Knockdown Of IRE1α Research Articles

A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

MHO7 (6-epi-ophiobolin G), a novel component extracted from a mangrove fungus, exhibits significant anticancer effects against breast cancer. However, the precise mechanism underlying the anticancer effects of MHO7 in prostate cancer (PCa) is yet to be fully elucidated. Therefore, this study was undertaken to assess the effect of MHO7 on PCa cells and elucidate its underlying mechanism. A series of in vitro experiments were conducted, including Cell Counting Kit-8, and plate clone formation assays, flow cytometry analysis, electron microscopy, immunofluorescence staining, western blotting, and molecular dynamics simulation. Additionally, in vivo tumor xenograft models were employed. Our findings revealed that MHO7 could induce cellular autophagy at low concentration (2 μM) and apoptosis at relatively high concentration (4 and 8 μM), leading to significant PCa cell growth inhibition. Furthermore, MHO7 triggered endoplasmic reticulum (ER) stress, which subsequently stimulated autophagy and apoptosis via IRE1α/XBP-1s signaling pathway activation. Notably, IRE1α knockdown markedly reduced MHO7-induced autophagy and apoptosis. Moreover, MHO7 targeted the IRE1α protein, thereby enhancing its stability. MHO7 also exhibited substantial anticancer activity in tumor xenograft models. Our study revealed that MHO7 holds considerable potential as an anticancer agent against PCa, attributable to its activation of ER stress-induced autophagy and apoptosis at different concentrations, facilitated by the upregulation of IRE1α expression.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

The total xanthones extracted from Gentianella acuta alleviates HFpEF by activating the IRE1α/Xbp1s pathway.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterized by pulmonary and systemic congestion resulting from left ventricular diastolic dysfunction and increased filling pressure. Currently, however, there is no evidence on effective pharmacotherapy for HFpEF. In this study, we aimed to investigate the therapeutic effect of total xanthones extracted from Gentianella acuta (TXG) on HFpEF by establishing an high-fat diet (HFD) + L-NAME-induced mouse model. Echocardiography was employed to assess the impact of TXG on the cardiac function in HFpEF mice. Haematoxylin and eosin staining, wheat germ agglutinin staining, and Masson's trichrome staining were utilized to observe the histopathological changes following TXG treatment. The results demonstrated that TXG alleviated HFpEF by reducing the expressions of genes associated with myocardial hypertrophy, fibrosis and apoptosis. Furthermore, TXG improved cardiomyocyte apoptosis by inhibiting the expression of apoptosis-related proteins. Mechanistic investigations revealed that TXG could activate the inositol-requiring enzyme 1α (IRE1α)/X-box-binding protein 1 (Xbp1s) signalling pathway, but the knockdown of IRE1α using the IRE1α inhibitor STF083010 or siRNA-IRE1α impaired the ability of TXG to ameliorate cardiac remodelling in HFpEF models. In conclusion, TXG alleviates myocardial hypertrophy, fibrosis and apoptosis through the activation of the IRE1α/Xbp1s signalling pathway, suggesting its potential beneficial effects on HFpEF patients.

Novel XBP1s-independent function of IRE1 RNase in HIF-1α-mediated glycolysis upregulation in human macrophages upon stimulation with LPS or saturated fatty acid.

In obesity, adipose tissue infiltrating macrophages acquire a unique pro-inflammatory polarization, thereby playing a key role in the development of chronic inflammation and Type 2 diabetes. Increased saturated fatty acids (SFAs) levels have been proposed to drive this specific polarization. Accordingly, we investigated the immunometabolic reprogramming in SFA-treated human macrophages. As expected, RNA sequencing highlighted a pro-inflammatory profile but also metabolic signatures including glycolysis and hypoxia as well as a strong unfolded protein response. Glycolysis upregulation was confirmed in SFA-treated macrophages by measuring glycolytic gene expression, glucose uptake, lactate production and extracellular acidification rate. Like in LPS-stimulated macrophages, glycolysis activation in SFA-treated macrophages was dependent on HIF-1α activation and fueled the production of pro-inflammatory cytokines. SFAs and LPS both induced IRE1α endoribonuclease activity, as demonstrated by XBP1 mRNA splicing, but with different kinetics matching HIF-1α activation and the glycolytic gene expression. Interestingly, the knockdown of IRE1α and/or the pharmacological inhibition of its RNase activity prevented HIF-1α activation and significantly decreased glycolysis upregulation. Surprisingly, XBP1s appeared to be dispensable, as demonstrated by the lack of inhibiting effect of XBP1s knockdown on glycolytic genes expression, glucose uptake, lactate production and HIF-1α activation. These experiments demonstrate for the first time a key role of IRE1α in HIF-1α-mediated glycolysis upregulation in macrophages stimulated with pro-inflammatory triggers like LPS or SFAs through XBP1s-independent mechanism. IRE1 could mediate this novel function by targeting other transcripts (mRNA or pre-miRNA) through a mechanism called regulated IRE1-dependent decay or RIDD. Deciphering the underlying mechanisms of this novel IRE1 function might lead to novel therapeutic targets to curtail sterile obesity- or infection-linked inflammation.

