kinases. A virus with UL148 disrupted showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV-infected cell.IMPORTANCE The unfolded protein response (UPR) is an ancient cellular response to ER stress that is of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shutoff, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER-resident glycoprotein UL148 contributes substantially to the UPR during infection and, moreover, is sufficient to activate the UPR in noninfected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell-mediated immunity.

The synthetic chalcone derivative 2-hydroxy-3′,5,5′-trimethoxychalcone induces unfolded protein response-mediated apoptosis in A549 lung cancer cells

The synthetic chalcone derivative 2-hydroxy-3′,5,5′-trimenthoxyochalcone (named DK-139) exhibits anti-inflammatory and anti-tumor invasion properties. However, effects of DK-139 on tumor cell growth remain unknown. In the present study, we evaluated the inhibitory activity of DK-139 against human lung cancer cells. Treatment with DK-139 inhibited clonogenicity in various lung cancers and stimulated the caspase cascade, leading to the apoptosis of A549 lung cancer cells. To investigate the mode of action of DK-139-induced apoptosis, we analyzed the effect of DK-139 on the endoplasmic reticulum (ER) stress response. DK-139 increased expression of ER stress sensors, including p-PERK, GRP78/BiP, and IRE1α. IRE1α-regulated XBP-1 mRNA splicing and PERK-induced ATF4 expression was also upregulated following DK-139 treatment. In addition, expression levels of the pro-apoptotic transcription factor CHOP and its downstream target Bim, which is involved in mitochondria-mediated apoptosis, were increased by DK-139 treatment. These results suggest that DK-139 triggers caspase-mediated apoptosis via the ER stress-activated unfolded protein response (UPR) pathway. We propose that the synthetic chalcone derivative DK-139 may be used as a potential agent for the prevention and/or treatment of human lung cancer.

Exendin-4 protects INS-1 cells against palmitate-induced apoptosis through the IRE1α-Xbp1 signaling pathway.

The anti-apoptotic effect of the incretin analog, exendin-4 (EX-4) on pancreatic β cells is mediated via the activation of protein kinase B (Akt) signaling, and its effect is partly produced through the inhibition of endoplasmic reticulum (ER) stress. However, the molecular mechanisms that underlie the effect of EX-4 on the suppression of ER stress and the upregulation of Akt signaling are poorly understood. Inositol-requiring enzyme 1 (IRE1), a member of the ER-localized transmembrane protein family, activates its downstream transcription factor X-box binding protein 1 (XBP1) to mediate a key part of the cellular unfolded protein response in order to cope with ER stress. Using the clonal rat pancreatic β cell line INS-1, the present study produced an in vitro model of ER stress using palmitate (PA) in order to determine whether the beneficial effect of EX-4 under ER stress was regulated by the IRE1α-Xbp1 signaling pathway. The results demonstrated that the reduction in ER stress and the activation Akt by EX-4 may be associated with the upregulation of IRE1α phosphorylation and the splicing of Xbp1 mRNA, which improved PA-reduced cell viability. This effect was partially abrogated by the knockdown of IRE1α with small interfering RNA. Additionally, cellular IRE1α was phosphorylated by the protein kinase A (PKA) associated with EX-4 and the activation of IRE1α, as IRE1α phosphorylation was attenuated by the inhibition of PKA with its inhibitor. In conclusion, the data identified the IRE1α-Xbp1 signaling pathway as an essential mediator that associates EX-4 with the intracellular mechanism that inhibits ER stress and activates Akt in order to regulate β cell survival. This may provide important evidence for the use of EX-4 in treatments for type 2 diabetes.