Inositol-requiring Enzyme 1 Pathway Research Articles

Grass Carp Reovirus (GCRV) infection activates the PERK-eIF2α pathway to promote the viral replication

Grass carp reovirus (GCRV) belongs to the genus Aquareovirus and is responsible for causing serious hemorrhagic disease in grass carp (Ctenopharyngodon idella), characterized by high mortality rates. Numerous animal viruses have been shown to activate endoplasmic reticulum stress (ERS). However, the potential for GCRV infection to induce ERS and its implications for viral infection remain unclear. In this study, we demonstrated that GCRV infection induces ERS, activates the protein kinase R-like ER kinase (PERK) pathway, and inhibits both the inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathways within the unfolded protein response (UPR). Additionally, we modulated the levels of ERS and UPR pathways in CIK cells through drug treatment and small interfering RNAs (siRNAs). Our findings revealed that the onset of ERS accelerated GCRV infection, while the ATF6 and IRE1 pathways within the UPR negatively regulated GCRV infection. Conversely, the PERK pathway facilitated GCRV infection. Furthermore, we showed that GCRV infection induced oxidative stress, with the production of reactive oxygen species (ROS) being positively regulated by the PERK pathway and the downstream gene endoplasmic reticulum oxidoreductase-1α (ERO1α). Notably, ROS promoted GCRV infection. Collectively, our findings indicate that GCRV infection activates ERS, which in turn promotes viral infection through the PERK-ERO1α-ROS signaling pathway. Thus, the PERK pathway may serve as a novel antiviral target for the prevention of GCRV infection.

Astaxanthin inhibits apoptosis in a cell model of tauopathy by attenuating endoplasmic reticulum stress and unfolded protein response

The accumulation of misfolded proteins is a common pathological characteristic shared by many neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. The disruption of proteostasis triggers endoplasmic reticulum (ER) stress, during which the unfolded protein response (UPR) is initiated by the activation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). These three branches of UPR signals act in concert to reduce the levels of abnormal proteins and restore ER homeostasis. However, the overactivation of UPR impairs cell function and induces apoptosis, which has been implicated in neurodegeneration. Astaxanthin is a xanthophyll carotenoid which has been shown to have neuroprotective effects in both cell and animal models; however, its effects on ER stress and UPR induced by disrupted proteostasis remain unclear. In this study, the effects of astaxanthin on ER stress and cytotoxicity were investigated in N2a cells stably expressing the pro-aggregant tau repeat domain carrying FTDP-17 mutation ΔK280 (Tau4RDΔK280). The results demonstrated that astaxanthin significantly inhibited Tau4RDΔK280-induced loss of cell viability and apoptosis, attenuating Tau4RDΔK280-induced caspase-3 activation and decrease of Bcl-2. Further studies revealed that astaxanthin treatment alleviated Tau4RDΔK280-induced ER stress and suppressed the activation of PERK, IRE1 and ATF6 signaling pathways. These findings suggested that astaxanthin might inhibit Tau4RDΔK280-induced cytotoxicity by attenuating UPR and ER stress. In addition, astaxanthin treatment resulted in a great reduction in the production of intracellular reactive oxygen species and a significant decrease in calcium influx induced by Tau4RDΔK280, which also contributed to the protective effects of astaxanthin against Tau4RDΔK280-induced cytotoxicity.

Kai-Xin-San Improves Cognitive Impairment via Wnt/β-Catenin and IRE1/XBP1s Signalings in APP/PS1 Mice.

