Endoplasmic Reticulum Stress Sensor Research Articles

Anticancer effect of caudatin in diethylnitrosamine‑induced hepatocarcinogenesis in rats.

An overwhelming endoplasmic reticulum stress (ERS) and the following unfolded protein response (UPR) can induce hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). Caudatin, one of the species of C-21 steroidal glycosides mainly isolated from the roots of Cynanchum bungei Decne, exhibits potent anticancer activities in vivo. However, the effect of caudatin on HCC remains unclear. In the present study, a diethylnitrosamine (DEN)-induced HCC model was established. Nodules and tumors in rat livers were monitored by T2-/T1-weighted-magnetic resonance imaging (MRI) using a 1.5 T scanner. Caudatin reduced the number and size of nodules and alleviated the inflammatory foci in the liver. In addition, the hepatic pro-inflammatory levels of interleukin (IL) 6, monocyte chemoattractant protein 1 and IL-1β were decreased in caudatin-treated rats. The DEN-induced surge in malondialdehyde, aspartate aminotransferase, alanine transaminase and TBIL were alleviated following caudatin treatment. The expression of ERS chaperones glucose-regulated protein, 94 kDa, glucose-regulated protein, 78 kDa and protein disulfide-isomerase A4 and the proliferation marker Ki-67 in liver nodules were all downregulated by caudatin as demonstrated by immunohistochemistry, reverse transcription-quantitative PCR and western blot analysis. Caudatin reduced the cytoprotective ERS sensor activating transcription factor 6-mediated signal transduction and inhibited the PKR-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway. However, the effect of caudatin on inositol requiring enzyme 1 signaling was negligible. In conclusion, restoration of the dysregulated UPR program was involved in the antitumor efficacy of caudatin without inducing cumulative hepatotoxicity.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity and promotes natural killer cell memory by regulating c-Myc and mitochondrial respiration

Abstract Natural killer (NK) cells are critical mediators of host immunity to pathogens. Though historically being considered as a part of the innate immune system, NK cells exhibit adaptive features and can ‘remember’ stimuli like inflammation or viral infection and hence are capable of responding more robustly upon re-encountering the stimulation. Successful design of an NK cell-based therapy would ideally rely on harnessing NK cell memory, the key drivers and regulators of which remain poorly understood. Here, we demonstrate that the endoplasmic reticulum stress sensor IRE1α and its substrate transcription factor XBP1 drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mTOR pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. NK cells with c-Myc haploinsufficency phenocopied IRE1α deficiency, while c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α also promotes oxidative phosphorylation in NK cells. Combined, our study identifies an IRE1α-XBP1-cMyc axis in NK cell immunity; and based on this work, we are currently investigating the role of IRE1 in NK cell memory using novel transgenic mouse models and small molecule drug treatment in primary human cells. We are also evaluating the therapeutic benefit of manipulating IRE1 function in CAR-NK cells to treat cancer. Our ongoing study should advance the current knowledge of NK cell memory and also open new opportunities in cancer therapy for patients with otherwise extremely poor prognoses.

Naringin induces endoplasmic reticulum stress-mediated apoptosis, inhibits β-catenin pathway and arrests cell cycle in cervical cancer cells.

Naringin is a promising anticancer bioflavonoid phytochemical, mainly extracted from citrus fruits. This study evaluates the antiproliferative effect and the cell death mechanism induced by naringin on cervical cancer (CC) cells. Our results demonstrated that naringin exerts significant inhibition in cell viability and exhibits IC50 value 745, 764, 793 µM against C33A, SiHa, and HeLa cells respectively. Annexin V FITC and immunoblotting analysis reveal significant apoptosis induction in cells exposed to higher doses naringin. Mechanistically, naringin induces endoplasmic reticulum (ER) stress-associated cell killing in CC cells. Naringin increases the protein expression of ER stress sensors, phosphorylates eIF2α by and activates apoptosis-associated protein CHOP and other associated proapoptotic proteins (PARP1 and caspase-3). Intriguingly, pre-treatment with of ER stress inhibitor (salubrinal), reverses the apoptotic effect exerted by naringin. Additionally, the naringin abrogates the β-catenin pathway by decreasing the protein expression as well as phosphorylation of β-catenin (Ser576) and GSK-3β (Ser9) and simultaneously triggers cell cycle arrest at a G0/G1 phase by increasing the expression of cell cycle checkpoint proteins p21/cip and p27/kip. Naringin induces ER stress-mediated apoptosis and simultaneously abrogates Wnt/β-catenin signaling which eventually triggers the arrest of the cell cycle at a G0/G1 phase in CC cells.

