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Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma. NEW & NOTEWORTHY: TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion. [ABSTRACT FROM AUTHOR]

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP: [ABSTRACT FROM AUTHOR]

In chronic diabetic neuropathy (DN), the cellular mechanisms of neuropathic pain remain unclear. Protein kinase C epsilon (PKCε) is an intracellular signaling molecule that mediates chronic pain. This paper addresses the long-term upregulated PKCε in DN associated with endoplasmic reticulum (ER) stress and autophagic formation and correlates to chronic neuropathic pain. We found that thermal hyperalgesia and mechanical allodynia course development were associated with PKCε upregulation after DN but not skin denervation. Pathologically, PKCε upregulation was associated with the expression of inositol-requiring enzyme 1α (IRE1α; ER stress–related molecule) and ubiquitin D (UBD), which are involved in the ubiquitin-proteasome system (UPS)-mediated degradation of misfolded proteins under ER stress. Manders coefficient analyses revealed an approximately 50% colocalized ratio for IRE1α(+):PKCε(+) neurons (0.34–0.48 for M1 and 0.40–0.58 for M2 Manders coefficients). The colocalized coefficients of UBD/PKCε increased (M1: 0.33 ± 0.03 vs. 0.77 ± 0.04, p < 0.001; M2: 0.29 ± 0.05 vs. 0.78 ± 0.04; p < 0.001) in the acute DN stage. In addition, the regulatory subunit p85 of phosphoinositide 3-kinase, which is involved in regulating insulin signaling, exhibited similar expression patterns to those of IRE1α and UBD; for example, it had highly colocalized ratios to PKCε. The ultrastructural examination further confirmed that autophagic formation was associated with PKCε upregulation. Furthermore, PKCεv1-2, a PKCε specific inhibitor, reverses neuropathic pain, ER stress, and autophagic formation in DN. This finding suggests PKCε plays an upstream molecule in DN-associated neuropathic pain and neuropathology and could provide a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons. [ABSTRACT FROM AUTHOR]

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress. Synopsis: This study describes a new technology for isolating organelle membranes from yeast, MemPrep. Combined with quantitative lipidomics, MemPrep establishes molecular fingerprints of membrane-based forms of endoplasmic reticulum (ER) stress. MemPrep provides quantitative insight into the compositions of the yeast vacuole and ER membrane. The ER membrane composition is significantly distinct from the whole cell lipidome. Lipid metabolic perturbation causes dramatic changes of the ER membrane composition, which sometimes trigger ER stress. Inositol depletion and prolonged proteotoxic stress lead to distinct lipid fingerprints and membrane proteomes. Increased membrane thickness and low levels of anionic lipids are common denominators of lipid bilayer stress. Combined with proteomics and quantitative lipidomics, this method for purification of specific yeast membranes establishes molecular fingerprints of membrane-based forms of endoplasmic reticulum (ER) stress. [ABSTRACT FROM AUTHOR]

Introduction: Neonatal hypoxic-ischemic brain damage (HIBD) refers to brain damage in newborns caused by hypoxia and reduced or even stopped cerebral blood flow during the perinatal period. Currently, there are no targeted treatments for neonatal ischemic hypoxic brain damage, primarily due to the incomplete understanding of its pathophysiological mechanisms. Especially, the role of NMDA receptors is less studied in HIBD. Therefore, this study explored the molecular mechanism of endogenous protection mediated by GluN2B-NMDAR in HIBD. Method: Hypoxic ischemia was induced in mice aged 9-11 days. The brain damage was examined by Nissl staining and HE staining, while neuronal apoptosis was examined by Hoechst staining and TTC staining. And cognitive deficiency of mice was examined by various behavior tests including Barnes Maze, Three Chamber Social Interaction Test and Elevated Plus Maze. The activation of ER stress signaling pathways were evaluated by Western blot. Results: We found that after HIBD induction, the activation of GluN2B-NMDAR attenuated neuronal apoptosis and brain damage. Meanwhile, the ER stress PERK/eIF2α signaling pathway was activated in a time-dependent manner after HIBE. Furthermore, after selective inhibiting GluN2B-NMDAR in HIBD mice with ifenprodil, the PERK/eIF2α signaling pathway remains continuously activated, leading to neuronal apoptosis, morphological brain damage. and aggravating deficits in spatial memory, cognition, and social abilities in adult mice. Discussion: The results of this study indicate that, unlike its role in adult brain damage, GluN2B in early development plays a neuroprotective role in HIBD by inhibiting excessive activation of the PERK/eIF2α signaling pathway. This study provides theoretical support for the clinical development of targeted drugs or treatment methods for HIBD. [ABSTRACT FROM AUTHOR]

Numerous studies have been conducted to investigate the genomic characterization of bZIP genes and their involvement in the cellular response to endoplasmic reticulum (ER) stress. These studies have provided valuable insights into the coordinated cellular response to ER stress, which is mediated by bZIP transcription factors (TFs). However, a comprehensive and systematic investigations regarding the role of bZIP genes and their involvement in ER stress response in pak choi is currently lacking in the existing literature. To address this knowledge gap, the current study was initiated to elucidate the genomic characteristics of bZIP genes, gain insight into their expression patterns during ER stress in pak choi, and investigate the protein-to-protein interaction of bZIP genes with the ER chaperone BiP. In total, 112 members of the BcbZIP genes were identified through a comprehensive genome-wide analysis. Based on an analysis of sequence similarity, gene structure, conserved domains, and responsive motifs, the identified BcbZIP genes were categorized into 10 distinct subfamilies through phylogenetic analysis. Chromosomal location and duplication events provided insight into their genomic context and evolutionary history. Divergence analysis estimated their evolutionary history with a predicted divergence time ranging from 0.73 to 80.71 million years ago (MYA). Promoter regions of the BcbZIP genes were discovered to exhibit a wide variety of cis-elements, including light, hormone, and stress-responsive elements. GO enrichment analysis further confirmed their roles in the ER unfolded protein response (UPR), while co-expression network analysis showed a strong relationship of BcbZIP genes with ER-stress-responsive genes. Moreover, gene expression profiles and protein–protein interaction with ER chaperone BiP further confirmed their roles and capacity to respond to ER stress in pak choi. [ABSTRACT FROM AUTHOR]