Alzheimer's disease (AD) is the most common type of dementia with an insidious onset and slow progression. Kai-Xin-San (KXS) has been reported to be effective in improving cognitive impairment in AD. However, the mechanism is still confused. In this study, we employed APP/PS1 mice to explore the neuroprotective mechanism of KXS. Forty-eight male APP/PS1 mice were randomly divided into model group, KXS groups (0.7, 1.4, and 2.8 g/kg/d, p.o.) and the wild-type mice were assigned to the normal control group (n = 12 in each group). Y-maze and novel object recognition tests were carried out after continuous intragastric administration for 2 months. The abilities of learning, memory, and new object recognition in the APP/PS1 mice were enhanced significantly after KXS treatment. KXS can reduce the deposition of Aβ40 and Aβ42 in APP/PS1 mice brain. KXS decreased the levels of serum inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6. KXS increased the activities of superoxide dismutase and glutathione peroxidase significantly, whereas it inhibited the contents of reactive oxygen species and malondialdehyde significantly. In addition, we also detected Wnt/β-catenin signaling related proteins, such as Wnt7a, β-catenin, low-density lipoprotein receptor-related protein 6 (LRP6), glycogen synthase kinase-3β (GSK-3β), nuclear factor kappa-B (NF-κB), postsynaptic density 95 (PSD95), microtubule associated protein-2 (MAP-2), and endoplasmic reticulum stress (IRE1 pathway) related proteins, such as inositol-requiring enzyme 1 (IRE1), phosphorylated IRE1(p-IRE1), spliced X-box-binding protein 1 (XBP1s), immunoglobulin binding protein (BIP), and protein disulfide isomerase (PDI) in the hippocampus. Results showed that KXS decreased the expression of GSK-3β, NF-kB, p-IRE1/IRE1 ratio, XBP1s, and BIP; increased the expression of Wnt7a, β-catenin, LRP6, PSD95, MAP2, and PDI. In conclusion, KXS improved cognitive impairment by activating Wnt/β-catenin signaling, inhibiting the IRE1/XBP1s pathway in APP/PS1 mice.

Role of IRE1 in autophagy induced by vitamin E succinate in human gastric cancer cell

To investigate the role of inositol-requiring enzyme 1(IRE1) in autophagy of human gastric cancer cells induced by vitamin E succinate(VES). Human gastric cancer SGC-7901 cells were cultured in vitro and divided into solvent control group(0.1% ethanol absolute), different doses(5, 10, 15 and 20 μg/mL) VES group, 4μ8C group, and VES + 4μ8C group. The endoplasmic reticulum stress-related molecules glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP), autophagy marker microtubule associated Protein1 light chain 3(LC3), Beclin-1, unfolded protein response branching pathway Inositol-requiring enzyme 1(IRE1), X box-binding protein 1(XBP1), c-Jun n-terminal kinase(JNK) and p-JNK were detected by Western blot in the solvent control group and different doses of VES group. IRE1 was inhibited by 4μ8C. The expressions of IRE1, XBP1, JNK, p-JNK, GRP78 and CHOP were detected by Western blot, and the expressions of LC3 and Beclin-1 were detected. The expression of GRP78(1.16±0.06) and CHOP(1.36±0.11) in 20 μg/mL VES group were significantly higher than those in solvent control group GRP78(0.36±0.10) and CHOP(0.48±0.05)(P<0.001). The expression of Beclin-1(1.09±0.20) and LC3-Ⅱ/LC3-Ⅰ(1.29±0.03) in 20 μg/mL VES group were significantly higher than those in solvent control group(0.27±0.07) and LC3-Ⅱ/LC3-Ⅰ(0.43±0.06)(P<0.001). The expression levels of IRE1(1.07±0.20), XBP1(1.33±0.07) and p-JNK/JNK(1.19±0.31) in 20 μg/mL VES group were significantly higher than those in the solvent control group(P<0.01). After IRE1 is inhibited: The expression level of IRE1(0.63±0.27), XBP1(0.74±0.09), p-JNK/JNK(0.35±0.04), GRP78(0.66±0.02), CHOP(0.51±0.02), LC3-Ⅱ/LC3-Ⅰ(0.72±0.01), Beclin-1(0.70±0.15) was significantly lower than that of VES group(P<0.05). VES may participate in the regulation of autophagy in gastric cancer cells by upregulating IRE1 pathway.

