Inositol-requiring Enzyme 1α Research Articles

The Ire of IRE1α: Overexpression of IRE1α at Myeloma Diagnosis Is Associated with Decreased Survival While Downregulation of IRE1α Expression Is Predictive of Therapy Resistance

Introduction: Inositol-requiring enzyme 1α (IRE1α) is one of three principal transmembrane sensors in the endoplasmic reticulum (ER) involved in regulating the unfolded protein response (UPR). It splices X-box protein 1 (XBP1) mRNA yielding a transcription factor intimately involved in the plasma cell differentiation and proliferation. The IRE1α-XBP1 axis exhibits positive feedback functions that promote the pro-survival UPR and allows plasma cells to survive the high biosynthetic burden of immunoglobulin production, although IRE1α also has functions that promote cell death depending on the chronicity and severity of ER stress. Functional IRE1α has been identified as critical for in vitro cytotoxicity of bortezomib by Leung-Hagesteijn et al. (Cancer Cell 2013), with loss of IRE1α leading to in vitro resistance to proteasome inhibition. However, Mimura et al (Blood 2012) identified in vitro synergy with IRE1α-inhibition and bortezomib, and Papandeou et al. (Blood 2011) identified in vitro and in vivo anti-myeloma activity of a different IRE1α endonuclease inhibitor. The clinical implications of IRE1α are still poorly understood. In this study, we sought to identify associations between IRE1α mRNA expression (the level at which it is principally regulated) with treatment resistance and patient outcomes. Methods: We performed a secondary analysis of data from the MMRF CoMMpass study (IA14). In the CoMMpass study, RNAseq on CD138-enriched bone marrow cells was performed using Illumina TruSeq RNA library kits. Cox Regression analysis was used to compare the association of IRE1α expression with progression-free and overall survival. To determine if IRE1α expression is modulated following disease progression, we also identified patients who had pre- and post-treatment RNAseq data. We analyzed expression of IRE1α from the pre- and post-treatment specimens using paired T-tests comparing patients who had relapsed disease (relapse following completion of therapy) and refractory disease (progression on or within 60 days of completing therapy). Results: We included 768 patients who had IRE1α expression data at myeloma diagnosis. The median age was 63 (range 27-93) and 50% were male. Thirty percent of patients (n = 231) were considered to have high IRE1a expression at myeloma diagnosis. High IRE1α expression was associated with worse clinical outcome. Those with high expression had a 37% (aHR 1.37; 95% CI 1.10-1.72; p = 0.006) increase hazard for progression and a 55% (aHR 1.55; 95% CI 1.13-2.15; p = 0.008) increase hazard for death. We then identified 46 patients who had pre- and post-treatment RNAseq data. Twenty-two had relapsed disease while off of treatment and 24 had developed refractory disease while on treatment. Patients who had refractory disease were noted to have a significant decrease in the expression of IRE1α (p = 0.008). This was similar when we performed subset analysis of patients who were refractory to PI-based treatments (n=8; p = 0.044) and IMID-based treatments (n = 13; p = 0.054). In contrast, there was not a significant difference between pre-treatment and post-treatment IRE1α in relapsed disease (p = 0.312). Expression of other UPR elements, specifically PERK and XBP1, was not significantly downregulated in relapsed or refractory disease. Conclusion: Our study demonstrates that high expression of IRE1α at diagnosis is associated with worse outcomes, but that resistance to therapy is associated with a significant decrease in IRE1α expression. While this seems counterintuitive, it highlights the complex nature of the UPR and the roles of IRE1α. More aggressive/proliferative disease relies heavily on an adapted pro-survival UPR, highlighted by increased expression of IRE1α de novo. However, IRE1α has previously been demonstrated to be required for anti-myeloma activity of novel therapies; potentially either by clonal selection, dynamic alterations in UPR signaling, or epigenetic modifications, treatment-refractory myeloma cells downregulate the expression of IRE1α, a phenomenon not seen in those who completed treatment but subsequently relapsed. IRE1α inhibition could initially potentiate the clinical effects of novel therapies in a higher-risk population, but may also promote later resistance to them. Importantly, IRE1α could serve as a prognostic biomarker for risk-stratification at diagnosis as well as one predictive of impending resistance to therapy. Disclosures Fiala: Incyte: Research Funding. Wildes:Janssen: Research Funding; Carevive: Consultancy. Vij:Takeda: Honoraria, Research Funding; Sanofi: Honoraria; Karyopharm: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria. Stockerl-Goldstein:AbbVie: Equity Ownership; Abbott: Equity Ownership.

