Endoplasmic Reticulum Stress Sensor Research Articles

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Coadministration with bendamustine restores the antitumor activity of obinutuzumab in obinutuzumab-resistant tumors.

The glycoengineered, humanized anti-CD20 antibody obinutuzumab is indicated for previously untreated or relapsed/refractory CD20-positive follicular lymphoma (FL). However, the effectiveness of obinutuzumab retreatment in relapsed/refractory FL after prior obinutuzumab-containing therapy is unclear. To address this issue, we investigated the antitumor activity of obinutuzumab plus bendamustine in obinutuzumab-resistant tumors established from a human non-Hodgkin lymphoma xenograft model. Obinutuzumab-resistant tumors (SU-DHL-4-OR-18-8) were established from an SU-DHL-4 xenograft model by repeated administration of obinutuzumab. Antitumor activity was evaluated based on tumor volume after treatment with obinutuzumab on Day 1, 8, and 15 and/or bendamustine on Day 1 and 2. Intratumoral natural killer (NK) cells/macrophages were evaluated by immunohistochemistry and flow cytometry. In SU-DHL-4-OR-18-8 xenografted tumors, intratumoral NK cells/macrophages after obinutuzumab treatment were significantly decreased compared with parent tumors on Day 4. The endoplasmic reticulum stress sensor phospho-IRE1 was also decreased. In SU-DHL-4-OR-18-8 tumors, bendamustine treatment increased phospho-IRE1 on Day 4 and intratumor NK cells/macrophages on Day 10. Obinutuzumab combined with bendamustine significantly increased antitumor activity compared with each single agent on Day 29, with an increase in chemoattractant CCL6 expression on Day 10. Coadministration of bendamustine in obinutuzumab retreatment may be effective against obinutuzumab-resistant tumors.

Self-association status-dependent inactivation of the endoplasmic reticulum stress sensor Ire1 by C-terminal tagging with artificial peptides.

Upon endoplasmic reticulum (ER) stress, eukaryotic cells commonly induce unfolded protein response (UPR), which is triggered, at least partly, by the ER stress sensor Ire1. Upon ER stress, Ire1 is dimerized or forms oligomeric clusters, resulting in the activation of Ire1 as an endoribonuclease. In ER-stressed Saccharomyces cerevisiae cells, HAC1 mRNA is spliced by Ire1 and then translated into a transcription factor that promotes the UPR. Herein, we report that Ire1 tagged artificially with irrelevant peptides at the C terminus is almost completely inactive when only dimerized, while it induced the UPR as well as untagged Ire1 when clustered. This finding suggests a fundamental difference between the dimeric and clustered forms of Ire1. By comparing UPR levels in S. cerevisiae cells carrying artificially peptide-tagged Ire1 to that in cells carrying untagged Ire1, we estimated the self-association status of Ire1 under various ER stress conditions.

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin – an inhibitor of the de novo sphingolipid synthesis pathway – inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

Kinin B1R Activation Induces Endoplasmic Reticulum Stress in Primary Hypothalamic Neurons

The endoplasmic reticulum (ER) is a key organelle involved in homeostatic functions including protein synthesis and transport, and the storage of free calcium. ER stress potentiates neuroinflammation and neurodegeneration and is a key contributor to the pathogenesis of neurogenic hypertension. Recently, we showed that kinin B1 receptor (B1R) activation plays a vital role in modulating neuroinflammation and hypertension. However, whether B1R activation results in the progression and enhancement of ER stress has not yet been studied. In this brief research report, we tested the hypothesis that B1R activation in neurons contributes to unfolded protein response (UPR) and the development of ER stress. To test this hypothesis, we treated primary hypothalamic neuronal cultures with B1R specific agonist Lys-Des-Arg9-Bradykinin (LDABK) and measured the components of UPR and ER stress. Our data show that B1R stimulation via LDABK, induced the upregulation of GRP78, a molecular chaperone of ER stress. B1R stimulation was associated with an increased expression and activation of transmembrane ER stress sensors, ATF6, IRE1α, and PERK, the critical components of UPR. In the presence of overwhelming ER stress, activated ER stress sensors can lead to oxidative stress, autophagy, or apoptosis. To determine whether B1R activation induces apoptosis we measured intracellular Ca2+ and extracellular ATP levels, caspases 3/7 activity, and cell viability. Our data show that LDABK treatment does increase Ca2+ and ATP levels but does not alter caspase activity or cell viability. These findings suggest that B1R activation initiates the UPR and is a key factor in the ER stress pathway.