Ursodeoxycholic Acid Inhibits Glioblastoma Progression via Endoplasmic Reticulum Stress Related Apoptosis and Synergizes with the Proteasome Inhibitor Bortezomib.

Ursodeoxycholic acid (UDCA) has demonstrated cancer suppressive potential in several tumors. Here, we investigated the antitumor potential and biochemical mechanism of UDCA on glioblastoma multiforme (GBM), the deadliest form of brain cancer with a median survival of 15 months. Cell viability was assessed using the CCK-8 and colony forming assays. Expression profiles were obtained using RNA sequencing, and PCR and Western blot were used to validate changes in related markers at the RNA and protein levels. Flow cytometry was used to examine cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). UDCA inhibited GBM cell viability in a dose- and time-dependent manner. Flow cytometry demonstrated that cells were arrested in the G1 phase and underwent apoptosis. The RNA sequencing results showed UDCA treatment in part targeted gene expression related to mitochondria and endoplasmic reticulum (ER). UDCA indeed led to decreased MMP, overproduction of ROS, and ER stress. Three critical ER stress sensors ATF6, IRE1α, and PERK were increased in the acute phase. Additionally, combining UDCA with the proteasome inhibitor bortezomib (BTZ) achieved a synergistic effect through enhancing the PERK/ATF4/CHOP pathway and protracting ER stress. UDCA inhibited GBM progression, and the combination with BTZ achieved a synergistic effect via protracted ER stress. Thus, UDCA, alone or with combination of BTZ, shows promise as a possible therapeutic agent for the treatment of GBM.

S1PR1 interacts with endoplasmic reticulum stress sensor, BiP, to regulate calcium homeostasis and endothelial barrier function

Endothelial–cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is well known to enhance barrier function upon ligating S1P. We recently showed that S1P phosphorylation of S1PR1 at tyrosine (Y) 143 residue induces S1PR1 internalization and compromises barrier function. Here, we assessed the mechanism internalizing the Y143‐S1PR1 and how Y143‐S1PR1 disrupts barrier function. Herein, using TIRF and confocal imaging, we show that phosphorylated (Y143D‐S1PR1) S1PR1 mutant localizes at endoplasmic reticulum (ER). Inhibition of dynamin activity, a GTP‐dependent pinchase, rescued Y143D‐S1PR1 expression at the cell surface. However, dynamin inhibition had no effect on cell‐surface retention of S1PR1 or Y143F‐S1PR1. In other studies, we show that Y143D‐S1PR1 bound WT‐dynamin strongly while weakly with GTPase defective dynamin. Cell‐surface retained S1PR1 induces signaling including calcium mobilization and activation of MAP kinases by binding Gi heterotrimeric protein. Intriguingly, we show that internalized Y143D‐S1PR1 augmented Ca2+ entry which was blocked by pertussis toxin. Furthermore, phosphorylated receptor bound Gi indicating that internalized S1PR1 binds cytosolic Gi which then increases Ca2+ entry. We further showed Y143D‐S1PR1 mutant augmented Ca2+ entry by organizing stromal‐interaction molecule 1 (STIM1) dependent ER‐PM junction formation. To address the mediator involved in retaining Y143D‐S1PR1 at the ER, we performed mass spectrometric analysis and identified GRP78 (BiP), an endoplasmic resident protein, as the novel interacting partner of S1PR1. Depletion of BiP or STIM1 rescued barrier function even in presence of Y143D‐S1PR1. Thus, BiP recruits phosphorylated Y143D‐S1PR1 at the ER where S1PR1 mutant organizes ER‐PM junctions to mediate Ca2+ entry disrupting endothelial barrier function.Support or Funding InformationNIH

Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells.

ObjectiveTitanium oxide (TiO2) acts as a photosensitizer in photodynamic therapy by mediating reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress. This study aimed to investigate the effect of TiO2 on ER stress in liver cancer cells.MethodsNormal human liver and human hepatocarcinoma cell lines were incubated with various concentrations of TiO2 nanotubes for 48 hours. Cell growth, apoptosis, cell cycle, and cellular ROS were detected. Expression levels of ER stress sensors (PERK and ATF6) and Bax were evaluated by western blot. The effect of TiO2 on liver cancer growth was also investigated in mice in vivo.ResultsTiO2 inhibited cell growth, increased apoptosis and cellular ROS levels, and arrested the cell cycle in G1 stage in liver cancer cells. TiO2 also increased PERK, ATF6, and Bax expression levels in liver cancer cells in dose-dependent manners. TiO2 had no significant effect on cell growth, apoptosis, ROS level, cell cycle distribution, or PERK, ATF6, or Bax expression in normal liver cells. TiO2 administration reduced tumor volume and increased PERK, Bax, and ATF6 expression levels in tumor tissues in vivo.ConclusionsTiO2 nanoparticles increased ROS-induced ER stress and activated the PERK/ATF6/Bax axis in liver cancer cells in vitro and in vivo.