Effect of Kinase Inhibiting RNase Attenuator (KIRA) Compounds on the Formation of Face-to-Face Dimers of Inositol-Requiring Enzyme 1: Insights from Computational Modeling.

Inositol-requiring enzyme 1α (IRE1α) is a transmembrane dual kinase/ribonuclease protein involved in propagation of the unfolded protein response (UPR). Inositol-requiring enzyme 1α is currently being explored as a potential drug target due to the growing evidence of its role in variety of disease conditions. Upon activation, IRE1 cleaves X-box binding protein 1 (XBP1) mRNA through its RNase domain. Small molecules targeting the kinase site are known to either increase or decrease RNase activity, but the allosteric relationship between the kinase and RNase domains of IRE1α is poorly understood. Subsets of IRE1 kinase inhibitors (known as “KIRA” compounds) bind to the ATP-binding site and allosterically impede the RNase activity. The KIRA compounds are able to regulate the RNase activity by stabilizing the monomeric form of IRE1α. In the present work, computational analysis, protein–protein and protein–ligand docking studies, and molecular dynamics simulations were applied to different IRE1 dimer systems to provide structural insights into the perturbation of IRE1 dimers by small molecules kinase inhibitors that regulate the RNase activity. By analyzing structural deviations, energetic components, and the number of hydrogen bonds in the interface region, we propose that the KIRA inhibitors act at an early stage of IRE1 activation by interfering with IRE1 face-to-face dimer formation thus disabling the activation of the RNase domain. This work sheds light on the mechanism of action of KIRA compounds and may assist in development of further compounds in, for example, cancer therapeutics. The work also provides information on the sequence of events and protein–protein interactions initiating the unfolded protein response.

Anti-Inflammatory Effects of HDL (High-Density Lipoprotein) in Macrophages Predominate Over Proinflammatory Effects in Atherosclerotic Plaques.

HDL (high-density lipoprotein) infusion reduces atherosclerosis in animal models and is being evaluated as a treatment in humans. Studies have shown either anti- or proinflammatory effects of HDL in macrophages, and there is no consensus on the underlying mechanisms. Here, we interrogate the effects of HDL on inflammatory gene expression in macrophages. Approach and Results: We cultured bone marrow-derived macrophages, treated them with reconstituted HDL or HDL isolated from APOA1Tg;Ldlr-/- mice, and challenged them with lipopolysaccharide. Transcriptional profiling showed that HDL exerts a broad anti-inflammatory effect on lipopolysaccharide-induced genes and proinflammatory effect in a subset of genes enriched for chemokines. Cholesterol removal by POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) liposomes or β-methylcyclodextrin mimicked both pro- and anti-inflammatory effects of HDL, whereas cholesterol loading by POPC/cholesterol-liposomes or acetylated LDL (low-density lipoprotein) before HDL attenuated these effects, indicating that these responses are mediated by cholesterol efflux. While early anti-inflammatory effects reflect reduced TLR (Toll-like receptor) 4 levels, late anti-inflammatory effects are due to reduced IFN (interferon) receptor signaling. Proinflammatory effects occur late and represent a modified endoplasmic reticulum stress response, mediated by IRE1a (inositol-requiring enzyme 1a)/ASK1 (apoptosis signal-regulating kinase 1)/p38 MAPK (p38 mitogen-activated protein kinase) signaling, that occurs under conditions of extreme cholesterol depletion. To investigate the effects of HDL on inflammatory gene expression in myeloid cells in atherosclerotic lesions, we injected reconstituted HDL into Apoe-/- or Ldlr-/- mice fed a Western-type diet. Reconstituted HDL infusions produced anti-inflammatory effects in lesion macrophages without any evidence of proinflammatory effects. Reconstituted HDL infusions in hypercholesterolemic atherosclerotic mice produced anti-inflammatory effects in lesion macrophages suggesting a beneficial therapeutic effect of HDL in vivo.