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling. Synopsis: Dual regulation of Cell Death Receptor CD95 signalling by ER stress sensor IRE1. IRE1, a mediator of the unfolded protein response (UPR), governs CD95/Fas expression and CD95L-induced cell death in opposing ways. IRE1 can induce CD95 expression to promote CD95L-induced cell death via the transcription factor XBP1s in triple-negative breast cancer (TNBC) and glioblastoma (GB) cells. IRE1 can also repress CD95 expression to limit CD95L-induced cell death through RIDD activity, which cleaves CD95 mRNA in both TNBC and GB cells. The net effect of these opposing mechanisms depends on the cellular context. IRE1 controls CD95 signalling in vivo. Activation of RIDD or XBP1s downstream of IRE1 dually correlates with CD95 mRNA expression in human TNBC and GB tumours. Dual regulation of Cell Death Receptor CD95 signaling by ER stress sensor IRE1. IRE1, a mediator of the unfolded protein response (UPR), governs CD95/Fas expression and CD95L-induced cell death in opposing ways. [ABSTRACT FROM AUTHOR]

Acute HSV-1 infection is associated with mild symptoms, such as fever and lesions of the mouth, face and skin. This phase is followed by a latency period before reactivation, which is associated with symptoms ranging from ulcers to encephalitis. Despite available anti-HSV-1 drugs, the development of new antiviral agents is sought due to the presence of resistant viruses. Melatonin, a molecule secreted by the pineal gland, has been shown to be an antioxidant, inducer of antioxidant enzymes, and regulator of various biological processes. Clinical trials have explored its therapeutic utility in conditions including infections. This study focuses on melatonin's role in HSV-1 replication and the underlying mechanisms. Melatonin was found to decrease the synthesis of HSV-1 proteins in infected Vero cells measured by immunofluorescence, indicating an inhibition of HSV-1 replication. Additionally, it regulates the activities of antioxidant enzymes and affects proteasome activity. Melatonin activates the unfolded protein response (UPR) and autophagy and suppresses apoptosis in HSV-1-infected cells. In summary, melatonin demonstrates an inhibitory role in HSV-1 replication by modulating various cellular responses, suggesting its potential utility in the treatment of viral infections. [ABSTRACT FROM AUTHOR]

The accumulation of misfolded and aggregated α-synuclein can trigger endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), leading to apoptotic cell death in patients with Parkinson's disease (PD). As the major ER chaperone, glucose-regulated protein 78 (GRP78/BiP/HSPA5) plays a key role in UPR regulation. GRP78 overexpression can modulate the UPR, block apoptosis, and promote the survival of nigral dopamine neurons in a rat model of α-synuclein pathology. Here, we explore the therapeutic potential of intranasal exogenous GRP78 for preventing or slowing PD-like neurodegeneration in a lactacystin-induced rat model. We show that intranasally-administered GRP78 rapidly enters the substantia nigra pars compacta (SNpc) and other afflicted brain regions. It is then internalized by neurons and microglia, preventing the development of the neurodegenerative process in the nigrostriatal system. Lactacystin-induced disturbances, such as the abnormal accumulation of phosphorylated pS129-α-synuclein and activation of the pro-apoptotic GRP78/PERK/eIF2α/CHOP/caspase-3,9 signaling pathway of the UPR, are substantially reversed upon GRP78 administration. Moreover, exogenous GRP78 inhibits both microglia activation and the production of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in model animals. The neuroprotective and anti-inflammatory potential of exogenous GRP78 may inform the development of effective therapeutic agents for PD and other synucleinopathies. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Defective insulin signalling and dysfunction of the endoplasmic reticulum (ER), driven by excessive lipid accumulation in the liver, is a characteristic feature in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). Thromboxane A2 (TXA2), an arachidonic acid metabolite, is significantly elevated in obesity and plays a crucial role in hepatic gluconeogenesis and adipose tissue macrophage polarization. However, the role of liver TXA2/TP receptors in insulin resistance and lipid metabolism is largely unknown. Experimental Approach: TP receptor knockout (TP−/−) mice were generated and fed a high‐fat diet for 16 weeks. Insulin sensitivity, ER stress responses and hepatic lipid accumulation were assessed. Furthermore, we used primary hepatocytes to dissect the mechanisms by which the TXA2/TP receptor axis regulates insulin signalling and hepatocyte lipogenesis. Key Results: TXA2 was increased in diet‐induced obese mice, and depletion of TP receptors in adult mice improved systemic insulin resistance and hepatic steatosis. Mechanistically, we found that the TXA2/TP receptor axis disrupts insulin signalling by activating the Ca2+/calcium calmodulin‐dependent kinase II γ (CaMKIIγ)–protein kinase RNA‐like endoplasmic reticulum kinase (PERK)–C/EBP homologous protein (Chop)–tribbles‐like protein 3 (TRB3) axis in hepatocytes. In addition, our results revealed that the TXA2/TP receptor axis directly promoted lipogenesis in primary hepatocytes and contributed to Kupffer cell inflammation. Conclusions and Implications: The TXA2/TP receptor axis facilitates insulin resistance through Ca2+/CaMKIIγ to activate PERK–Chop–TRB3 signalling. Inhibition of hepatocyte TP receptors improved hepatic steatosis and inflammation. The TP receptor is a new therapeutic target for NAFLD and metabolic syndrome. [ABSTRACT FROM AUTHOR]

Ulcerative colitis (UC), a prevalent form of inflammatory bowel disease (IBD), may result from immune system dysfunction, leading to the sustained overproduction of reactive oxygen species (ROS) and subsequent cellular oxidative stress damage. Recent studies have identified both peroxisome proliferator-activated receptor-γ (PPARγ) and endoplasmic reticulum (ER) stress as critical targets for the treatment of IBD. Oroxyloside (C 22 H 20 O 11), derived from the root of Scutellaria baicalensis Georgi, has traditionally been used in treating inflammatory diseases. In this study, we investigated the molecular mechanisms by which oroxyloside mitigates dextran sulfate sodium (DSS)-induced colitis. We examined the effects of oroxyloside on ROS-mediated ER stress in colitis, including the protein expressions of GRP78, p-PERK, p-eIF2α, ATF4, and CHOP, which are associated with ER stress. The beneficial impact of oroxyloside was reversed by the PPARγ antagonist GW9662 (1 mg·kg−1, i.v.) in vivo. Furthermore, oroxyloside decreased pro-inflammatory cytokines and ROS production in both bone marrow-derived macrophages (BMDM) and the mouse macrophage cell line RAW 264.7. However, PPARγ siRNA transfection blocked the anti-inflammatory effect of oroxyloside and even abolished ROS generation and ER stress activation inhibited by oroxyloside in vitro. In conclusion, our study demonstrates that oroxyloside ameliorates DSS-induced colitis by inhibiting ER stress via PPARγ activation, suggesting that oroxyloside might be a promising effective agent for IBD. [ABSTRACT FROM AUTHOR]