Inhibition of IRE1 suppresses the catabolic effect of IL-1β on nucleus pulposus cell and prevents intervertebral disc degeneration in vivo

Neck pain and low back pain are two of the major diseases, which causes patients a low quantify of life and a heavy economic burden, intervertebral disc degeneration (IDD) contributes to them, and the mechanism is not totally clear. The increased inflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)α and downstream signaling pathways are involved. Inositol requiring enzyme 1 (IRE1) is a crucial enzyme that regulates endoplasmic reticulum (ER) stress. It is reported that IRE1 plays an important role in the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. Considering this, we performed a series of experiments in vitro and in vivo to evaluate the role of IRE1 in the progress of IDD. We demonstrated that IRE1 pathway was induced by IL-1β, inhibition of IRE1 suppressed the matrix degeneration of NP cells and ameliorated IDD grade in the punctured rat model. Further results indicated that inhibition of IRE1 suppressed H2O2 induced cell senescence, IL-1β-induced cellular reactive oxygen species (ROS) level and the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. It also played a crucial role in the apoptosis of NP cells and the progress of macrophage polarization. Our findings demonstrated that inhibition of IRE1 could suppress the degeneration of NP cells and prevent IDD in vivo. IRE1 may be a potential target for IDD treatment.

Coronavirus transmissible gastroenteritis virus antagonizes the antiviral effect of the microRNA miR-27b via the IRE1 pathway.

Although the functional parameters of microRNAs (miRNAs) have been explored to some extent, the roles of these molecules in coronavirus infection and the regulatory mechanism of miRNAs in virus infection are still unclear. Transmissible gastroenteritis virus (TGEV) is an enteropathgenic coronavirus and causes high morbidity and mortality in suckling piglets. Here, we demonstrated that microRNA-27b-3p (miR-27b-3p) suppressed TGEV replication by directly targeting porcine suppressor of cytokine signaling 6 (SOCS6), while TGEV infection downregulated miR-27b-3p expression in swine testicular (ST) cells and in piglets. Mechanistically, the decrease of miR-27b-3p expression during TGEV infection was mediated by the activated inositol-requiring enzyme 1 (IRE1) pathway of the endoplasmic reticulum (ER) stress. Further studies showed that when ER stress was induced by TGEV, IRE1 acted as an RNase activated by autophosphorylation and unconventionally spliced mRNA encoding a potent transcription factor, X-box-binding protein 1 (Xbp1s). Xbp1s inhibited the transcription of miR-27 and ultimately reduced the production of miR-27b-3p. Therefore, our findings indicate that TGEV inhibits the expression of an anti-coronavirus microRNA through the IRE1 pathway and suggest a novel way in which coronavirus regulates the host cell response to infection.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-1967-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Isoginkgetin attenuates endoplasmic reticulum stress-induced autophagy of brain after ischemic reperfusion injury

Isoginkgetin is characterized by properties of potent anticancer and anti-inflammation. To explore its effect on ischemic stroke, a rat model of ischemia/reperfusion (I/R) injury was established and induced by transient middle cerebral artery occlusion/reperfusion (MCAO/R). Different doses of isoginkgetin were intraperitoneally injected into each rat. Expressions of ER stress activation-related makers including phosphorylated inositol-requiring enzyme 1 (IRE1), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), activating transcription factor-6 (ATF6), and two autophagy markers (ratio of LC3II/I and Beclin-1) were detected by western blot. Infarct volume, neurological deficits, and brain water content were detected. The results showed that ER stress and autophagy were activated by cerebral (I/R) injury, which could be effectively attenuated following pre-ischemia isoginkgetin administration. Moreover, autophagy induced by ER stress was triggered by the activation of PERK and IRE1 pathways. ER stress inhibitor (4-PBA) and ER related signaling inhibitors including PERK, GSK, IRE1, and DBSA markedly inhibited ER stress and autophagy induced by I/R. In addition, isoginkgetin markedly mitigated cerebral infarction, edema, neuronal apoptosis as well as neurological impairment induced by I/R injury, while tunicamycin (ER stress activator TM) and rapamycin (autophagy activator RAPA) could eliminate these lesions. This research identified a novel therapeutic agent isoginkgetin, which could effectively attenuate I/R injury by blocking autophagy induced by ER stress.