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.

The IRE1α Arm of UPR Regulates Muscle Cells Immune Characters by Restraining p38 MAPK Activation

Skeletal muscle repair and systemic inflammation/immune responses are linked to endoplasmic reticulum stress (ER stress) pathways in myopathic muscle, and muscle cells play an active role in muscular immune reactions by exhibiting immunological characteristics under persistent proinflammation stimuli. Whether ER stress affects the intrinsic immunological capacities of myocytes in the inflammatory milieu, as it does to immune cells, and which arms of the unfolded protein response (UPR) mainly participate in these processes remain mostly unknown. We investigated this issue and showed that inflammatory stimuli can induce the activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α) arms of the UPR in myocytes both in vivo and in vitro. UPR stressor administration reversed the increased IFN-γ-induced expression of the MHC-II molecule H2-Ea, the MHC-I molecule H-2Kb, toll-like receptor 3 (TLR3) and some proinflammatory myokines in differentiated primary myotubes in vitro. However, further IRE1α inhibition thoroughly corrected the trend in the UPR stressor-triggered suppression of immunobiological molecules. In IFN-γ-treated myotubes, dramatic p38 MAPK activation was observed under IRE1α inhibitory conditions, and the pharmacological inhibition of p38 reversed the immune molecule upregulation induced by IRE1α inhibition. In parallel, our coculturing system verified that the ovalbumin (OVA) antigen presentation ability of inflamed myotubes to OT-I T cells was enhanced by IRE1α inhibition, but was attenuated by further p38 inhibition. Thus, the present findings demonstrated that p38 MAPK contributes greatly to IRE1α arm-dependent immunobiological suppression in myocytes under inflammatory stress conditions.

Mitochondrial retrograde signaling to the endoplasmic-reticulum regulates unfolded protein responses

ABSTRACTUnfolded protein response (UPRs) directs adaption or apoptosis depending on the severity of endoplasmic-reticulum (ER) stress. We found that apoptotic signaling by inositol requiring enzyme 1α (IRE1α), a transducer of UPRs, is suppressed by mitochondrial ubiquitin ligase MITOL/MARCH5 on ER-mitochondria contacts, suggesting that mitochondria regulate cell fate under ER stress.

Dual role of Endoplasmic Reticulum Stress-Mediated Unfolded Protein Response Signaling Pathway in Carcinogenesis.

Cancer constitutes a grave problem nowadays in view of the fact that it has become one of the main causes of death worldwide. Poor clinical prognosis is presumably due to cancer cells metabolism as tumor microenvironment is affected by oxidative stress. This event triggers adequate cellular response and thereby creates appropriate conditions for further cancer progression. Endoplasmic reticulum (ER) stress occurs when the balance between an ability of the ER to fold and transfer proteins and the degradation of the misfolded ones become distorted. Since ER is an organelle relatively sensitive to oxidative damage, aforementioned conditions swiftly cause the activation of the unfolded protein response (UPR) signaling pathway. The output of the UPR, depending on numerous factors, may vary and switch between the pro-survival and the pro-apoptotic branch, and hence it displays opposing effects in deciding the fate of the cancer cell. The role of UPR-related proteins in tumorigenesis, such as binding the immunoglobulin protein (BiP) and inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6) or the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), has already been specifically described so far. Nevertheless, due to the paradoxical outcomes of the UPR activation as well as gaps in current knowledge, it still needs to be further investigated. Herein we would like to elicit the actual link between neoplastic diseases and the UPR signaling pathway, considering its major branches and discussing its potential use in the development of a novel, anti-cancer, targeted therapy.