Retinal ischemia exists in various ischemic retinopathies including glaucoma, contributing to the death of retinal neurons. Calcium binding protein S100A4 is important in tumors, and our previous study found that S100A4 protects retinal ganglion cells (RGCs) against retinal ischemia-reperfusion (I/R) injury. This study was aimed to further discuss the neuroprotection and mechanisms of S100A4 in retinal I/R of mice. The rAAV-EF1α-s100a4-EGFP-WPRE or rAAV-EF1α-EGFP-WPRE-Pa was injected intravitreally 4 weeks before I/R. S100A4, molecules in TLR4 signaling pathway and endoplasmic reticulum (ER) stress branches, inflammatory molecules, and surviving RGCs and cholinergic amacrine (ChAT) cells were determined by quantitative PCR, western blot, or immunofluorescent staining. The apoptosis and necrosis of retinal neurons induced by I/R were inhibited by overexpressed S100A4. RGCs, ChAT cells, and the retinal function were preserved by S100A4 overexpressing 7 days after I/R. Mechanistically, the beneficial effects of S100A4 may be mediated by inhibiting the activation of TLR4 signaling pathway and alleviating ER stress, leading to the attenuation of inflammatory response of the retina after I/R. Our findings indicated that S100A4 has neuroprotective effect against retinal I/R injury, and promoting S100A4 expression may be an effective strategy to inhibit retinal neurons from degeneration in ischemic retinopathy. [ABSTRACT FROM AUTHOR]

Neuronal cell death is acknowledged as the primary pathological basis underlying developmental neurotoxicity in response to sevoflurane exposure, but the exact mechanism remains unclear. Ferroptosis is a form of programmed cell death characterized by iron-dependent lipid peroxidation that is driven by hydrogen peroxide (H2O2) and ferrous iron through the Fenton reaction and participates in the pathogenesis of multiple neurological diseases. As stress response factor, activating transcription factor 3 (ATF3) can be activated by the PERK/ATF4 pathway during endoplasmic reticulum (ER) stress, followed by increased intracellular H2O2, which is involved in regulation of apoptosis, autophagy, and ferroptosis. Here, we investigated whether ferroptosis and ATF3 activation were implicated in sevoflurane-induced neuronal cell death in the developing brain. The results showed that sevoflurane exposure induced neuronal death as a result of iron-dependent lipid peroxidation damage secondary to H2O2 accumulation and ferrous iron increase, which was consistent with the criteria for ferroptosis. Furthermore, we observed that increases in iron and H2O2 induced by sevoflurane exposure were associated with the upregulation and nuclear translocation of ATF3 in response to ER stress. Knockdown of ATF3 expression alleviated iron-dependent lipid peroxidation, which prevented sevoflurane-induced neuronal ferroptosis. Mechanistically, ATF3 promoted sevoflurane-induced H2O2 accumulation by activating NOX4 and suppressing catalase, GPX4, and SLC7A11 expression. Additionally, an increase in H2O2 was accompanied by the upregulation of TFR and TF and downregulation of FPN, which linked iron overload to ferroptosis induced by sevoflurane. Taken together, our results demonstrated that ER stress-mediated ATF3 activation contributed to sevoflurane-induced neuronal ferroptosis via H2O2 accumulation and the resultant iron overload. [ABSTRACT FROM AUTHOR]

Cholestane-3β,5α,6β-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer, characterized by a five-year survival rate of 15% and poor prognosis. Accumulating evidence indicates a prominent role of endoplasmic reticulum (ER) stress and the protein kinase RNA-like ER kinase (PERK)-dependent pathway of the unfolded protein response (UPR) in the pathogenesis of NSCLC. Increased expression of downstream targets of PERK was observed in various subtypes of NSCLC, and it was associated with a more aggressive phenotype, high risk of recurrence, and poor prognosis. Therefore, the present study aimed to investigate the biological effect of the selective PERK inhibitor NCI 159456 on A549 NSCLC cells and Human Pulmonary Fibroblasts (HPF) in vitro. Treatment of both normal and ER-stressed A549 cells with NCI 159456 resulted in a significant increase in the mRNA expression level of pro-apoptotic genes like activating transcription factor 4 (ATF4), DNA damage inducible transcript 3 (DDIT3), and BCL2 Associated X, Apoptosis Regulator (BAX) as well as a decreased level of the anti-apoptotic gene B-cell lymphoma 2 (Bcl-2). Cytotoxicity and genotoxicity analyses revealed that NCI 159456 significantly decreased viability and increased DNA damage in A549 cells under normal and ER stress conditions. Caspase-3 and reactive oxygen species (ROS) detection assays demonstrated that NCI 159456 significantly induced apoptosis and increased the ROS level in normal and ER-stressed A549 cells. Importantly, treatment with the inhibitor did not affect substantially normal HPF cells at any used concentration. The results indicate that PERK inhibitors could potentially be applied as a targeted therapy for NSCLC. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson's disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade. [ABSTRACT FROM AUTHOR]

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly folded secretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis. [ABSTRACT FROM AUTHOR]

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2–proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15–21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair. [ABSTRACT FROM AUTHOR]