MicroRNA-322 overexpression reduces neural tube defects in diabetic pregnancies

Hyperglycemia from pregestational diabetes mellitus induces neural tube defects in the developing fetus. Folate supplementation is the only effective way to prevent neural tube defects; however, some cases of neural tube defects are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes mellitus-induced neural tube defects can identify potential targets for preventing such defects. Despite not yet being in clinical use, growing evidence suggests that microRNAs are important intermediates in embryonic development and can serve as both biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes mellitus invivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo and that a high glucose condition invitro reduces microRNA-322 (miR-322) levels. IRE1α is an RNA endonuclease; however, it is unknown whether IRE1α targets and degrades miR-322 specifically or whether miR-322 degradation leads to neural tube defects via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes mellitus-induced neural tube defects and that restoring miR-322 expression in developing neuroepithelium ameliorates neural tube defects. This study aimed to identify potential targets for preventing maternal diabetes mellitus-induced neural tube defects and to investigate the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes mellitus. To determine whether miR-322 reduction is necessary for neural tube defect formation in pregnancies complicated by diabetes mellitus, male mice carrying a transgene expressing miR-322 were mated with nondiabetic or diabetic wide-type female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 when the neural tube is not yet closed, embryos were harvested for the assessment of 3 miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (TRAF3), and neuroepithelium cell survival. Neural tube defect incidences were determined in embryonic day 10.5 embryos when the neural tube should be closed if there is no neural tube defect formation. To identify which miR-322 transcript is affected by maternal diabetes mellitus and high glucose conditions, 3 miR-322 transcripts were assessed in embryos from dams with or without diabetes mellitus and in C17.2 mouse neural stem cells treated with different concentrations of glucose and at different time points. To determine whether the endonuclease IRE1α targets miR-322, small interfering RNA knockdown of IRE1α or overexpression of inositol-requiring transmembrane kinase/endoribonuclease 1α by DNA plasmid transfection was used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression. RNA immunoprecipitation was performed to reveal the direct targets of inositol-requiring transmembrane kinase/endoribonuclease 1α. Maternal diabetes mellitus suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes mellitus-induced caspase-3 and caspase-8 cleavage and cell apoptosis, leading to a neural tube defect reduction. Reversal of maternal diabetes mellitus-inhibited miR-322 via transgenic overexpression prevented TRAF3 up-regulation in embryos exposed to maternal diabetes mellitus. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction. This study supports the crucial role of the IRE1α-microRNA-TRAF3 circuit in the induction of neuroepithelial cell apoptosis and neural tube defect formation in pregnancies complicated by diabetes mellitus and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes mellitus-induced neural tube defects.

The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection.

The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that induces the expression of ER-related targets. Activation of the IRE1α-XBP1 pathway occurs in association with risk factors for severe human coronavirus infection. In this study, we found that the human coronaviruses HCoV-OC43 (human coronavirus OC43) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) both robustly activate the IRE1α-XBP1 branch of the unfolded protein response in cultured cells. Using IRE1α nuclease inhibitors and genetic knockdown of IRE1α and XBP1, we found that these host factors are required for optimal replication of both viruses. Our data suggest that IRE1α supports infection downstream of initial viral attachment and entry. In addition, we found that ER stress-inducing conditions are sufficient to enhance human coronavirus replication. Furthermore, we found markedly increased XBP1 in circulation in human patients with severe coronavirus disease 2019 (COVID-19). Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection. IMPORTANCE There is a critical need to understand the cellular processes co-opted during human coronavirus replication, with an emphasis on identifying mechanisms underlying severe disease and potential therapeutic targets. Here, we demonstrate that the host proteins IRE1α and XBP1 are required for robust infection by the human coronaviruses, SARS-CoV-2 and HCoV-OC43. IRE1α and XBP1 participate in the cellular response to ER stress and are activated during conditions that predispose to severe COVID-19. We found enhanced viral replication with exogenous IRE1α activation, and evidence that this pathway is activated in humans during severe COVID-19. Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.