Mining Data From Plasma Cell Differentiation Identified Novel Genes for Engineering of a Yeast Antibody Factory.

Saccharomyces cerevisiae is a common platform for production of therapeutic proteins, but it is not intrinsically suited for the manufacturing of antibodies. Antibodies are naturally produced by plasma cells (PCs) and studies conducted on PC differentiation provide a comprehensive blueprint for the cellular transformations needed to create an antibody factory. In this study we mined transcriptomics data from PC differentiation to improve antibody secretion by S. cerevisiae. Through data exploration, we identified several new target genes. We tested the effects of 14 genetic modifications belonging to different cellular processes on protein production. Four of the tested genes resulted in improved antibody expression. The ER stress sensor IRE1 increased the final titer by 1.8-fold and smaller effects were observed with PSA1, GOT1, and HUT1 increasing antibody titers by 1. 6-, 1. 4-, and 1.4-fold. When testing combinations of these genes, the highest increases were observed when co-expressing IRE1 with PSA1, or IRE1 with PSA1 and HUT1, resulting in 3.8- and 3.1-fold higher antibody titers. In contrast, strains expressing IRE1 alone or in combination with the other genes produced similar or lower levels of recombinantly expressed endogenous yeast acid phosphatase compared to the controls. Using a genetic UPR responsive GFP reporter construct, we show that IRE1 acts through constitutive activation of the unfolded protein response. Moreover, the positive effect of IRE1 expression was transferable to other antibody molecules. We demonstrate how data exploration from an evolutionary distant, but highly specialized cell type can pinpoint new genetic targets and provide a novel concept for rationalized cell engineering.

Nucleotide-binding oligomerization domain protein 2 deficiency enhances CHOP expression and plaque necrosis in advanced atherosclerotic lesions.

Endoplasmic reticulum (ER) stress‐induced cell death of vascular smooth muscle cells (VSMCs) is extensively involved in atherosclerotic plaque stabilization. We previously reported that nucleotide‐binding oligomerization domain protein 2 (NOD2) participated in vascular homeostasis and tissue injury. However, the role and underlying mechanisms of NOD2 remain unknown in ER stress‐induced cell death of VSMC during vascular diseases, including advanced atherosclerosis. Here, we report that NOD2 specifically interacted with ER stress sensor activating transcription factor 6 (ATF6) and suppressed the expression of proapoptotic transcription factor CHOP (C/EBP homologous protein) during ER stress. CHOP‐positive cells were increased in neointimal lesions after femoral artery injury in NOD2‐deficient mice. In particular, a NOD2 ligand, MDP, and overexpression of NOD2 decreased CHOP expression in wild‐type VSMCs. NOD2 interacted with an ER stress sensor molecule, ATF6, and acted as a negative regulator for ATF6 activation and its downstream target molecule, CHOP, that regulates ER stress‐induced apoptosis. Moreover, NOD2 deficiency promoted disruption of advanced atherosclerotic lesions and CHOP expression in NOD2−/−ApoE−/− mice. Our findings indicate an unsuspected critical role for NOD2 in ER stress‐induced cell death.

PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

Palmitate induces cardiomyocyte death via inositol requiring enzyme-1 (IRE1)-mediated signaling independent of X-box binding protein 1 (XBP1)