Background: Ferroptosis is known to play a crucial role in diabetic osteopathy. However, key genes and molecular mechanisms remain largely unclear. This study aimed to identify a crucial ferroptosis-related differentially expressed gene (FR-DEG) in diabetic osteopathy and investigate its potential mechanism. Methods: We identified fibronectin type III domain-containing protein 5 (FNDC5)/irisin as an essential FR-DEG in diabetic osteopathy using the Ferroptosis Database (FerrDb) and GSE189112 dataset. Initially, a diabetic mouse model was induced by intraperitoneal injection of streptozotocin (STZ), followed by intraperitoneal injection of irisin. MC3T3-E1 cells treated with high glucose (HG) were used as an in vitro model. FNDC5 overexpression plasmid was used to explore underlying mechanisms in vitro experiments. Femurs were collected for micro-CT scan, histomorphometry, and immunohistochemical analysis. Peripheral serum was collected for ELISA analysis. Cell viability was assessed using a CCK-8 kit. The levels of glutathione (GSH), malondialdehyde (MDA), iron, reactive oxygen species (ROS), and lipid ROS were detected by the corresponding kits. Mitochondria ultrastructure was observed through transmission electron microscopy (TEM). Finally, mRNA and protein expressions were examined by quantitative real‐time PCR (qRT‐PCR) and western blot analysis. Results: The expression of FNDC5 was found to be significantly decreased in both in vivo and in vitro models. Treatment with irisin significantly suppressed ferroptosis and improved bone loss. This was demonstrated by reduced lipid peroxidation and iron overload, increased antioxidant capability, as well as the inhibition of the ferroptosis pathway in bone tissues. Furthermore, in vitro studies demonstrated that FNDC5 overexpression significantly improved HG-induced ferroptosis and promoted osteogenesis. Mechanistic investigations revealed that FNDC5 overexpression mitigated ferroptosis in osteoblasts by inhibiting the eukaryotic initiation factor 2 alpha (eIF2α)/activated transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) pathway. Conclusions: Collectively, our study uncovered the important role of FNDC5/irisin in regulating ferroptosis of diabetic osteopathy, which might be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Background: The aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear. Methods and results: RNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson's analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM. Conclusions: The findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion. [ABSTRACT FROM AUTHOR]

The Arabidopsis pi4kβ1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) β1 and PI4Kβ2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kβ1,2 plants display autoimmunity and shorter roots. Though the pi4kβ1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kβ1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kβ1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kβ and SPP in ER stress and plant immunity. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) pathogenesis involves complex inflammatory events and cell death. Although IBD involves mainly necrosis in the digestive tract, pyroptosis has also been recognized. Nonetheless, the underlying basis is elusive. Gα12/13 overexpression may affect endoplasmic reticulum (ER) stress. This study examined how Gα12/13 and ER stress affect pyroptosis using dextran sulfate sodium (DSS)-induced colitis models. Gα12/13 levels were increased in the distal and proximal colons of mice exposed to a single cycle of DSS, as accompanied by increases of IRE1α, ATF6, and p-PERK. Moreover, Il-6, Il-1β, Ym1, and Arg1 mRNA levels were increased with caspase-1 and IL-1β activation, supportive of pyroptosis. In the distal colon, RIPK1/3 levels were enhanced to a greater degree, confirming necroptosis. By contrast, the mice subjected to three cycles of DSS treatments showed decreases of Gα12/13, as accompanied by IRE1α and ATF6 suppression, but increases of RIPK1/3 and c-Cas3. AZ2 treatment, which inhibited Gα12, has an anti-pyroptotic effect against a single cycle of colitis. These results show that a single cycle of DSS-induced colitis may cause ER stress-induced pyroptosis as mediated by Gα12 overexpression in addition to necroptosis, but three cycles model induces only necroptosis, and that AZ2 may have an anti-pyroptotic effect. [ABSTRACT FROM AUTHOR]

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) mediate the communication between the Endoplasmic Reticulum (ER) and the mitochondria, playing a fundamental role in steroidogenesis. This study aimed to understand how D-aspartate (D-Asp), a well-known stimulator of testosterone biosynthesis and spermatogenesis, affects the mechanism of steroidogenesis in rat testes. Our results suggested that D-Asp exerts this function through MAMs, affecting lipid trafficking, calcium signaling, ER stress, and mitochondrial dynamics. After 15 days of oral administration of D-Asp to rats, there was an increase in both antioxidant enzymes (SOD and Catalase) and in the protein expression levels of ATAD3A, FACL4, and SOAT1, which are markers of lipid transfer, as well as VDAC and GRP75, which are markers of calcium signaling. Additionally, there was a decrease in protein expression levels of GRP78, a marker of aging that counteracts ER stress. The effects of D-Asp on mitochondrial dynamics strongly suggested its active role as well. It induced the expression levels of proteins involved in fusion (MFN1, MFN2, and OPA1) and in biogenesis (NRF1 and TFAM), as well as in mitochondrial mass (TOMM20), and decreased the expression level of DRP1, a crucial mitochondrial fission marker. These findings suggested D-Asp involvement in the functional improvement of mitochondria during steroidogenesis. Immunofluorescent signals of ATAD3A, MFN1/2, TFAM, and TOMM20 confirmed their localization in Leydig cells showing an intensity upgrade in D-Asp-treated rat testes. Taken together, our results demonstrate the involvement of D-Asp in the steroidogenesis of rat testes, acting at multiple stages of both MAMs and mitochondrial dynamics, opening new opportunities for future investigation in other steroidogenic tissues. [ABSTRACT FROM AUTHOR]

Inflammation and cardiac fibrosis are prevalent pathophysiologic conditions associated with hypertension, cardiac remodeling, and heart failure. Endoplasmic reticulum (ER) stress triggers the cells to activate unfolded protein responses (UPRs) and upregulate the ER stress chaperon, enzymes, and downstream transcription factors to restore normal ER function. The mechanisms that link ER stress-induced UPRs upregulation and NF-κB activation that results in cardiac inflammation and collagen production remain elusive. N-Acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a natural tetrapeptide that negatively regulates inflammation and fibrosis, has been reported. Whether it can inhibit ER stress-induced collagen production in cardiac fibroblasts remains unclear. Thus, we hypothesized that Ac-SDKP attenuates ER stress-stimulated collagen production in cardiac fibroblasts by inhibiting CHOP-mediated NF-κB expression. We aimed to study whether Ac-SDKP inhibits tunicamycin (TM)- induced ER stress signaling, NF-κB signaling, the release of inflammatory cytokine interleukin-6, and collagen production in human cardiac fibroblasts (HCFs). HCFs were pre-treated with Ac-SDKP (10 nM) and then stimulated with TM (0.25 μg/mL). We found that Ac-SDKP inhibits TM-induced collagen production by attenuating ER stress-induced UPRs upregulation and CHOP/NF-κB transcriptional signaling pathways. CHOP deletion by specific shRNA maintains the inhibitory effect of Ac-SDKP on NF-κB and type-1 collagen (Col-1) expression at both protein and mRNA levels. Attenuating ER stress-induced UPR sensor signaling by Ac-SDKP seems a promising therapeutic strategy to combat detrimental cardiac inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Breast cancer is significantly influenced by endoplasmic reticulum (ER) stress, impacting both its initiation and progression. When cells experience an accumulation of misfolded or unfolded proteins, they activate the unfolded protein response (UPR) to restore cellular balance. In breast cancer, the UPR is frequently triggered due to challenging conditions within tumors. The UPR has a dual impact on breast cancer. On one hand, it can contribute to tumor growth by enhancing cell survival and resistance to programmed cell death in unfavorable environments. On the other hand, prolonged and severe ER stress can trigger cell death mechanisms, limiting tumor progression. Furthermore, ER stress has been linked to the regulation of non-coding RNAs (ncRNAs) in breast cancer cells. These ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play essential roles in cancer development by influencing gene expression and cellular processes. An improved understanding of how ER stress and ncRNAs interact in breast cancer can potentially lead to new treatment approaches. Modifying specific ncRNAs involved in the ER stress response might interfere with cancer cell survival and induce cell death. Additionally, focusing on UPR-associated proteins that interact with ncRNAs could offer novel therapeutic possibilities. Therefore, this review provides a concise overview of the interconnection between ER stress and ncRNAs in breast cancer, elucidating the nuanced effects of the UPR on cell fate and emphasizing the regulatory roles of ncRNAs in breast cancer progression. [ABSTRACT FROM AUTHOR]