Abstract 2029: The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1

Abstract UV induced DNA damage is the primary driver of human skin cancer. UV can also cause endoplasmic reticulum (ER) stress, but the role of unfolded protein response (UPR) in the cellular response to UV exposure is not clear. Inositol-requiring enzyme 1α (IRE1α) is one of the key regulators of the UPR but its function in the UV response has not been resolved. Here we show that IRE1α, is transiently dephosphorylated and its RNase activity downregulated by UVB resulting in reduced XBP1 splicing. Keratinocytes with either IRE1α knockout or knockdown have elevated cytosolic calcium and ROS resulting in increased apoptosis after UV. Pharmacological inhibition of the ER inositol triphosphate receptor (IP3R) or phospholipase C (PLC) restored normal cytosolic calcium and ROS levels to cells lacking IRE1α. While loss of IRE1α did not alter the frequency of cells with UV-induced cyclo-pyrimidine dimers (CPD), it significantly reduced the percent of γH2AX positive keratinocytes 6 hours after UVB irradiation. This percent of CPD positive cells and the level of γH2AX 6 hours after UV suggesting a defect in the DNA repair response. Pharmacological inhibition of IRE1α RNase or Xbp1 knockdown did not phenocopy IRE1α loss, but overexpression of IRE1α in IRE1α knockdown cells restored γH2AX expression and reduced keratinocyte apoptosis. IRE1α overexpression also enhanced the survival of keratinocytes deficient in p53 function after UV irradiation. Additionally, mice with an epidermal deletion of IRE1α showed more rapid loss of UV-induced CPD positive keratinocytes compared to wildtype mice, primarily through accelerated sloughing of UV damaged cells from the skin. Knockdown of Calcium and integrin binding protein 1 (CIB1), which binds and inhibits the IP3R, elevated cytosolic calcium in keratinocytes and suppressed UV-induced γH2AX. Knockdown of Apoptosis signal-regulating kinase 1 (ASK1), which can interact with IRE1α through TRAF2 as well as CIB1, restored normal levels of UV-induced γH2AX to IRE1α knockdown keratinocytes. Together these results show that IRE1α is essential for maintaining normal cellular calcium and ROS levels and the UV-induced repair response, and suggest a model where IRE1a activation state drives CIB1 binding either to the IP3R or ASK1 to regulate ER calcium efflux, ROS and DNA repair responses following UV. Citation Format: Jeongin Son, Saie Mogre, Adam Glick. The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2029.

The Role of Unfolded Protein Response in Human Intervertebral Disc Degeneration: Perk and IRE1-α as Two Potential Therapeutic Targets.

Inflammation plays a key role in intervertebral disc degeneration (IDD). The association between inflammation and endoplasmic reticulum (ER) stress has been observed in many diseases. However, whether ER stress plays an important role in IDD remains unclear. Therefore, this study is aimed at investigating the expression of ER stress in IDD and at exploring the underlying mechanisms of IDD, ER stress, and inflammation. The expression of ER stress was activated in nucleus pulposus cells from patients who had IDD (D-NPCs) compared with patients without IDD (N-NPCs); and both the proliferation and synthesis capacity were decreased by inducer tunicamycin (Tm) and proinflammatory cytokines. Pretreatment of NPCs with 4-phenyl butyric acid (4-PBA) prevented the inflammatory cytokine-induced upregulation of unfolded protein response- (UPR-) related proteins and recovered cell synthetic ability. Furthermore, proinflammatory cytokine treatment significantly upregulated the expression of inositol-requiring protein 1 (IRE1-α) and protein kinase RNA-like ER kinase (PERK), but not activating transcription factor 6 (ATF6). Finally, knockdown of IRE1-α and PERK also restored the biological activity of NPCs. Our findings identified that IRE1-α and PERK might be the potential targets for IDD treatment, which may help illustrate the underlying mechanism of ER stress in IDD.