IRE1-Mediated Unfolded Protein Response Promotes the Replication of Tick-Borne Flaviviruses in a Virus and Cell-Type Dependent Manner.

Tick-borne flaviviruses (TBFV) can cause severe neurological complications in humans, but differences in tissue tropism and pathogenicity have been described for individual virus strains. Viral protein synthesis leads to the induction of the unfolded protein response (UPR) within infected cells. The IRE1 pathway has been hypothesized to support flavivirus replication by increasing protein and lipid biogenesis. Here, we investigated the role of the UPR in TBFV infection in human astrocytes, neuronal and intestinal cell lines that had been infected with tick-borne encephalitis virus (TBEV) strains Neudoerfl and MucAr-HB-171/11 as well as Langat virus (LGTV). Both TBEV strains replicated better than LGTV in central nervous system (CNS) cells. TBEV strain MucAr-HB-171/11, which is associated with gastrointestinal symptoms, replicated best in intestinal cells. All three viruses activated the inositol-requiring enzyme 1 (IRE1) pathway via the X-box binding protein 1 (XBP1). Interestingly, the neurotropic TBEV strain Neudoerfl induced a strong upregulation of XBP1 in all cell types, but with faster kinetics in CNS cells. In contrast, TBEV strain MucAr-HB-171/11 failed to activate the IRE1 pathway in astrocytes. The low pathogenic LGTV led to a mild induction of IRE1 signaling in astrocytes and intestinal cells. When cells were treated with IRE1 inhibitors prior to infection, TBFV replication in astrocytes was significantly reduced. This confirms a supporting role of the IRE1 pathway for TBFV infection in relevant viral target cells and suggests a correlation between viral tissue tropism and the cell-type dependent induction of the unfolded protein response.

CCDC170 affects breast cancer apoptosis through IRE1 pathway.

Genome-wide association studies have revealed that multiple single-nucleotide polymorphisms in the intergenic region between estrogen receptor 1 and coiled-coil domain containing 170 (CCDC170) are associated with breast cancer risk. We performed microarray and bioinformatics analyses to identify genes that were induced upon CCDC170 overexpression, and confirmed our findings by evaluating paraffin-embedded breast cancer tissues and conducting cellular assays. In CCDC170-overexpressing MCF7 breast cancer cells, microarray analyses revealed that inositol-requiring enzyme 1 (IRE1) was the most elevated gene in enriched pathways. In breast cancer tissues, IRE1 expression correlated positively with CCDC170 and X-box binding protein 1 expression at both the mRNA and protein levels. In a survival analysis, patients with higher CCDC170 levels exhibited better disease-free survival. Western blotting indicated that overexpressing CCDC170 in MCF7 cells increased protein levels of IRE1α, estrogen receptor α and X-box binding protein 1, while silencing CCDC170 reduced them. CCDC170 overexpression promoted apoptosis in MCF7 cells, and this effect was more obvious under endoplasmic reticulum stress. MCF7 cells overexpressing CCDC170 were more sensitive to paclitaxel. Our study showed that higher CCDC170 expression is associated with a better prognosis in breast cancer patients and that CCDC170 may promote apoptosis through the IRE1α pathway.

Celecoxib-Dependent Neuroprotection in a Rat Model of Transient Middle Cerebral Artery Occlusion (tMCAO) Involves Modifications in Unfolded Protein Response (UPR) and Proteasome.

Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1h of tMCAO and treated with celecoxib or vehicle 1 and 24h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.Graphical abstract.

Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes great economic losses in the cattle industry. Herpesvirus infection generally induces endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in infected cells. However, it is not clear whether ER stress and UPR can be induced by BoHV-1 infection. Here, we found that ER stress induced by BoHV-1 infection could activate all three UPR sensors (the activating transcription factor 6 (ATF6), the inositol-requiring enzyme 1 (IRE1), and the protein kinase RNA-like ER kinase (PERK)) in MDBK cells. During BoHV-1 infection, the ATF6 pathway of UPR did not affect viral replication. However, both knockdown and specific chemical inhibition of PERK attenuated the BoHV-1 proliferation, and chemical inhibition of PERK significantly reduced the viral replication at the post-entry step of the BoHV-1 life cycle. Furthermore, knockdown of IRE1 inhibits BoHV-1 replication, indicating that the IRE1 pathway may promote viral replication. Further study revealed that BoHV-1 replication was enhanced by IRE1 RNase activity inhibition at the stage of virus post-entry in MDBK cells. Furthermore, IRE1 kinase activity inhibition and RNase activity enhancement decrease BoHV1 replication via affecting the virus post-entry step. Our study revealed that BoHV-1 infection activated all three UPR signaling pathways in MDBK cells, and BoHV-1-induced PERK and IRE1 pathways may promote viral replication. This study provides a new perspective for the interactions of BoHV-1 and UPR, which is helpful to further elucidate the mechanism of BoHV-1 pathogenesis.

Endoplasmic Reticulum Stress Induction of Yeast Bax Inhibitor (BXI1) Gene Expression Does Not Entirely Require IRE1

Bax Inhibitor (BI‐1/TMBIM6) is a regulator protein within the Unfolded Protein Response (UPR) that influences rates of apoptosis within cells. Studies have shown that BI‐1 is upregulated in human cancer cells like prostate and breast, therefore it is important to study the factors that control BI‐1 within a cell. In this paper, the Saccharomyces cerevisiae homolog of BI‐1, BXI1, is studied to better understand the signaling pathway that influences concentrations of BXI1, specifically the Inositol Requiring Enzyme 1 (IRE1) pathway. Ire1p is an Endoplasmic Reticulum (ER) bound transmembrane ligand receptor which detects the UPR and activates downstream targets which play further roles in regaining homeostasis. Here, our lab used two genetically modified S. cerevisiae strains, both with Green Fluorescent Protein (GFP) bound to BXI1 (Bxi1p‐GFP) and one lacking genetic information to code for IRE1 (ΔIre1) to test the effect of the IRE1 pathway on the expression of BXI1. When cells were cultured in media containing either tunicamycin or DDT, we observed an increase in the expression of BXI1 that was not entirely dependent on the IRE1 pathway. This suggests that there may be other signaling pathways that influence the expression of BXI1.Support or Funding InformationOur laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.

Leukemia-Associated HSC Vascular Niche Is Negatively Regulated By PERK of Unfolded Protein Response (UPR)

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from early T cell progenitors. Diffuse infiltration of the bone marrow by T-ALL is associated with worse prognosis. We previously reported that actively proliferating leukemia cells inhibit normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. We found that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. However, the underlying molecular mechanism that leads to the suppression of hematopoiesis and decreased HSPC in the vascular niche is unclear. It has been demonstrated that rapid cellular proliferation associated with oncogenic activity such as MYC in T-ALL leads to a global increase in protein synthesis and an increase in misfolded/unfolded polypeptides in the endoplasmic reticulum (ER), referred to as unfolded protein response (UPR) or ER stress. Elevated ER stress leads to activation of at least three types of ER stress transducers through the release of inhibitory binding by glucose-regulated chaperone protein (GRP78/BIP): the protein kinase RNA-like ER kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription factor 6 (ATF6). Activation of PERK phosphorylates eIF2 to repress global translation with the exception of a small number of proteins including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis and genes in apoptosis. Here, we studied the role of UPR in the regulation of HSC niche function in the setting of T-ALL progression. Using in vitro assays in which T-ALL leukemia cells driven by activated Notch1 (ICN1) were co-cultured with endothelial cells (MILE SVEN 1, MS1), and in vivo ICN1-driven T-ALL model, we found that PERK-eIF2a-ATF4 pathway was activated in both MS1 cells and BM endothelial cells isolated from T-ALL mice, while IRE1 and ATF6 pathways were only mildly altered. The activation of PERK was accompanied with the increased expression of Jagged1 and suppressed expression of CXCL12 in both cultured endothelial cells and bone marrow endothelial cells from leukemia mice. PERK inhibitor (GSK2606414) treatment of co-cultured cells largely restored CXCL12 expression, which was also negatively regulated by Jagged1, and accelerated the leukemia cell apoptosis as indicated by the enhanced annexin staining. These findings suggest that PERK is the upstream regulator of Jagged1 and CXCL12 in the endothelial cells; however, the function of cell-autonomous PERK on leukemia cell survival needs to be further clarified. To understand the role of PERK in bone marrow endothelium during leukemia development in vivo, we examined T-ALL leukemia progression and its effect on vascular niche function in VE-CadherinERT2/PERKF/F mice in which Perk was specifically deleted in endothelial cells. Consistent with in vitro findings, T-ALL development induced endothelial PERK-eIF2a-ATF4 activation, while up-regulated Jagged1 and down-regulated CXCL12 were also identified in isolated BM endothelial cells. Compared to the wild type mice, VE-CadherinERT2/PERKF/F mice showed attenuated leukemia progression, increased HSPC (Lin-Sca-1+c-kit+) frequency, and improved survival. Taken together, our findings suggest that PERK activation in BM endothelial cells is a key regulator of the leukemia vascular niche to promote leukemia progression and to suppress normal hematopoiesis. Therefore, targeting PERK may offer an effective strategy in restoring normal HSPC homeostasis and limiting leukemia progression. Disclosures No relevant conflicts of interest to declare.

Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro.

Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.

Gallotannin attenuates 2‑deoxy‑D‑glucose‑induced dedifferentiation and endoplasmic reticulum stress through inhibition of inositol‑requiring enzyme 1 downstream p38 kinase pathway in chondrocytes.

Gallotannin (GT) is a class of polyphenols with antioxidant, anticancer, and antiviral activities. 2‑Deoxy‑D‑glucose (2DG), a glucose‑derived molecule, can inhibit glucose metabolism and induce endoplasmic reticulum (ER) stress. GT in primary‑cultured chondrocytes enhances expression of typeII collagen, an indicator of differentiation, and cyclooxygenase‑2 (COX‑2), which mediates inflammatory reactions. In contrast, 2DG reduces typeII collagen and COX‑2 expression while driving ER‑stress‑induced unglycosylation. In the present study, it was investigated whether GT could attenuate 2DG‑induced dedifferentiation and ER‑stress. Following treatment with GT and 2DG, chondrocytes were assessed using western blotting, RT‑PCR, immunofluorescence, and alcian blue staining. GT restored typeII collagen expression that was reduced by 2DG, inhibited ER‑stress‑induced COX‑2 unglycosylation, and induced COX‑2 expression. The expression of a glucose‑regulated protein, GRP78, which is an indicator of reduced ER‑stress, was decreased. To link the GT signaling pathway with pathways that inhibit 2DG‑induced dedifferentiation and ER‑stress, inhibitors were treated in chondrocytes. The results revealed that, among the different signaling pathways triggered by ER‑stress, the p38 kinase pathway was involved in the inositol‑requiring enzyme 1 (IRE1) downstream signaling pathway. Following inhibition of the IRE1 pathway, typeII collagen expression was increased and COX‑2 expression was decreased. In addition, after examining the splicing of X‑box binding protein 1 (XBP‑1) which is dependent on IRE1 activation induced by ER‑stress, it was revealed that GT inhibited the increase of XBP‑1s after splicing due to 2DG‑induced ER stress. GT in chondrocytes inhibited 2DG‑induced dedifferentiation and ER‑stress‑induced COX‑2 unglycosylation while regulating differentiation and inflammation via the ER‑stress‑induced p38 kinase pathway downstream from the IRE1 pathway.