Mechanistic Insights into Ameliorating Effect of Geraniol on d-Galactose Induced Memory Impairment in Rats

Geraniol (GE), an important ingredient in several essential oils, displayed pleiotropic biological activities through targeting multiple signaling cascades. In the current study, we aimed to examine the protective effect of GE on d-galactose (d-gal) induced cognitive impairment and explore the underlying mechanisms. Forty male Wistar rats (8 weeks old) were randomly categorized into 4 groups; Group I (saline + vehicle [edible oil]), group II (saline + geraniol) (100 mg/kg/day orally), group III (d-galactose) (100 mg/kg/day subcutaneously injected), and group IV (d-galactose + geraniol). Behavioral impairments were evaluated. Brain levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as superoxide dismutase (SOD) and acetylcholinesterase (AchE) activities were estimated. The levels of inflammatory markers [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and nuclear factor kappa beta (NF-kβ)], endoplasmic reticulum stress sensors [inositol requiring protein 1(IRE1) and protein kinase RNA–like endoplasmic reticulum kinase (PERK)], brain-derived neurotrophic factor (BDNF), and mitogen-activated protein kinases (MAPK) pathway were measured by ELISA. Also, hippocampal histopathological assessment and immunohistochemical analysis of glial fibrillary acidic protein (GFAP) and caspase-3 were performed. Glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) mRNA expression and protein levels were assessed. GE effectively ameliorated aging-related memory impairment through increasing GSH, BDNF, Ach levels, and SOD activity. Additionally, GE treatment caused a decrease in the levels of MDA, inflammatory mediators, and ER stress sensors as well as the AchE activity together with concomitant down-regulation of GRP78 and CHOP mRNA expression. Moreover, GE improved neuronal architecture and rat's spatial memory; this is evidenced by the shortened escape latency and increased platform crossing number. Therefore, GE offers a unique pharmacological approach for aging-associated neurodegenerative disorders.Graphical

PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway.

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1α (IRE1α) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

Reactivation of dysfunctional dendritic cells by a stress-relieving nanosystem resets anti-tumor immune landscape

Dendritic cells (DCs), particularly tumor-infiltrating DCs (TIDCs), are critical to the orchestration of T cell-dependent anti-tumor immune response. However, immunosuppressive tumor microenvironment (TME) conspires to pose multifarious challenges that hinder the function of TIDCs and promote the development of immune evasion. The TIDCs with functional defects usually accumulate excessive lipid, characterized with both aberrantly endoplasmic reticulum (ER) stress and oxidative stress. In this study, we generate a KIRA6 loaded α-Tocopherol nanoemulsion (KT-NE), which concurrently inhibits ER stress sensor inositol-requiring kinase 1α (IRE1α)-X-box binding protein 1 (XBP1) and reactive oxygen species (ROS) in dysfunctional lipid-laden TIDCs. As a result, KT-NE prominently mitigates the XBP1- and ROS-triggered lipid accumulation of TIDCs, which reactivates TIDCs and rehabs their capacity of expanding and stimulating tumor-specific T cells. Moreover, both KT-NE treated TIDCs (adoptive transfer) and KT-NE itself could significantly restore the efficacy of DC-mediated immunotherapy and ease tumor burden in malignant ovarian cancer (OvCa)-bearing mice. In conclusion, co-inhibiting hyperactivated XBP1 and redundant intracellular ROS though KT-NE may be a potent therapeutic strategy to ameliorate the ineffective DCs, reverse hostile TME, evoke robust anti-tumor immunity, and extend host progression-free survival ultimately.