Gambogenic acid induces Noxa-mediated apoptosis in colorectal cancer through ROS-dependent activation of IRE1α/JNK

BackgroundGambogenic acid (GNA), an active component of Garcinia hanburyi Hook.f. (Clusiaceae) (common name gamboge), exerts anti-inflammatory and antitumor properties. However, the underlying mechanism of GNA in colorectal cancer (CRC) is still not well understood. PurposeThis study aimed to investigate the antitumor effects and mechanisms of GNA on CRC in vitro and in vivo. MethodsCell viability, colony formation and cell apoptosis assays were performed to determine the antitumor effects of GNA. qRT-PCR and Western blotting were performed to evaluate the expression of genes or proteins affected by GNA in vitro and in vivo. HCT116 colon cancer xenografts and the APCmin/+ mice model were used to confirm the antitumor effects of GNA on CRC in vivo. ResultsGNA induced Noxa-mediated apoptosis by inducing reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation. Moreover, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme-1α (IRE1α) leading to JNK phosphorylation. ROS scavenger attenuated GNA-induced IRE1α activation and JNK phosphorylation. Knockdown of IRE1α also prevented GNA-induced JNK phosphorylation. In vivo, GNA suppressed tumor growth and progression in HCT116 colon cancer xenografts and the APCmin/+ mices model. ConclusionThese findings revealed that GNA induced Noxa-mediated apoptosis by activating the ROS/IRE1α/JNK signaling pathway in CRC both in vitro and in vivo. GNA is therefore a promising antitumor agent for CRC treatment.

Abstract 5840: IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation

Abstract IRE1α is required for ROS homeostasis and PERK protein stability in response to UVB irradiation UV induced stress is one of the primary driving factors of human skin cancer. UV irradiation can activate Endoplasmic Reticulum (ER) stress and the Unfolded Protein Response (UPR). Inositol-requiring enzyme 1a (IRE1α) is one of the key players in the UPR. Previous studies have shown that UVB irradiation can activate IRE1α in human and mouse skin cells, but the function of IRE1α in the UV response has not yet been characterized. Here we show that IRE1α expression is transiently downregulated after UVB irradiation and that in the absence of IRE1α the stability and activation of PKR-like endoplasmic reticulum kinase (PERK) is compromised. Basal levels of PERK are reduced in the absecence of IRE1α. Following UV irradiation, loss of IRE1α not only causes rapid degradation of PERK protein but also suppresses phosphorylation of the PERK target, Nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2 dependent antioxidant gene expression. Similarly, PERK protein is rapidly degraded following thapsigargin treatment in the absence of IRE1α, suggesting a general regulation of PERK stability by IRE1α. Additionally, keratinocytes with either knockout or knockdown of IRE1α have increased intracellular ROS and cytosolic calcium. Overexpression of both wildtype and RNase defective human IRE1α can rescue the increased ROS levels. Additionally, the increased ROS is also suppressed by treatment with an inhibitor of the ER localized Inositol triphosphate receptor (InsP3R) which regulates calcium efflux from the ER. Because PERK regulates cytosolic calcium through inhibiting InsP3R, our result suggests that IRE1α is critical for suppressing intracellular ROS through stabilization of PERK. We conclude that crosstalk between IRE1α and PERK is essential for the basal and UVB- induced oxidative stress response, and this may have important implications for UV related damage and skin cancer development. Citation Format: Jeongin Son, Stephen Worrel, Adam Glick. IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5840.

Suppression of IRE1α Attenuated the Fatty Degeneration in Parenteral Nutrition-Related Liver Disease (PNALD) Cell Model.

Aims To model the parenteral nutrition-associated liver disease (PNALD) in rat normal hepatocytes BRL and investigate the role of endoplasmic reticulum stress- (ERS-) related IRE1α signal in the process of PNALD. Methods The BRL cells were treated with different concentrations of soybean oil emulsion (SO) to induce hepatocyte fatty degeneration. The PNALD cell disease model was further confirmed by analysis of Oil Red O staining and biochemical parameters. Next, the IRE1α signal in the process of PNALD. α signal in the process of PNALD. α signal in the process of PNALD. α signal in the process of PNALD. Results The results of Oil Red O staining indicated that the PNALD was successfully established in BRL cells and the CCK-8 data indicated which 0.6% that SO was further applied to the experiment owing to its better induction of PNALD and less toxicity to the cells. Besides, the value of biochemical parameters (TBIL, DBIL, ALT, and AST) was also elevated in the SO group compared with the NG group. After knockdown of IRE1α signal in the process of PNALD. α signal in the process of PNALD. Conclusion IRE1α was induced in PNALD cell model and suppression of IRE1α resulted in reduced steatosis in this cell disease model. Taken together, our data suggested that the IRE1α pathway may be involved in the development of PNALD.α signal in the process of PNALD. α signal in the process of PNALD. α signal in the process of PNALD.