Overloading of the saturated fatty acid (SFA) palmitate induces cardiomyocyte death. The purpose of this study is to elucidate signaling pathways contributing to palmitate-induced cardiomyocyte death.Palmitate-induced cardiomyocyte death was induced in Toll-like receptor 2/4 double-knockdown cardiomyocytes to a similar extent as wild-type cardiomyocytes, while cardiomyocyte death was canceled out by triacsin C, a long-chain acyl-CoA synthetase inhibitor. These results indicated that palmitate induced cytotoxicity after entry and conversion into palmitoyl-CoA. Palmitoyl-CoA is not only degraded by mitochondrial oxidation but also taken up as a component of membrane phospholipids. Palmitate overloading causes cardiomyocyte membrane fatty acid (FA) saturation, which is associated with the activation of endoplasmic reticulum (ER) unfolded protein response (UPR) signaling. We focused on the ER UPR signaling as a possible mechanism of cell death. Palmitate loading activates the UPR signal via membrane FA saturation, but not via unfolded protein overload in the ER since the chemical chaperone 4-phenylbutyrate failed to suppress palmitate-induced ER UPR. The mammalian UPR relies on three ER stress sensors named inositol requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Palmitate loading activated only IRE1 and PERK. Knockdown of PERK did not affect palmitate-induced cardiomyocyte death, while knockdown of IRE1 suppressed palmitate-induced cardiomyocyte death. However, knockdown of X-box binding protein 1 (XBP1), the downstream effector of IRE1, did not affect palmitate-induced cardiomyocyte death. These results were validated by pharmacological inhibitor experiments.In conclusion, we identified that palmitate-induced cardiomyocyte death was triggered by IRE1-mediated signaling independent of XBP1.

ER Stress Responses: An Emerging Modulator for Innate Immunity.

The endoplasmic reticulum (ER) is a critical organelle, storing the majority of calcium and governing protein translation. Thus, it is crucial to keep the homeostasis in all ER components and machineries. The ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2α and ATF6, orchestrate the major regulatory circuits to ensure ER homeostasis. The embryonic or postnatal lethality that occurs upon genetic depletion of these sensors reveals the essential role of the ER stress pathway in cell biology. In contrast, the impairment or excessive activation of ER stress has been reported to cause or aggravate several diseases such as atherosclerosis, diabetes, NAFDL/NASH, obesity and cancer. Being part of innate immunity, myeloid cells are the first immune cells entering the inflammation site. Upon entry into a metabolically stressed disease environment, activation of ER stress occurs within the myeloid compartment, leading to the modulation of their phenotype and functions. In this review, we discuss causes and consequences of ER stress activation in the myeloid compartment with a special focus on the crosstalk between ER, innate signaling and metabolic environments.

Ninjurin-1 upregulated by TNFα receptor 1 stimulates monocyte adhesion to human TNFα-activated endothelial cells; benefic effects of amlodipine

AimsThe objectives of the present study were to investigate the mechanisms of Ninj-1 regulation in TNFα-activated human endothelial cells (HEC), and to test if Amlodipine (AML) ameliorates the inflammatory stress by decreasing Ninj-1 expression. Main methodsTNFα-activated HEC with/without AML (0.1 μM and 1 μM) were used. TNFα-receptor 1 (TNFR1) was silenced and inhibitors for oxidative stress (N-acetyl cysteine), endoplasmic reticulum stress (salubrinal, 4-phenyl butyric acid), or NF-kB (Bay 11-7085) and p38 MAPK (SB203580) were used. Levels of Ninj-1, TNFR1, monocyte adhesion, endoplasmic reticulum stress (ERS) sensors, NADPH oxidase- and mitochondria-derived oxidative species were evaluated. Key findingsThe novel findings that we report here are: (i) silencing the endothelial TNFR1 leads to decreased Ninj-1 expression and diminished monocyte adhesion; (ii) increased oxidative stress, ERS and NF-kB activation enhance Ninj-1 expression and monocyte adhesion; (iii) up-regulation of endothelial Ninj-1 expression stimulates monocytes adhesion to TNFα – activated HEC; (iv) AML diminishes monocyte adhesion by reducing Ninj-1 expression through mechanisms involving the decrease of NADPH oxidase and mitochondria-dependent oxidative stress, ERS and NF-kB. In addition, AML alleviates apoptosis by reducing the pro-apoptotic CHOP expression and re-establishing the mitochondrial transmembrane potential. SignificanceThe results of the present study suggest that Ninj-1 and the proteins involved in its regulation can be considered therapeutic targets for the alleviation of inflammation- dependent disorders. In addition, we demonstrate that some of the benefic effects of AML can be achieved through regulation of Ninj-1.

Mir-331 negatively regulates thyroglobulin secretion via ERp29

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

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway.In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity.Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson’s disease (PD) and of those with Alzheimer’s disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

Endoplasmic Reticulum Stress in Subepithelial Myofibroblasts Increases the TGF-β1 Activity That Regulates Fibrosis in Crohn's Disease.