Changes in endoplasmic reticulum stress in spinal cord of rats with diabetic neuropathic pain

Objective To evaluate the changes in the endoplasmic reticulum stress in the spinal cord of rats with diabetic neuropathic pain (DNP). Methods Clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 120-160 g, were fed a high-fat and high-glucose diet for 8 weeks, then diabetes mellitus was induced by intraperitoneal streptozotocin 35 mg/kg and confirmed by blood glucose level >16.7 mmol/L 3 days later.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at day 14 after injection.The establishment of DNP model was considered successful when MWT and TWL were lower than 85% of the baseline value.Fifteen rats in which the DNP model was successfully established served as DNP group, 15 rats in which the DNP model was not successfully established served as non-NDNP group (NDNP), and another 15 normal rats were selected and served as control group (group C). The MWT and TWL were measured at 3, 7 and 14 days after successful establishment of the model.The rats were then sacrificed, and the lumbar enlargement segments (L4-6) of the spinal cord were harvested to detect the expression of inositol-requiring enzyme-1α, phosphorylated JNK (p-JNK) and Beclin1 by Western blot. Results Compared with C and NDNP groups, the MWT was significantly decreased and the TWL was shortened at 3, 7 and 14 days after successful establishment of the model, and the expression of inositol-requiring enzyme-1α, p-JNK and Beclin1 was up-regulated in DNP group (P<0.05). Conclusion The mechanism of development and maintenance of DNP may be related to aggravated endoplasmic reticulum stress in the spinal cord and to autophagy induced by up-regulated expression of p-JNK in rats. Key words: Diabetic neuropathies; Endoplasmic reticulum stress; Spinal cord

Structural distortions induced by Kinase Inhibiting RNase Attenuator (KIRA) compounds prevent the formation of face-to-face dimers of Inositol Requiring Enzyme 1α

Structural distortions induced by Kinase Inhibiting RNase Attenuator (KIRA) compounds prevent the formation of face-to-face dimers of Inositol Requiring Enzyme 1α

The Increased RNase Activity of IRE1α in PBMCs from Patients with Rheumatoid Arthritis.

Purpose: Despite recent advances in the diagnosis and treatment of rheumatoid arthritis (RA), this inflammatory disease remains a challenge to patients and physicians. Recent evidence highlights the contribution of endoplasmic reticulum (ER) stress in the pathogenesis and treatment of RA. Herein, we study the expression of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), as well as XBP1 splicing and the regulated IRE1-dependent decay (RIDD), in peripheral blood mononuclear cells (PBMCs) from patients with RA compared with healthy controls. Methods: The PBMCs from blood samples of RA patients and healthy volunteers were isolated by a density gradient centrifugation method using Ficoll. The gene expression levels of GRP78/ Bip, IRE1, XBP1s, micro-RNAs (miRNAs) were evaluated by real-time PCR. Results: The expression of GRP78, IRE1, and XBP1s were increased in PBMCs of RA patients compared with healthy controls. We further show that the RIDD targets (miRNA-17, -34a, -96, and -125b) were downregulated in RA samples. Conclusion: This study can expand our knowledge on the importance of RNase activity of IRE1α in RA and may offer new potentials for developing novel diagnostic and/or therapeutic biomarkers.

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.