A firm link between endoplasmic reticulum (ER) stress and tumors has been wildly reported. Endoplasmic reticulum oxidoreductase 1 alpha (ERO1α), an ER-resident thiol oxidoreductase, is confirmed to be highly upregulated in various cancer types and associated with a significantly worse prognosis. Of importance, under ER stress, the functional interplay of ERO1α/PDI axis plays a pivotal role to orchestrate proper protein folding and other key processes. Multiple lines of evidence propose ERO1α as an attractive potential target for cancer treatment. However, the unavailability of specific inhibitor for ERO1α, its molecular inter-relatedness with closely related paralog ERO1β and the tightly regulated processes with other members of flavoenzyme family of enzymes, raises several concerns about its clinical translation. Herein, we have provided a detailed description of ERO1α in human cancers and its vulnerability towards the aforementioned concerns. Besides, we have discussed a few key considerations that may improve our understanding about ERO1α in tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: Activating transcription factor 4 (ATF4) is a transcription factor known to regulate genes associated with the sensing of cellular stress such as amino acid deprival, protein misfolding, growth arrest, and cell death. Despite its key role at the crossroads of immune and stress responses, the precise impact of ATF4 during viral infections remains unclear. Thus, ATF4 has a dual role in promoting cell survival or cell death, but also in limiting infection or participating in viral replication. In this review, we focus specifically on ATF4-mediated signaling during human immunodeficiency virus 1 (HIV-1) infection and examine the multifaceted role of ATF4 in this context. We also explore the potential role of its paralogue ATF5. This review also discusses the possible hijacking of ATF4 activation by viruses to modulate host immune responses. By highlighting the involvement of ATF4 in viral infections, this study gives valuable insights that could be used for further strategies to tackle viral infections, providing a deeper understanding of host–pathogen interactions. Cellular integrated stress response (ISR), the mitochondrial unfolded protein response (UPRmt), and IFN signaling are associated with viral infections. Activating transcription factor 4 (ATF4) plays a pivotal role in these pathways and controls the expression of many genes involved in redox processes, amino acid metabolism, protein misfolding, autophagy, and apoptosis. The precise role of ATF4 during viral infection is unclear and depends on cell hosts, viral agents, and models. Furthermore, ATF4 signaling can be hijacked by pathogens to favor viral infection and replication. In this review, we summarize the ATF4-mediated signaling pathways in response to viral infections, focusing on human immunodeficiency virus 1 (HIV-1). We examine the consequences of ATF4 activation for HIV-1 replication and reactivation. The role of ATF4 in autophagy and apoptosis is explored as in the context of HIV-1 infection programmed cell deaths contribute to the depletion of CD4 T cells. Furthermore, ATF4 can also participate in the establishment of innate and adaptive immunity that is essential for the host to control viral infections. We finally discuss the putative role of the ATF4 paralogue, named ATF5, in HIV-1 infection. This review underlines the role of ATF4 at the crossroads of multiple processes reflecting host–pathogen interactions. [ABSTRACT FROM AUTHOR]

Many pathologic states can lead to the accumulation of unfolded/misfolded proteins in cells. This causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR), which encompasses three main adaptive branches. One of these UPR branches is mediated by protein kinase RNA-like ER kinase (PERK), an ER stress sensor. The primary consequence of PERK activation is the suppression of global protein synthesis, which reduces ER workload and facilitates the recovery of ER function. Ischemic stroke induces ER stress and activates the UPR. Studies have demonstrated the involvement of the PERK pathway in stroke pathophysiology; however, its role in stroke outcomes requires further clarification. Importantly, considering mounting evidence that supports the therapeutic potential of the PERK pathway in aging-related cognitive decline and neurodegenerative diseases, this pathway may represent a promising therapeutic target in stroke. Therefore, in this review, our aim is to discuss the current understanding of PERK in ischemic stroke, and to summarize pharmacologic tools for translational stroke research that targets PERK and its associated pathways. [ABSTRACT FROM AUTHOR]

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1−/− fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1−/− but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1−/− cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1−/− than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress. [ABSTRACT FROM AUTHOR]

Anticancer agents are playing an increasing role in the treatment of gastric cancer (GC); however, novel anticancer agents have not been fully developed. Therefore, it is important to investigate compounds that improve sensitivity to the existing anticancer drugs. We have reported that pterostilbene (PTE), a plant stilbene, enhances the antitumor effect of low doses of sunitinib in gastric cancer cells accumulating mitochondrial iron (II) (mtFe) at low doses. In this study, we investigated the relationship between the mtFe deposition and the synergistic effect of PTE and different anticancer drugs. For this study, we used 5-fluorouracil (5FU), cisplatin (CPPD), and lapatinib (LAP), which are frequently used in the treatment of GC, and doxorubicin (DOX), which is known to deposit mtFe. A combination of low-dose PTE and these drugs suppressed the expression of PDZ domain-containing 8 (PDZD8) and increased mtFe accumulation and mitochondrial H2O2. Consequently, reactive oxygen species-associated hypoxia inducible factor-1α activation induced endoplasmic reticulum stress and led to apoptosis, but not ferroptosis. In contrast, 5FU and CDDP did not show the same changes as those observed with PTE and DOX or LAP, and there was no synergistic effect with PTE. These results indicate that the combination of PTE with iron-accumulating anticancer drugs exhibits a strong synergistic effect. These findings would help in developing novel therapeutic strategies for GC. However, further clinical investigations are required. [ABSTRACT FROM AUTHOR]