The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

Interactions between the mitochondrial inner and outer membranes and between mitochondria and other organelles closely correlates with the sensitivity of ovarian cancer to cisplatin and other chemotherapeutic drugs. However, the underlying mechanism remains unclear. Recently, the mitochondrial protease OMA1, which regulates internal and external signals in mitochondria by cleaving mitochondrial proteins, was shown to be related to tumor progression. Therefore, we evaluated the effect of OMA1 on the response to chemotherapeutics in ovarian cancer cells and the mouse subcutaneous tumor model. We found that OMA1 activation increased ovarian cancer sensitivity to cisplatin in vivo and in vitro. Mechanistically, in ovarian cancer, OMA1 cleaved optic atrophy 1 (OPA1), leading to mitochondrial inner membrane cristae remodeling. Simultaneously, OMA1 induced DELE1 cleavage and its cytoplasmic interaction with EIF2AK1. We also demonstrated that EIF2AK1 cooperated with the ER stress sensor EIF2AK3 to amplify the EIF2S1/ATF4 signal, resulting in the rupture of the mitochondrial outer membrane. Knockdown of OMA1 attenuated these activities and reversed apoptosis. Additionally, we found that OMA1 protease activity was regulated by the prohibitin 2 (PHB2)/stomatin-like protein 2 (STOML2) complex. Collectively, OMA1 coordinates the mitochondrial inner and outer membranes to induce ovarian cancer cell death. Thus, activating OMA1 may be a novel treatment strategy for ovarian cancer.

Endoplasmic Reticulum Stress and miRNA Impairment in Aging and Age-Related Diseases.

Aging is a physiological process defined by decreased cellular and tissue functions. Reduced capacity of protein degradation is one of the important hallmarks of aging that may lead to misfolded protein accumulation and progressive loss of function in organ systems. Recognition of unfolded/misfolded protein aggregates via endoplasmic reticulum (ER) stress sensors activates an adaptive mechanism, the unfolded protein response (UPR). The initial step of UPR is defined by chaperone enhancement, ribosomal translation suppression, and misfolded protein degradation, while prolonged ER stress triggers apoptosis. MicroRNAs (miRNAs) are non-coding RNAs affecting various signaling pathways through degradation or translational inhibition of targeted mRNAs. Therefore, UPR and miRNA impairment in aging and age-related diseases is implicated in various studies. This review will highlight the recent insights in ER stress–miRNAs alterations during aging and age-related diseases, including metabolic, cardiovascular, and neurodegenerative diseases and several cancers.

Glibenclamide Elicits Endoplasmic Reticulum Stress and Myokine Expression in the L6 Skeletal Muscle Cells

Although glibenclamide is an oral hypoglycemic agent used in type 2 diabetes, skeletal muscle wasting has been reported as a side effect. To understand how to reduce this side effect, we determined whether glibenclamide induces endoplasmic reticulum (ER) stress in skeletal muscle cells and which myokine expression changes at this time. The ER chaperone genes do not show a significant change by glibenclamide, but the ER stress sensor genes are upexpressed approximately twice, and those downstream [ATF6 (activating transcription factor 6) fragmentation, eIF2α (eukaryotic initiation factor-2α) phosphorylation, and XBP1 (Xbox DNA-binding protein) mRNA splicing] are activated. Additionally, the myokine gene expression was up- or downregulated by glibenclamide. These results will serve as useful data for overcoming the side effects of sarcopenia caused by glibenclamide.

Induction and Aggravation of the Endoplasmic-Reticulum Stress by Membrane-Lipid Metabolic Intermediate Phosphatidyl-N-Monomethylethanolamine.

Phosphatidylcholine (PC) is produced via two distinct pathways in both hepatocytes and yeast, Saccharomyces cerevisiae. One of these pathways involves the sequential methylation of phosphatidylethanolamine (PE). In yeast cells, the methyltransferase, Cho2, converts PE to phosphatidylmonomethylethanolamine (PMME), which is further modified to PC by another methyltransferase, Opi3. On the other hand, free choline is utilized for PC production via the Kennedy pathway. The blockage of PC production is well known to cause endoplasmic reticulum (ER) stress and activate the ER-stress sensor, Ire1, to induce unfolded protein response (UPR). Here, we demonstrate that even when free choline is sufficiently supplied, the opi3Δ mutation, but not the cho2 Δ mutation, induces the UPR. The UPR was also found to be induced by CHO2 overexpression. Further, monomethylethanolamine, which is converted to PMME probably through the Kennedy pathway, caused or potentiated ER stress in both mammalian and yeast cells. We thus deduce that PMME per se is an ER-stressing molecule. Interestingly, spontaneously accumulated PMME seemed to aggravate ER stress in yeast cells. Collectively, our findings demonstrate the multiple detrimental effects of the low-abundance phospholipid species, PMME.