BDE-209 induces autophagy and apoptosis via IRE1α/Akt/mTOR signaling pathway in human umbilical vein endothelial cells

Recently, the essentiality and fatalness of cardiovascular diseases is attracting much attention. Polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants, which could induce the toxic effect and have been implicated in the occurrence and development of cardiovascular diseases. However, it is unclear how autophagy and apoptosis induced by BDE-209 in endothelial cells are regulated. The aim of the present study was to investigate the effects of BDE-209 on human umbilical vein endothelial cells (HUVECs) and elucidate the mechanisms involved. HUVECs were treated with a wide range concentration of BDE-209 for 24 h. The appearance of autophagy was tested by the testing index such as outcomes of monodansylcadaverine (MDC) staining and lysotracker staining, observation of autophagosomes and conversion between autophagy marker light chain 3 (LC3)-I and LC3-II. Besides, the apoptotic cell rate was detected with flow cytometry. In addition, BDE-209 induced endoplasmic reticulum (ER) stress was detected by transmission electron microscopy (TEM). Our data suggest that the exposure of BDE-209 could induce autophagy, which was confirmed by MDC staining, transmission electron microscopy observation, lysotracker staining and LC3-I/LC3-II conversion. Besides, the ER stress-related inositol-requiring enzyme 1α (IRE1α)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway could be activated by reactive oxygen species (ROS) to regulate autophagy. Moreover, the apoptosis of endothelial cells was alleviated when autophagy was blocked by 3-Methyladenine (3-MA). The results demonstrated that BDE-209 could induce the production of ROS and ER stress, activate autophagy through IRE1α/AKT/mTOR signaling pathway and ultimately induce apoptosis of vascular endothelial cells. These findings indicate that exposure to PBDE is possible to be a potential risk factor for cardiovascular diseases.

Human placenta-derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice.

Previous studies have shown that the ovarian failure in autoimmune-induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta-derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol-requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X-box binding protein 1 (XBP1), up-regulated 78 kDa glucose-regulated protein (GRP78) and caspase-12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis-induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation-mediating ovarian function recovery.

Interferon Regulatory Factor 3 Deficiency Induces Age-Related Alterations of the Retina in Young and Old Mice.

Age-related changes in visual function and retina structure are very common in aged animals, but the underlying mechanisms of these changes remain unclear. Here we report that the expression of interferon regulatory factor 3 (IRF3), a critical immune regulatory factor, is dramatically down-regulated in mouse retinas during aging. To address the role of IRF3 in the retina, we examined the structure and function of retinas in young (3–4 months) and old (22–24 months) Irf3-/- mice in comparison to age-matched wildtype (WT) mice. We found that IRF3 deletion resulted in impaired electroretinogram (ERG) responses and decreased retinal thickness in both young and old mice. In addition, numerous synapses of the outer plexiform layer (OPL) were found obviously extending into outer nuclear layer (ONL) in Irf3-/- mice, along with a reduction of the average synapse density in the OPL. These changes suggest that IRF3 deletion may accelerate retinal senescence. In support of this hypothesis, a number of classic senescence-associated markers were found in remarkably elevated level in Irf3-/- retina, including p53, p16INK4a, inositol-requiring enzyme 1α (IREα), p-H2A.X and promyelocytic leukemia protein (PML). Overall, our results indicate that maintenance normal IRF3 levels is necessary for retinal structure and function and suggest that IRF3 is an important regulator of retinal senescence.

Gambogic Acid Shows Anti-Proliferative Effects on Non-Small Cell Lung Cancer (NSCLC) Cells by Activating Reactive Oxygen Species (ROS)-Induced Endoplasmic Reticulum (ER) Stress-Mediated Apoptosis.