Knockdown of IRE1ɑ suppresses metastatic potential of colon cancer cells through inhibiting FN1-Src/FAK-GTPases signaling.

The inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-resident transmembrane protein and senses cellular unfolded/misfolded proteins. Upon activation, IRE1α removes a 26-bp nucleotide from the mRNA encoding X-box binding protein (XBP) 1 to generate a spliced active form of this transcription factor (XBP1s). Though IRE1α is implicated in development of cancer, the role and underlying mechanism remain unclear. Here, we demonstrate that IRE1α regulates colon cancer cell metastasis through regulating the expression of fibronectin-1 (FN1). We found that knockdown of IRE1α inhibited colon cancer cell migration and invasion in vitro and metastasis in vivo. Knockdown of IRE1α decreased the formation of XBP1s and attenuated the expression of FN1, leading to inhibition of phosphorylation of Src and FAK and inactivation the downstream effector GTPases including RhoA, Rac1 and CDC42. Addition of exogenous FN1 reversed Src/FAK phosphorylation and cell migration inhibited by IRE1α knockdown. We found that XBP1s bound FN1 promoter and acted as a transcription factor to initiate FN1 expression. Our results suggest that IRE1α modulates metastatic potential of colon cancer cells through regulating the expression of FN1.

T-2 toxin inhibits the production of mucin via activating the IRE1/XBP1 pathway

T-2 toxin is a trichothecene mycotoxin that widely contaminates food and has a variety of toxic effects. However, the underlying mechanism of T-2 toxin on intestinal mucin remains unclear. In present study, human intestinal Caco-2 cells and HT-29 cells were treated with 100 ng/mL T-2 toxin at one-quarter of the IC50 for 24 h, which caused the inhibition of MUC2 and adhesion of E. coli O157:H7. We found T-2 toxin induced endoplasmic reticulum stress and activated the IRE1/XBP1 pathway, which may be related to the inhibition of MUC2. Interestingly, T-2 toxin activated IRE1α to inhibit IRE1β, which optimized mucin production. Furthermore, overexpression of IRE1β in the cells apparently alleviated the inhibition of MUC2 caused by T-2 toxin. IRE1α knock-down blocked the down-regulation of IRE1β and MUC2 induced by T-2 toxin. We revealed the critical role of IRE1α in the inhibition of intestinal mucin. This finding was confirmed in BALB/c mice which were exposed to T-2 toxin (0.5 mg/kg bw) for 4 weeks. T-2 toxin activated the IRE1/XBP1 pathway to disrupt intestinal mucin, which lead to the imbalance of gut microbiota and an increased risk of host infection by E. coli O157:H7. T-2 toxin exposure also increased the expressions of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in mice, which might respond to IRE1α activation. Importantly, IRE1α activation was a therapeutic target for intestinal inflammation caused by T-2 toxin. This study provided a new perspective to understand the intestinal toxicity of T-2 toxin.

Naringenin ameliorates insulin resistance by modulating endoplasmic reticulum stress in hepatitis C virus-infected liver

Hepatitis C virus (HCV) infection may lead to hepatic insulin resistance (IR), and endoplasmic reticulum (ER) stress has been found to induce IR. In our previous study, naringenin (NGEN) had an insulin sensitization effect on the HCV core protein (HCVCP) infected mouse livers. In the present study, we examined the effects of NGEN on HCVCP infection-induced ER stress and investigated the insulin sensitization mechanism involved. We found that XBP1s was up-regulated in the livers of HCV-infected patients, in hepatocytes with HCV infection, and in HCVCP-infected mice. HCVCP induces ER stress in the mouse liver and hepatocytes by increasing XBP1s and downstream gene expression. Pre-treatment with NGEN inhibited the ER stress and downstream gene expression both in vivo and in vitro. Similar to the HCVCP infection results, NGEN also inhibited the ER stress in tunicamycin-treated Huh-7.5.1 cells. In addition, the role of IRE1α in HCVCP-induced IR was detected, and knockdown of IRE1α abolished HCVCP-stimulated IR. Overexpression induced IR but could be abolished by NGEN. NGEN also blocked the HCVCP-induced IRE1α expression levels that were up-regulated in vivo. Our data reveal that ER stress may be associated with HCV-induced IR, and NGEN treatment inhibited ER stress activity and increased insulin sensitivity by decreasing the expression of IRE1α.