Assays to Study IRE1 Activation and Signaling.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known asregulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress.Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Cell Cycle Analysis of ER Stress and Autophagy.

Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.

The interplay of GTP-binding protein AGB1 with ER stress sensors IRE1a and IRE1b modulates Arabidopsis unfolded protein response and bacterial immunity

ABSTRACT In eukaryotic cells, the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in ER stress that induces a cascade of reactions called the unfolded protein response (UPR). In Arabidopsis, the most conserved UPR sensor, Inositol-requiring enzyme 1 (IRE1), responds to both abiotic- and biotic-induced ER stress. Guanine nucleotide-binding proteins (G proteins) constitute another universal and conserved family of signal transducers that have been extensively investigated due to their ubiquitous presence and diverse nature of action. Arabidopsis GTP-binding protein β1 (AGB1) is the only G-protein β-subunit encoded by the Arabidopsis genome that is involved in numerous signaling pathways. Mounting evidence suggests the existence of a crosstalk between IRE1 and G protein signaling during ER stress. AGB1 has previously been shown to control a distinct UPR pathway independently of IRE1 when treated with an ER stress inducer tunicamycin. Our results obtained with combinatorial knockout mutants support the hypothesis that both IRE1 and AGB1 synergistically contribute to ER stress responses chemically induced by dithiothreitol (DTT) as well as to the immune responses against a phytopathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000. Our study highlights the crosstalk between the plant UPR transducers under abiotic and biotic stress.

The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

Hepatic ischemia/reperfusion (I/R) injury is an inflammation-mediated process arising from ischemia/reperfusion-elicited stress in multiple cell types, causing liver damage during surgical procedures and often resulting in liver failure. Endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response (UPR) and is implicated in tissue injuries, including hepatic I/R injury. However, the cellular mechanism that links the UPR signaling to local inflammatory responses during hepatic I/R injury remains largely obscure. Here, we report that IRE1α, a critical ER-resident transmembrane signal transducer of the UPR, plays an important role in promoting Kupffer-cell-mediated liver inflammation and hepatic I/R injury. Utilizing a mouse model in which IRE1α is specifically ablated in myeloid cells, we found that abrogation of IRE1α markedly attenuated necrosis and cell death in the liver, accompanied by reduced neutrophil infiltration and liver inflammation following hepatic I/R injury. Mechanistic investigations in mice as well as in primary Kupffer cells revealed that loss of IRE1α in Kupffer cells not only blunted the activation of the NLRP3 inflammasome and IL-1β production, but also suppressed the expression of the inducible nitric oxide synthase (iNos) and proinflammatory cytokines. Moreover, pharmacological inhibition of IRE1α′s RNase activity was able to attenuate inflammasome activation and iNos expression in Kupffer cells, leading to alleviation of hepatic I/R injury. Collectively, these results demonstrate that Kupffer cell IRE1α mediates local inflammatory damage during hepatic I/R injury. Our findings suggest that IRE1α RNase activity may serve as a promising target for therapeutic treatment of ischemia/reperfusion-associated liver inflammation and dysfunction.