BackgroundGambogic acid (AG) is believed to be a potent anti-cancer agent. ER (endoplasmic reticulum) stress-induced cell apoptosis was identified as one of the anti-proliferative mechanisms of several anti-cancer agents. In this study, we investigated the involvement of ER stress-induced apoptosis in the anti-proliferative effect of GA on NSCLC (non-small cell lung cancer) cells.Material/MethodsGA at 0, 0.5, and 1.0 μmol/l was used to treat A549 cells. We also used the ER stress-specific inhibitor 4-PBA (4-phenylbutyric acid) (1 μmol/l) to co-treat the cells incubated with GA. Cell viability was assessed by MTT (methyl thiazolyl tetrazolium) assay. Cell apoptosis was evaluated by MTT (methyl thiazolyl tetrazolium) assay. Intracellular ROS (reactive oxygen species) production was detected by DCFH-DA (2,7- dichloro-dihydrofluorescein diacetate) florescent staining. Western blotting was used to assess the expression and phosphorylation levels of protein.ResultsGA treatment significantly reduced cell viabilities of NSCLC cells in a concentration-dependent manner. GA treatment increased intracellular ROS level, expression levels of GRP (glucose-regulated protein) 78, CHOP (C/EBP-homologous protein), ATF (activating transcription factor) 6 and caspase 12, as well as the phosphorylation levels of PERK (protein kinase R-like ER kinase) and IRE (inositol-requiring enzyme) 1α. Co-treatment of 4-PBA dramatically impaired the inhibitory effect of GA on cell viability. 4PBA co-treatment also decreased expression levels of GRP78, CHOP, ATF6, and caspase12, as well as the phosphorylation levels of PERK and IRE1α, in GA-treated NSCLC cells, without affecting ROS levels.ConclusionsGA inhibited NSCLC cell proliferation by inducing ROS-induced ER stress-medicated apoptosis of NSCLC cells.

Dual effects of gossypol on human hepatocellular carcinoma via endoplasmic reticulum stress and autophagy.

Treatment outcomes for hepatocellular carcinoma (HCC) remain unsatisfactory, and effective new therapeutic methods are urgently needed. Gossypol has been shown to have an anti-HCC effect, but the underlying mechanism requires further study. In this study, we found gossypol inhibited HCC cells in vitro and in vivo. Typical apoptosis was induced in HCC cells. Dilated ER and autophagosomes were observed by electron microscopy, and the activation of the unfolded protein response and autophagy markers suggested that gossypol induced both ER stress and autophagy. C/EBP homologous protein was the key factor that led to apoptotic cell death, whereas inositol-requiring enzyme 1α and eukaryotic initiation factor 2α played a protective role. Autophagy protected the cells from ER stress-related apoptosis. Both in vitro and in vivo studies indicated that inhibition of autophagy enhanced the anti-HCC effect of gossypol. Taken together, ER stress is the molecular mechanism underlying gossypol-induced apoptosis and autophagy. Gossypol exhibits anti-HCC activity primarily through the activation of apoptosis. However, gossypol-induced autophagy protects HCC cells from ER stress. Therefore, a combination therapy of gossypol and autophagy inhibitors may lead to an enhanced anti-HCC effect.

Coupling of COPII vesicle trafficking to nutrient availability by the IRE1α-XBP1s axis

The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)-XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking.

Exosomes from Microglia Attenuate Photoreceptor Injury and Neovascularization in an Animal Model of Retinopathy of Prematurity

The role of microglia in the pathophysiology of ischemic retinal diseases has been studied extensively. Exosomes from microglial cells exert protective effects during several nervous system diseases, but their roles in hypoxia-induced retinopathy remain unclear. In our study, exosomes derived from microglial cells were injected into the vitreous body of mice with oxygen-induced retinopathy (OIR). Results showed that exosome-treated OIR mice exhibited smaller avascular areas and fewer neovascular tufts in addition to decreased vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) expression. Moreover, photoreceptor apoptosis was suppressed by exosome injection. Mechanistically, exosomes from microglial cells were incorporated into photoreceptors in vitro and inhibited the inositol-requiring enzyme 1α (IRE1α)-X-box binding protein 1 (XBP1) cascade, which contributes to hypoxia-induced photoreceptor apoptosis. Furthermore, the exosomes also downregulated the mRNA and protein levels of VEGF and TGF-β in hypoxia-exposed photoreceptors. A microRNA assay showed that microRNA-24-3p (miR-24-3p) levels were extremely high in exosomes from microglial cells, suggesting that this could be the key molecule that inhibits the hypoxia-induced expression of IRE1α in photoreceptors. These findings delineate a novel exosome-mediated mechanism of microglial cell-photoreceptor crosstalk that facilitates normal angiogenesis and visual function in OIR mice; thus, our results also suggest a potential therapeutic approach for retinopathy of prematurity.