IRE1α is critical for Kaempferol-induced neuroblastoma differentiation.

Neuroblastoma is an embryonic malignancy that arises out of the neural crest cells of the sympathetic nervous system. It is the most common childhood tumor known for its spontaneous regression via the process of differentiation. The induction of differentiation using small molecules such as retinoic acid is one of the therapeutic strategies to treat the residual disease. In this study, we have reported the effect of kaempferol (KFL) in inducing differentiation of neuroblastoma cells invitro. Treatment of neuroblastoma cells with KFL reduced the proliferation and enhanced apoptosis along with the induction of neuritogenesis. Analysis of the expression of neuron-specific markers such as β-III tubulin, neuron-specific enolase, and N-myc downregulated gene 1 revealed the process of differentiation accompanying KFL-induced apoptosis. Further analysis to understand the molecular mechanism of action showed that the effect of KFL is mediated by the activation of the endoribonuclease activity of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum-resident transmembrane protein. Insilico docking analysis and biochemical assays using recombinant human IRE1α confirm the binding of KFL to the ATP-binding site of IRE1α, which thereby activates IRE1α ribonuclease activity. Treatment of cells with the small molecule STF083010, which specifically targets and inhibits the endoribonuclease activity of IRE1α, showed reduced expression of neuron-specific markers and curtailed neuritogenesis. The knockdown of IRE1α using plasmid-based shRNA lentiviral particles also showed diminished changes in the morphology of the cells upon KFL treatment. Thus, our study suggests that KFL induces differentiation of neuroblastoma cells via the IRE1α -XBP1 pathway.

IRE1α aggravates ischemia reperfusion injury of fatty liver by regulating phenotypic transformation of kupffer cells

Fatty liver is one of the widely accepted marginal donor for liver transplantation, but is also more sensitive to ischemia and reperfusion injury (IRI) and produces more reactive oxygen species (ROS). Moreover, so far, no effective method has been developed to alleviate it. Endoplasmic reticulum stress (ER-stress) of hepatocyte is associated with the occurrence of fatty liver disease, but ER-stress of kupffer cells (KCs) in fatty liver is not clear at all. This study evaluates whether ER-stress of KCs is activated in fatty liver and accelerate IRI of fatty livers.ER-stress of KCs was activated in fatty liver, especially the IRE1α signal pathway. KCs with activated ER-stress secreted more proinflammatory cytokine to induce its M1-phenotypic shift in fatty liver, resulting in more severe IRI. Also, activated ER-stress of BMDMs in vitro by tunicamycin can induce its pro-inflammatory shift and can be reduced by 4-PBA, an ER-stress inhibitor. Knockdown of IRE1α could regulate the STAT1 and STAT6 pathway of macrophage to inhibit the M1-type polarization and promote M2-phenotypic shift. Furthermore, transfusion of IRE1α-knockdown KCs significantly reduced the liver IRI as well as the ROS of HFD feeding mice. Altogether, these data demonstrated that IRE1α of KCs may be a potential target to reduce the fatty liver associated IRI in liver transplantation.

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.

Autophagy mediated by endoplasmic reticulum stress enhances the caffeine-induced apoptosis of hepatic stellate cells.