Cisplatin nephrotoxicity is a critical limitation of solid cancer treatment. Until now, the complex interplay of various pathophysiological mechanisms leading to proximal tubular cell apoptosis after cisplatin exposure has not been fully understood. In our study, we assessed the role of the autophagy-related protein BECLIN1 (ATG6) in cisplatin-induced acute renal injury (AKI)—a candidate protein involved in autophagy and with putative impact on apoptosis by harboring a B-cell lymphoma 2 (BCL2) interaction site of unknown significance. By using mice with heterozygous deletion of Becn1, we demonstrate that reduced intracellular content of BECLIN1 does not impact renal function or autophagy within 12 months. However, these mice were significantly sensitized towards cisplatin-induced AKI, and by using Becn1+/−;Sglt2-Cre;Tomato/EGFP mice with subsequent primary cell analysis, we confirmed that nephrotoxicity depends on proximal tubular BECLIN1 content. Mechanistically, BECLIN1 did not impact autophagy or primarily the apoptotic pathway. In fact, a lack of BECLIN1 sensitized mice towards cisplatin-induced ER stress. Accordingly, the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blunted cisplatin-induced cell death in Becn1 heterozygosity. In conclusion, our data first highlight a novel role of BECLIN1 in protecting against cellular ER stress independent from autophagy. These novel findings open new therapeutic avenues to intervene in this important intracellular stress response pathway with a promising impact on future AKI management. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the digestive tract and is closely associated with the homeostasis of the gut microbiota. Inulin, as a natural prebiotic, displays anti-inflammatory activity and maintains equilibrium of the intestinal microbiota. In this study, our research aimed to explore the potential of inulin in enhancing intestinal immunity and reducing inflammation in stress-recurrent IBD. In this study, a co-culture intestinal epithelium model and a stress-recurrent IBD mouse model was used to examine the protective effects of inulin. It was observed that inulin digesta significantly reduced pro-inflammatory cytokine expression (CXCL8/IL8 and TNFA) and increased MUC2 expression in intestinal epithelial cells. In vivo, our findings showed that Inulin intake significantly prevented IBD symptoms. This was substantiated by a decrease in serum inflammatory markers (IL-6, CALP) and a downregulation of inflammatory cytokine (Il6) in colon samples. Additionally, inulin intake led to an increase in short-chain fatty acids (SCFAs) in cecal contents and a reduction in the expression of endoplasmic reticulum (ER) stress markers (CHOP, BiP). Our results highlight that inulin can improve stress-recurrent IBD symptoms by modulating microbiota composition, reducing inflammation, and alleviating ER stress. These findings suggested the therapeutic potential of inulin as a dietary intervention for ameliorating stress-recurrent IBD. [ABSTRACT FROM AUTHOR]

Background and Aims: To systematically review the literature for reports on Wolcott‐Rallison syndrome, focusing on the spectrum and natural history, genotype‐phenotype correlations, patient and native liver survival, and long‐term outcomes. Methods: PubMed, Livio, Google Scholar, Scopus and Web of Science databases were searched. Data on genotype, phenotype, therapy, cause of death and follow‐up were extracted. Survival and correlation analyses were performed. Results: Sixty‐two studies with 159 patients met the inclusion criteria and additional 30 WRS individuals were collected by personal contact. The median age of presentation was 2.5 months (IQR 2) and of death was 36 months (IQR 50.75). The most frequent clinical feature was neonatal diabetes in all patients, followed by liver impairment in 73%, impaired growth in 72%, skeletal abnormalities in 59.8%, the nervous system in 37.6%, the kidney in 35.4%, insufficient haematopoiesis in 34.4%, hypothyroidism in 14.8% and exocrine pancreas insufficiency in 10.6%. Episodes of acute liver failure were frequently reported. Liver transplantation was performed in six, combined liver‐pancreas in one and combined liver‐pancreas‐kidney transplantation in two individuals. Patient survival was significantly better in the transplant cohort (p =.0057). One‐, five‐ and ten‐year patient survival rates were 89.4%, 65.5% and 53.1%, respectively. Liver failure was reported as the leading cause of death in 17.9% of cases. Overall survival was better in individuals with missense mutations (p =.013). Conclusion: Wolcott‐Rallison syndrome has variable clinical courses. Overall survival is better in individuals with missense mutations. Liver‐ or multi‐organ transplantation is a feasible treatment option to improve survival. [ABSTRACT FROM AUTHOR]

Ischemia‐reperfusion injury is an event concerning any organ under a procedure of transplantation. The early result of ischemia is hypoxia, which causes malfunction of mitochondria and decrease in cellular ATP. This leads to disruption of cellular metabolism. Reperfusion also results in cell damage due to reoxygenation and increased production of reactive oxygen species, and later by induced inflammation. In damaged and hypoxic cells, the endoplasmic reticulum (ER) stress pathway is activated by increased amount of damaged or misfolded proteins, accumulation of free fatty acids and other lipids due to inability of their oxidation (lipotoxicity). ER stress is an adaptive response and a survival pathway, however, its prolonged activity eventually lead to induction of apoptosis. Sustaining cell functionality in stress conditions is a great challenge for transplant surgeons as it is crucial for maintaining a desired level of graft vitality. Pathways counteracting negative consequences of ischemia‐reperfusion are autophagy and lipid droplets (LD) metabolism. Autophagy remove damaged organelles and molecules driving them to lysosomes, digested simpler compounds are energy source for the cell. Mitophagy and ER‐phagy results in improvement of cell energetic balance and alleviation of ER stress. This is important in sustaining metabolic homeostasis and thus cell survival. LD metabolism is connected with autophagy as LD are degraded by lipophagy, a source of free fatty acids and glycerol—thus autophagy and LD can readily remove lipotoxic compounds in the cell. In conclusion, monitoring and pharmaceutic regulation of those pathways during transplantation procedure might result in increased/improved vitality of transplanted organ. [ABSTRACT FROM AUTHOR]