IRE1 promotes neurodegeneration through autophagy-dependent neuron death in the Drosophila model of Parkinson\u2019s disease

Abnormal aggregation of misfolded pathological proteins in neurons is a prominent feature of neurodegenerative disorders including Parkinson’s disease (PD). Perturbations of proteostasis at the endoplasmic reticulum (ER) triggers ER stress, activating the unfolded protein response (UPR). Chronic ER stress is thought to underlie the death of neurons during the neurodegenerative progression, but the precise mechanism by which the UPR pathways regulate neuronal cell fate remains incompletely understood. Here we report a critical neurodegenerative role for inositol-requiring enzyme 1 (IRE1), the evolutionarily conserved ER stress sensor, in a Drosophila model of PD. We found that IRE1 was hyperactivated upon accumulation of α-synuclein in the fly photoreceptor neurons. Ectopic overexpression of IRE1 was sufficient to trigger autophagy-dependent neuron death in an XBP1-independent, JNK-dependent manner. Furthermore, IRE1 was able to promote dopaminergic neuron loss, progressive locomotor impairment, and shorter lifespan, whereas blocking IRE1 or ATG7 expression remarkably ameliorated the progression of α-synuclein-caused Parkinson’s disease. These results provide in vivo evidence demonstrating that the IRE1 pathway drives PD progression through coupling ER stress to autophagy-dependent neuron death.

VceC Mediated IRE1 Pathway and Inhibited CHOP-induced Apoptosis to Support Brucella Replication in Goat Trophoblast Cells

The effectors of the type IV secretion system (T4SS) of bacteria play important roles in mediating bacterial intracellular proliferation and manipulating host-related pathway responses to bacterial infection. Brucella Spp. inhibit the apoptosis of host cells to benefit their own intracellular proliferation. However, the underlying mechanisms between T4SS effectors and Brucella-inhibited apoptosis in goat trophoblast cells remain unclear. Here, based on Brucella suis vaccine strain 2, the VceC was deleted by allelic exchange. We show that ΔVceC was able to infect and proliferate to high titers in goat trophoblast cells (GTCs) and increase C/EBP-homologous protein (CHOP)-mediated apoptosis. GRP78 expression decreased upon ΔVceC infection. In addition, we discovered that the inositolrequiring enzyme 1 (IRE1) pathway was inhibited in this process. Changing endoplasmic reticulum (ER) stress affected Brucella intracellular replication in GTCs. The replication of ΔVceC was more sensitive under the different ERstress conditions in the GTC line after treatment with ER stress inhibitors 4 phenyl butyric acid (4-PBA) or ER stress activator Tm. Together, our findings show that VceC has a protective effect on the intracellular persistence of Brucella infection, and inhibits ER stress-induced apoptosis in the CHOP pathway. The present work provides new insights for understanding the mechanism of VceC in the establishment of chronic Brucella infection.

Non-Structural Protein 2B of Human Rhinovirus 16 Activates Both PERK and ATF6 Rather Than IRE1 to Trigger ER Stress.

To understand the underlying mechanisms of endoplasmic reticulum (ER) stress caused by human rhinovirus (HRV) 16 and non-structural transmembrane protein 2B, the expressions of ER chaperone glucose-regulated protein 78 (GRP78) and three signal transduction pathways, including protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1), were evaluated after HRV16 infection and 2B gene transfection. Our results showed that both HRV16 infection and 2B gene transfection increased the expression of ER chaperone GRP78, and induced phosphorylation of PERK and cleavage of ATF6 in a time-dependent manner. Our data also revealed that the HRV16 2B protein was localized to the ER membrane. However, both HRV16 infection and HRV16 2B gene transfection did not induce ER stress through the IRE1 pathway. Moreover, our results showed that apoptosis occurred in H1-HeLa cells infected with HRV16 or transfected with 2B gene accompanied with increased expression of CHOP and cleaved caspase-3. Taken together, non-structural protein 2B of HRV16 induced an ER stress response through the PERK and ATF6 pathways rather than the IRE1 pathway.

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like inhibitory protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the endoplasmic reticulum (ER) and that c-FLIP-deficient mouse embryonic fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP−/− MEFs. Surprisingly, c-FLIP−/− MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP−/− cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP−/− MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP−/− MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP−/− cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like Inhibitory Protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the Endoplasmic Reticulum (ER) and that c-FLIP-deficient Mouse Embryonic Fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.