The multiple roles of the unfolded protein response regulator IRE1α in cancer.

Cancer is associated with a number of conditions such as hypoxia, nutrient deprivation, cellular redox, and pH changes that result in accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) and trigger a stress response known as the unfolded protein response (UPR). The UPR is a conserved cellular survival mechanism mediated by the ER transmembrane proteins activating transcription factor 6, protein kinase-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1α (IRE1α) that act to resolve ER stress and promote cell survival. IRE1α is a kinase/endoribonuclease (RNase) with multiple activities including unconventional splicing of the messenger RNA (mRNA) for the transcription factor X-Box Binding Protein 1 (XBP1), degradation of other mRNAs in a process called regulated IRE1α-dependent decay (RIDD) and activation of a pathway leading to c-Jun N-terminal kinase phosphorylation. Each of these outputs plays a role in the adaptive and cell death responses to ER stress. Many studies indicate an important role for XBP1 and RIDD functions in cancer and new studies suggest that these two functions of the IRE1α RNase can have opposing functions in the early and later stages of cancer pathogenesis. Finally, as more is learned about the context-dependent role of IRE1α in cancer development, specific small molecule inhibitors and activators of IRE1α could play an important role in counteracting the protective shield provided by ER stress signaling in cancer cells.

Endoplasmic Reticulum Stress Causes Liver Cancer Cells to Release Exosomal miR-23a-3p and Up-regulate Programmed Death Ligand 1 Expression in Macrophages.

Endoplasmic reticulum (ER) stress promotes tumor cell escape from immunosurveillance. However, the underlying mechanisms remain unknown. We hypothesized that ER stress induces hepatocellular carcinoma (HCC) cells to release exosomes, which attenuate antitumor immunity by modulating the expression of programmed death ligand 1 (PD-L1) in macrophages. In this study, we demonstrated that expression of several ER stress markers (glucose-regulated protein 78, activating transcription factor 6, protein kinase R-like ER kinase, and inositol-requiring enzyme 1α) was up-regulated in HCC tissues and negatively correlated with the overall survival and clinicopathological scores in patients with HCC. Expression of ER stress-related proteins positively correlated with CD68+ macrophage recruitment and PD-L1 expression in HCC tissues. High-throughput sequencing analysis identified miR-23a-3p as one of the most abundant microRNAs in exosomes derived from tunicamycin (TM)-treated HCC cells (Exo-TMs). miR-23a-3p levels in HCC tissues negatively correlated with overall survival. Treatment with Exo-TMs up-regulated the expression of PD-L1 in macrophages in vitro and in vivo. Bioinformatics analysis suggests that miR-23a-3p regulates PD-L1 expression through the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway. This notion was confirmed by in vitro transfection and coculture experiments, which revealed that miR-23a-3p inhibited PTEN expression and subsequently elevated phosphorylated AKT and PD-L1 expression in macrophages. Finally, coculture of T cells with Exo-TM-stimulated macrophages decreased CD8+ T-cell ratio and interleukin-2 production but increased T-cell apoptosis in vitro. Conclusion: ER-stressed HCC cells release exosomes to up-regulate PD-L1 expression in macrophages, which subsequently inhibits T-cell function through an exosome miR-23a-PTEN-AKT pathway. Our findings provide insight into the mechanism how tumor cells escape from antitumor immunity.