The Calreticulin Workshop, initiated in 1994 by Marek Michalak in Banff (Alberta, Canada), was first organized to be an informal scientific meeting attended by researchers working on diverse biological questions related to functions associated with the endoplasmic reticulum (ER)‐resident lectin‐like chaperone and applied to a wide range of biological systems and models. Since then, this workshop has broadened the range of topics to cover all ER‐related functions, has become international and has been held in Canada, Chile, Denmark, Italy, Switzerland, UK, USA, Greece and this year in France. Each conference, which is organized every other year (pending world‐wide pandemic), generally attracts between 50 and 100 participants, including both early career researchers and international scientific leaders to favour discussions and exchanges. Over the years, the International Calreticulin Workshop has become an important gathering of the calreticulin and ER communities as a whole. The 14th International Calreticulin Workshop occurred from May 9–12 in St‐Malo, Brittany, France, and has been highlighted by its rich scientific content and open‐minded discussions held in a benevolent atmosphere. The 15th International Calreticulin Workshop will be organized in 2025 in Brussels, Belgium. [ABSTRACT FROM AUTHOR]

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Hexavalent chromium [Cr(VI)] exists widely in occupational environments. The mechanistic target of rapamycin (mTOR) has been well-documented to regulate autophagy negatively. However, we found that low concentration of Cr(VI) (0.2 μM) elevated both mTOR and autophagy and promote cell survival. Conversely, high concentration of Cr(VI) (6 μM) caused cell death by inhibiting mTOR and subsequently inducing autophagy. Tunicamycin (Tm), as an Endoplasmic reticulum (ER) stress activator was used to induce mild ER stress at 0.1 μg/ml and it activated both autophagy and mTOR, which also caused cell migration in a similar manner to that observed with low concentration of Cr(VI). Severe ER stress caused by Tm (2 μg/ml) decreased mTOR, increased autophagy and then inhibited cell migration, which was the same as 6 μM Cr(VI) treatment, although Cr(VI) in high concentration inhibited ER stress. Activating transcription factor 4 (ATF4), a downstream target of ER stress, only increased under mild ER stress but decreased under severe ER stress and 6 μM Cr(VI) treatment. Chromatin immunoprecipitation (ChIP) experiment indicated that ATF4 could bind to the promoter of ATG4B and AKT1. To sum up, our data revealed that mild ER stress induced by low concentration of Cr(VI) could enhance transcriptional regulation of ATG4B and AKT1 by ATF4, which induced both autophagy and mTOR to promote cell viability.

Abstract Objective Acute pancreatitis (AP) stands as a frequent cause for clinical emergency hospital admissions. The X‐box binding protein 1 (XBP1) was found to be implicated in pancreatic acinar cell apoptosis. The objective is to unveil the potential mechanisms governed by XBP1 and SIRT6 in the context of AP. Methods Caerulein‐treated human pancreatic duct epithelial (HPDE) cells to establish an in vitro research model. The levels and regulatory role of SIRT6 in the treated cells were evaluated, including its effects on inflammatory responses, oxidative stress, apoptosis, and endoplasmic reticulum stress. The relationship between XBP1 and SIRT6 was explored by luciferase and ChIP experiments. Furthermore, the effect of XBP1 overexpression on the regulatory function of SIRT6 on cells was evaluated. Results Caerulein promoted the decrease of SIRT6 and the increase of XBP1 in HPDE cells. Overexpression of SIRT6 slowed down the secretion of inflammatory factors, oxidative stress, apoptosis level, and endoplasmic reticulum stress in HPDE cells. However, XBP1 negatively regulated SIRT6, and XBP1 overexpression partially reversed the regulation of SIRT6 on the above aspects. Conclusion Our study illuminates the role of XBP1 in downregulating SIRT6 in HPDE cells, thereby promoting cellular injury. Inhibiting XBP1 or augmenting SIRT6 levels holds promise in preserving cell function and represents a potential therapeutic avenue in the management of AP.

Endoplasmic reticulum-associated degradation (ERAD) is a principal quality-control mechanism responsible for targeting misfolded ER proteins for cytosolic degradation. Evidence suggests that impairment of ERAD contributes to neuron dysfunction and death in neurodegenerative diseases, many of which are characterized by accumulation and aggregation of misfolded proteins. However, the physiological role of ERAD in neurons remains unclear. The Sel1L-Hrd1 complex consisting of the E3 ubiquitin ligase Hrd1 and its adaptor protein Sel1L is the best-characterized ERAD machinery. Herein, we showed that Sel1L deficiency specifically in neurons of adult mice impaired the ERAD activity of the Sel1L-Hrd1 complex and led to disruption of ER homeostasis, ER stress and activation of the unfold protein response (UPR). Adult mice with Sel1L deficiency in neurons exhibited weight loss and severe motor dysfunction, and rapidly succumbed to death. Interestingly, Sel1L deficiency in neurons caused global brain atrophy, particularly cerebellar and hippocampal atrophy, in adult mice. Moreover, we found that cerebellar and hippocampal atrophy in these mice resulted from degeneration of Purkinje neurons and hippocampal neurons, respectively. These findings indicate that ERAD is required for maintaining ER homeostasis and the viability and function of neurons in adults under physiological conditions.

Excessive alcohol exposure can cause neurobehavioral deficits and structural alterations in the brain. Emerging research evidence suggests that endoplasmic reticulum (ER) stress plays an important role in alcohol-induced neurotoxicity. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress inducible protein and is responsible to maintain ER homeostasis. MANF is highly expressed in both the developing and mature brain. We have previously shown that MANF deficiency exacerbated alcohol induced neurodegeneration and ER stress in the developing brain. However, little is known regarding the role of MANF in alcohol induced neuronal damage in the adult brain. In this study, we used a neuron-specific MANF knockout (KO) mouse model to investigate the effect of MANF deficiency on acute binge alcohol exposure-induced neurobehavioral deficits and ER stress. Adult male and female MANF KO mice and littermate controls received daily alcohol gavage (5 g/kg) for 10 days and then subjected to a battery of neurobehavioral tests including rotarods, balance beam, DigiGait, open field, elevated plus maze, Barnes maze, and three-chamber sociability task. Female MANF KO animals were more susceptible to alcohol-induced body weight loss. Alcohol exposure did not affect motor function, however female but not male MANF KO mice exhibited an increased locomotor activity in open field test. Learning and memory was not significantly impaired, but it was altered by MANF deficiency in females while it was affected by alcohol treatment in males. Both alcohol-exposed male and female MANF KO mice displayed increased sociability. Alcohol induced the expression of ER chaperones GRP78 and GRP94 and altered the levels of several unfolded protein response (UPR) and neuroinflammation markers in MANF KO mice in a sex-specific manner. The expression of MANF interacting proteins neuroplastin, PDIA1, and PDIA6 was increased in MANF KO mice, and was further induced by alcohol. In conclusion, alcohol exposure and neuronal MANF deficiency interacted to alter neurobehavioral outcomes, ER homeostasis and neuroinflammation in a sex-specific manner.