Caffeine has been identified to have beneficial effects against chronic liver diseases, particularly liver fibrosis. Many studies have reported that caffeine ameliorates liver fibrosis by directly inducing hepatic stellate cell (HSC) apoptosis; however, the molecular mechanisms involved in this process remain unclear. The presents study aimed to detect the underlying mechanisms by which caffeine mediates HSC apoptosis and eliminates activated HSCs. For this purpose, the LX-2 cell line was applied in this study and the cells were exposed to various concentrations of caffeine for the indicated times. The effects of caffeine on cell viability and apoptosis were assessed by Cell Counting Kit-8 assay and flow cytometry, respectively. Autophagy and endoplasmic reticulum (ER) stress were explored by morphological assessment and western blotting. In the present study, caffeine was found to inhibit the viability and increase the apoptosis of the LX-2 cells in dose- and time-dependent manners. ER stress was stimulated by caffeine as demonstrated by increased levels of GRP78/BiP, CHOP and inositol-requiring enzyme 1 (IRE1)-α as well as many enlarged ERs detected by electron microscopy. Caffeine induced autophagy as shown by increased p62 and LC3II accumulation and the number of GFP/RFP-LC3 puncta and autophagosomes/autolysosomes. Moreover, IRE1-α knockdown decreased the level of autophagic flux, and inhibition of autophagy protected LX-2 cells from apoptotic death. In conclusion, our study showed that the caffeine-enhanced autophagic flux in HSCs was stimulated by ER stress via the IRE1-α signaling pathway, which further weakened HSC viability via the induction of apoptosis. These findings provide new insight into the mechanism of caffeine's anti-fibrotic effects.

Knockdown of IRE1α inhibits colonic tumorigenesis through decreasing β-catenin and IRE1α targeting suppresses colon cancer cells.

The endoplasmic reticulum (ER) stress occurs frequently in cancers. The unfolded protein response (UPR) is activated to cope with ER stress. This has generated widespread interest in targeting UPR as therapeutic strategies. Inositol-requiring transmembrane kinase/endonuclease 1α (IRE1α), an ER stress sensor, is a key component of UPR. However, the role of IRE1α in tumorigenesis remains unclear. The purpose of this work is to investigate the role of IRE1α in colon cancer and to determine whether IRE1α could serve as a target for therapy. We found that knockdown of IRE1α suppressed the proliferation of colon cancer cells in vitro and xenograft growth in vivo. Inhibition of expression of IRE1α decreased stemness of colon cancer stem cells (CSCs) and attenuated growth of intestinal organoids. Genetic ablation of IRE1α prevented the colitis-associated colonic tumorigenesis in mice. The mechanistic study indicates that knockdown of IRE1α repressed the expression of β-catenin, a key factor that drives colonic tumorigenesis, through activating pancreatic ER kinase/eukaryotic translation initiation factor 2α signaling. We found that the IRE1a-specific inhibitor 4μ8C could suppress the production of β-catenin, inhibited the proliferation of colon cancer cells, repressed colon CSCs and prevented xenograft growth. The results suggest that IRE1α has a critical role in colonic tumorigenesis and IRE1α targeting might be a strategy for treatment of colon cancers.

O43 The effects and mechanisms of two main constituents from Glycyrrhiza uralensis Fisch-induced autophagy in non-small cell lung cancer

The Glycyrrhiza uralensis Fisch, one of the famous Chinese medicinal herbs, has been widely used in anti-cancer prescription. We found that glycerrhetinic acid (GA, triterpenoid) and licochalcone A (LCA, flavonoid), two primary constituents of Glycyrrhiza uralensis Fisch, induced cell proliferative inhibition, apoptosis, and autophagy in non-small cell lung cancer (NSCLC) A549 and NCI-H1299 cells. Combined treatment with chloroquine further enhanced GA- or LCA-induced expression of LC3-II and more red-fluorescent puncta than green ones were observed in GA- or LCA-treated cells with transfection of mRFP-EGFP-LC3, suggesting that GA and LCA induces autophagic flux in NSCLC cells. Inhibition of autophagy enhanced GA-induced cell proliferative inhibition and apoptosis in NSCLC cells. In contrast, LCA-induced cell proliferative inhibition and apoptosis were not significantly altered after autophagy inhibition in NSCLC cells. The JNK and IRE1 α were activated after incubation with GA. Pretreatment with the JNK inhibitor SP600125 or silencing of the JNK pathway by siRNA of JNK or c-jun decreased GA-induced autophagy. Moreover, the GA-induced JNK pathway activation and autophagy were decreased by IRE1α knockdown, suggesting that GA induces autophagy through activation of IRE1α-JNK/c-jun pathway. LCA increased the expression of CHOP, and knockdown of CHOP reversed LCA-induced autophagy, suggesting that LCA-induced autophagy is due to induction of CHOP expression. Collectively, both GA and LCA induced cell proliferative inhibition, apoptosis, and autophagy in NSCLC, and inhibition of autophagy might be an effective strategy to enhance the anti-NSCLC effects of Glycyrrhiza uralensis Fisch contained prescriptions.