Endoplasmic reticulum (ER) stress is an essential response of epithelial and immune cells to inflammation in Crohn's disease. The presence and mechanisms that might regulate the ER stress response in subepithelial myofibroblasts (SEMFs) and its role in the development of fibrosis in patients with Crohn's disease have not been examined. Subepithelial myofibroblasts were isolated from the affected ileum and normal ileum of patients with each Montreal phenotype of Crohn's disease and from normal ileum in non-Crohn's subjects. Binding of GRP78 to latent TGF-β1 and its subcellular trafficking was examined using proximity ligation-hybridization assay (PLA). The effects of XBP1 and ATF6 on TGF-β1 expression were measured using DNA-ChIP and luciferase reporter assay. Endoplasmic reticulum stress components, TGF-β1, and collagen levels were analyzed in SEMF transfected with siRNA-mediated knockdown of DNMT1 and GRP78 or with DNMT1 inhibitor 5-Azacytidine or with overexpression of miR-199a-5p. In SEMF of strictured ileum from patients with B2 Crohn's disease, expression of ER stress sensors increased significantly. Tunicamycin elicited time-dependent increase in GRP78 protein levels, direct interaction with latent TGF-β1, and activated TGF-β1 signaling. The TGFB1 DNA-binding activity of ATF-6α and XBP1 were significantly increased and elicited increased TGFB1 transcription in SEMF-isolated from affected ileum. The levels of ER stress components, TGF-β1, and collagen expression in SEMF were significantly decreased following knockdown of DNMT1 or GRP78 by 5-Azacytidine treatment or overexpression of miR-199a-5p. Endoplasmic reticulum stress is present in SEMF of patients susceptible to fibrostenotic Crohn's disease and can contribute to development of fibrosis. Targeting ER stress may represent a novel therapeutic target to prevent fibrosis in patients with fibrostenotic Crohn's disease.

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress.

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1β can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1β has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1β to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host-environment interface.

Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

Mode of action of granulocyte-colony stimulating factor (G-CSF) as a novel therapy for stroke in a mouse model

BackgroundThe FDA approved drug granulocyte-colony stimulating factor (G-CSF) displays anti-apoptotic and immunomodulatory properties with neurogenesis and angiogenic functions. It is known to demonstrate neuroprotective mechanisms against ischemic global stroke. Autophagy is a method for the degradation of intracellular components and in particular, unrestrained autophagy may lead to uncontrolled digestion of affected neurons as well as neuronal death in cerebral ischemia. Mitochondrial dynamics is vital for the regulation of cell survival and death after cerebral ischemia and an early upstream event in neuronal death is mitochondrial fission. We examined the pro-survival mechanisms of G-CSF against apoptosis resulting from autophagy, mitochondrial stress and endoplasmic reticulum (ER) stress.MethodsMale Swiss Webster mice (20 weeks of age) were subjected to bilateral common carotid artery occlusion (BCAO) for 30 min. After occlusion, mice were injected with G-CSF (50 μg/kg) subcutaneously for 4 days. Behavioral analysis was carried out using the corner test and locomotor activity test before animals were sacrificed on day 4 or day 7. Key proteins in ER stress, autophagy and mitochondrial stress induced apoptosis were analyzed by immunoblotting.ResultsG-CSF improved neurological deficits and improved behavioral performance on corner and locomotor test. G-CSF binds to G-CSF receptors and its activation leads to upregulation of Akt phosphorylation (P-Akt) which in turn decreases levels of the ER stress sensor, GRP 78 and expression of proteins involved in ER stress apoptosis pathway; ATF6, ATF4, eIF2α, XBP1, Caspase 12 and CHOP. G-CSF treatment significantly decreased Beclin-1, an autophagy marker, and decreased mitochondrial stress biomarkers DRP1 and P53. G-CSF also up-regulated the mitochondrial fusion protein, OPA1 and anti-apoptotic protein Bcl-2 while down-regulating the pro-apoptotic proteins Bax, Bak and PUMA.ConclusionsG-CSF is an endogenous ligand in the CNS that has a dual activity that is beneficial both in reducing acute neuronal degeneration and adding to long-term plasticity after cerebral ischemia. G-CSF treatment exerts neuroprotective effects on damaged neurons through the suppression of the ER stress and mitochondrial stress and maintains cellular homeostasis by decreasing pro-apoptotic proteins and increasing of anti-apoptotic proteins.

Activation of endoplasmic reticulum stress mediates oxidative stress-induced apoptosis of granulosa cells in ovaries affected by endometrioma.

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress-induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

Molecular Insight Into the IRE1α-Mediated Type I Interferon Response Induced by Proteasome Impairment in Myeloid Cells of the Brain.

Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNβ1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.