ObjectiveBeta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity. Among the underlying mechanisms, secretion of extracellular vesicles (EVs) by beta cells has been suggested to mediate the crosstalk with the immune cell compartment.MethodsTo study the role of cellular stress in the early events of T1D development, we generated a novel cellular model for constitutive ER stress by modulating the expression of HSPA5, which encodes BiP/GRP78, in EndoC-βH1 cells. To investigate the role of EVs in the interaction between beta cells and the immune system, we characterized the EV miRNA cargo and evaluated their effect on innate immune cells.ResultsAnalysis of the transcriptome showed that HSPA5 knockdown resulted in the upregulation of signaling pathways involved in the unfolded protein response (UPR) and changes the miRNA content of EVs, including reduced levels of miRNAs involved in IL-1β signaling. Treatment of primary human monocytes with EVs from stressed beta cells resulted in increased surface expression of CD11b, HLA-DR, CD40 and CD86 and upregulation of IL-1β and IL-6.ConclusionThese findings indicate that the content of EVs derived from stressed beta cells can be a mediator of islet inflammation.

The average lifespan of humans has been increasing, resulting in a rapidly rising percentage of older individuals and high morbidity of aging-associated diseases, especially cardiovascular diseases (CVDs). Diverse intracellular and extracellular factors that interrupt homeostatic functions in the endoplasmic reticulum (ER) induce ER stress. Cells employ a dynamic signaling pathway of unfolded protein response (UPR) to buffer ER stress. Recent studies have demonstrated that ER stress triggers various cellular processes associated with aging and many aging-associated diseases, including CVDs. Autophagy is a conserved process involving lysosomal degradation and recycling of cytoplasmic components, proteins, organelles, and pathogens that invade the cytoplasm. Autophagy is vital for combating the adverse influence of aging on the heart. The present report summarizes recent studies on the mechanism of ER stress and autophagy and their overlap in aging and on CVD pathogenesis in the context of aging. It also discusses possible therapeutic interventions targeting ER stress and autophagy that might delay aging and prevent or treat CVDs.

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by increased LDL-cholesterol levels. About 85% of FH cases are caused by LDLR mutations encoding the low-density lipoprotein receptor (LDLR). LDLR is synthesized in the endoplasmic reticulum (ER) where it undergoes post-translational modifications and then transported through Golgi apparatus to the plasma membrane. Over 2900 LDLR variants have been reported in FH patients with limited information on the pathogenicity and functionality of many of them. This study aims to elucidate the cellular trafficking and functional implications of LDLR missense variants identified in suspected FH patients using biochemical and functional methods.MethodsWe used HeLa, HEK293T, and LDLR-deficient-CHO-ldlA7 cells to evaluate the subcellular localization and LDL internalization of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients. We also analyzed the functional impact of three variants (p.D445E, p.D482H, and p.C677F), two of which previously shown to be retained in the ER.ResultsWe show that p.D622G, p.D482H, and p.C667F are largely retained in the ER whereas p.R744Q is partially retained. The other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 cells indicate that p.D482H, p.C243Y, p.D622G, and p.C667F have quantitatively lost their ability to internalize Dil-LDL with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q and p.R814Q) showing significant losses except for p.E277K which retained full activity. However, the LDL internalization assay is only to able evaluate the impact of the variants on LDL internalization and not the exact functional defects such as failure to bind LDL. The data represented illustrate the hypomorphism nature of variants causing FH which may explain some of the variable expressivity of FH.ConclusionOur combinatorial approach of in silico, cellular, and functional analysis is a powerful strategy to determine pathogenicity and FH disease mechanisms which may provide opportunitites for novel therapeutic strategies.

Many neurodegenerative diseases (NDD) appear to share commonality of origin, chronic ER stress. The endoplasmic reticulum (ER) is a dynamic organelle, functioning as a major site of protein synthesis and protein posttranslational modifications, required for proper folding. ER stress can occur because of external stimuli, such as oxidative stress or neuroinflammatory cytokines, creating the ER luminal environment permissive for the accumulation of aggregated and misfolded proteins. Unresolvable ER stress upregulates a highly conserved pathway, the unfolded protein response (UPR). Maladaptive chronic activation of UPR components leads to apoptotic neuronal death. In addition to other factors, physiological responses to stressors are emerging as a significant risk factor in the etiology and pathogenesis of NDD. Owned cats share a common environment with people, being exposed to many of the same stressors as people and additional pressures due to their “quasi” domesticated status. Feline Cognitive Dysfunction Syndrome (fCDS) presents many of the same disease hallmarks as human NDD. The prevalence of fCDS is rapidly increasing as more people welcome cats as companions. Barely recognized 20 years ago, veterinarians and scientists are in infancy stages in understanding what is a very complex disease. This review will describe how cats may represent an unexplored animal mimetic phenotype for human NDD with stressors as potential triggering mechanisms. We will consider how multiple variations of stressful events over the short-life span of a cat could affect neuronal loss or glial dysfunction and ultimately tip the balance towards dementia.

Vacuole membrane protein 1 (VMP1) is an integral membrane protein that plays a pivotal role in cellular processes, particularly in the regulation of autophagy. Autophagy, a self-degradative mechanism, is essential for maintaining cellular homeostasis by degradation and recycling damaged organelles and proteins. VMP1 involved in the autophagic processes include the formation of autophagosomes and the subsequent fusion with lysosomes. Moreover, VMP1 modulates endoplasmic reticulum (ER) calcium levels, which is significant for various cellular functions, including protein folding and cellular signaling. Recent studies have also linked VMP1 to the cellular response against viral infections and lipid droplet (LD). Dysregulation of VMP1 has been observed in several pathological conditions, including neurodegenerative diseases such as Parkinson’s disease (PD), pancreatitis, hepatitis, and tumorogenesis, underscoring its potential as a therapeutic target. This review aims to provide an overview of VMP1’s multifaceted roles and its implications in disease pathology.