A Benzenesulfonamide-Based Mitochondrial Uncoupler Induces Endoplasmic Reticulum Stress and Immunogenic Cell Death in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and requires new therapeutic strategies to improve clinical outcomes. EOC metastasizes in the abdominal cavity through dissemination in the peritoneal fluid and ascites, efficiently adapt to the nutrient-deprived microenvironment, and resist current chemotherapeutic agents. Accumulating evidence suggests that mitochondrial oxidative phosphorylation is critical for the adaptation of EOC cells to this otherwise hostile microenvironment. Although chemical mitochondrial uncouplers can impair mitochondrial functions and thereby target multiple, essential pathways for cancer cell proliferation, traditional mitochondria uncouplers often cause toxicity that precludes their clinical application. In this study, we demonstrated that a mitochondrial uncoupler, specifically 2,5-dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide, hereinafter named Y3, was an antineoplastic agent in ovarian cancer models. Y3 treatment activated AMP-activated protein kinase and resulted in the activation of endoplasmic reticulum stress sensors as well as growth inhibition and apoptosis in ovarian cancer cells in vitro Y3 was well tolerated in vivo and effectively suppressed tumor progression in three mouse models of EOC, and Y3 also induced immunogenic cell death of cancer cells that involved the release of damage-associated molecular patterns and the activation of antitumor adaptive immune responses. These findings suggest that mitochondrial uncouplers hold promise in developing new anticancer therapies that delay tumor progression and protect patients with ovarian cancer against relapse.

The function role of ubiquitin proteasome pathway in the ER stress-induced AECII apoptosis during hyperoxia exposure

BackgroundBronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar dysplasia and pulmonary microvascular remodeling. In the present study, we have investigated the functional roles of ubiquitin proteasome pathway (UPP) in BPD, and its relationship with endoplasmic reticulum stress (ERS) mediated type II alveolar epithelial cell (AECII) apoptosis.MethodsA hyperoxia-induced BPD rat model was constructed and the pathologic changes of lung tissues were evaluated by hematoxylin–eosin staining. Cell apoptosis and protein expression were determined by TUNEL assay and Western blotting, respectively. Further reagent kit with specific fluorescent substrate was utilized to measure the activity of 20 s proteasome. Meanwhile, AECII were cultured in vitro and exposed to hyperoxia. AECII apoptosis were measured by flow cytometry. In contrast, MG132 treatment was induced to explore UPP during hyperoxia exposure on AECII apoptosis and ERS sensors expression.ResultsA significant increase in apoptosis and total ubiquitinated proteins expression were observed in BPD rats and AECII culture, and the change of UPP was associated with ERS. In order to confirm the role of UPP in AECII apoptosis of BPD, AECII cells were treated by MG132 with the concentration of 10 μmol/L under hyperoxia exposure. We found that the proteins expression of glucose-regulated protein 78 (GRP-78), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), as well as AECII apoptosis were increased following MG132 treatment. Furthermore, the relatively up-regulated in the levels of total ubiquitinated proteins expression and 20 s proteasome activity were correlated with increased ERS sensors expression.ConclusionsOur findings indicate that UPP may participate in the ERS-induced AECII apoptosis under hyperoxia condition.

The Endoplasmic Reticulum Stress Sensor IRE1α Regulates the UV DNA Repair Response through the Control of Intracellular Calcium Homeostasis

The unfolded protein response is activated by UVB irradiation, but the role of a key mediator, IRE1α, is not clear. In this study, we show that mice with an epidermal IRE1α deletion are sensitized to UV with increased apoptosis, rapid loss of UV-induced cyclopyrimidine dimer‒positive keratinocytes, and sloughing of the epidermis. Invitro, Ire1α-deficient keratinocytes have increased UVB sensitivity, reduced cyclopyrimidine dimer repair, and reduced accumulation of γH2AX and phosphorylated ATR, suggesting defective activation of nucleotide excision repair. Knockdown of XBP1 or pharmacologic inhibition of the IRE1α ribonuclease did not phenocopy Ire1α deficiency. The altered UV response was linked to elevated intracellular calcium levels and ROS, and this was due to dysregulation of the endoplasmic reticulum calcium channel InsP3R. Pharmacologic, genetic, and biochemical studies linked the regulation of the Ins3PR, intracellular calcium, and normal UV DNA damage response to CIB1 and the IRE1α‒TRAF2‒ASK1 complex. These results suggest a model where IRE1α activation state drives CIB1 binding either to the InsP3R or ASK1 to regulate endoplasmic reticulum calcium efflux, ROS, and DNA repair responses after UV irradiation.