Endoplasmic Reticulum Stress-mediated Signaling Research Articles

Dioscin pretreatment ameliorates ferroptosis in cardiomyocytes after myocardial infarction via inhibiting endoplasmic reticulum stress

BackgroundMyocardial infarction (MI) remains a leading cause of mortality globally, often resulting in irreversible damage to cardiomyocytes. Ferroptosis, a recently identified form of regulated cell death driven by iron-dependent lipid peroxidation, has emerged as a significant contributor to post-MI cardiac injury. The endoplasmic reticulum (ER) stress response has been implicated in exacerbating ferroptosis.MethodsHere, we investigated the potential of Dioscin, a natural compound known for its diverse pharmacological properties, in mitigating ferroptosis in cardiomyocytes following MI by targeting ER stress.ResultsIn animal models subjected to MI, administration of Dioscin notably improved cardiac function, reduced infarct size by approximately 24%, and prevented adverse remodeling, highlighting its therapeutic potential. Through in vitro and in vivo models of MI, we demonstrated that Dioscin treatment significantly attenuates ferroptosis in cardiomyocytes, as evidenced by a decrease in lipid peroxidation by about 19% and preserved mitochondrial integrity. Moreover, Dioscin exerted its protective effects by inhibiting ER stress markers, such as the phosphorylation levels of PERK and eIF2α proteins, and the expression levels of BIP and ATF4 proteins, thus disrupting the ER stress-mediated signaling cascade associated with ferroptosis.ConclusionOverall, our findings suggested that Dioscin holds promise as a therapeutic agent against post-MI cardiac injury by mitigating ferroptosis via the suppression of ER stress. Further investigations into the precise molecular mechanisms and clinical translation of Dioscin’s cardioprotective effects are warranted, offering a potential avenue for novel therapeutic interventions in MI-related cardiac complications.Graphical

Folic Acid Prevents High-Fat Diet-Induced Postpartum Weight Retention in Rats, Which Is Associated with a Reduction in Endoplasmic Reticulum Stress-Mediated Hepatic Lipogenesis.

Proactively preventing postpartum weight retention (PPWR) is one of the effective intervention strategies to reduce the occurrence of obesity in women. Population studies have shown that serum folate levels are closely related to body weight. The regulation of folic acid on lipid metabolism has been fully confirmed in both in vivo and in vitro studies. For many years, folic acid supplementation has been widely used in periconceptional women due to its role in preventing fetal neural tube defects. However, whether folic acid supplementation prior to and throughout pregnancy exerts preventive effects on PPWR remains uncertain. This study aims to investigate the preventive effect of folic acid on PPWR in rats and further explore the underlying mechanisms. In this study, pregnant rats were administered one of the dietary schedules: control diet (CON), high-fat diet (HF), control diet combined with folic acid (FA) and high-fat diet combined with folic acid (HF + FA). We discovered that folic acid supplementation inhibited high-fat diet-induced elevations in body weight, visceral fat weight, liver weight, hepatic lipid levels and serum lipid levels at 1 week post-weaning (PW). Western blot analysis showed that folic acid supplementation inhibited the expression of endoplasmic reticulum (ER) stress-specific proteins including GRP78, PERK, eIF2α, IRE1α, XBP1 and ATF6, subsequently decreasing the expression of proteins related to lipid synthesis including SREBP-1c, ACC1 and FAS. In conclusion, folic acid supplementation prior to and throughout pregnancy exerts preventive effects on high-fat diet-induced PPWR in rats, and the mechanism is associated with the inhibition of ER stress-mediated lipogenesis signaling pathways in the liver. Folic acid supplementation may serve as a potential strategy for preventing PPWR. In the future, the effectiveness of folic acid in PPWR prevention can be further verified by population studies.

Gestational Exposure to Cyfluthrin through Endoplasmic Reticulum (ER) Stress-Mediated PERK Signaling Pathway Impairs Placental Development.

Cyfluthrin, a typical type II pyrethroid pesticide, is widely used in house hygiene and agricultural pest control. Several epidemiological investigations have found that maternal pyrethroid exposure is connected to adverse pregnancy outcomes. However, the underlying mechanisms remain to be elucidated. Thus, we evaluated the effect of cyfluthrin exposure during pregnancy on placenta development in vivo. In the current study, Pregnant SD rats were randomly divided into four groups and administered 6.25, 12.5, and 25 mg/kg body weight cyfluthrin or an equivalent volume of corn oil by gavage from GD0 to GD19. The results have shown that gestational exposure to cyfluthrin exerted no effect on the fetal birth defect, survival to PND4, or fetal resorption and death. However, live fetuses and implantation sites significantly decreased in the high-dose cyfluthrin-treated group. Moreover, a significant reduction in placenta weight and diameter was observed in rats. Correspondingly, the fetal weight and crown-rump length from dams exposed to cyfluthrin were reduced. Cyfluthrin-treat groups, the total area of the placenta, spongiotrophoblast area, and labyrinth area had abnormal changes. Meanwhile, the area of blood sinusoid and CD34-positive blood vessel numbers in the placenta were considerably reduced, as well as abnormal expression of placental pro-angiogenic and anti-angiogenic factors in dams exposed to cyfluthrin. Further observation by transmission electron microscopy revealed significant changes in the ultrastructure of the medium-dose and high-dose groups. Additional experiments showed gestational exposure to cyfluthrin inhibited proliferation and induced apoptosis of placentas, as decreased PCNA-positive cells and increased TUNEL-positive cells. Furthermore, western blot and qPCR analysis revealed that gestational exposure to medium-dose and high-dose cyfluthrin increased the expression of GRP78, and three downstream mRNA and proteins (p-eIF2α, ATF4, and CHOP) of the PERK signaling, indicating that endoplasmic reticulum (ER) stress-mediated PERK/eIF2α/ATF4/CHOP signaling pathway in rat placentas was activated. Our study demonstrated that gestational exposure to cyfluthrin leads to placental developmental disorder, which might be associated with ER stress-mediated PERK signaling pathway.

Exposure to BDE-47 causes female infertility risk and induces oxidative stress and lipotoxicity-mediated ovarian hormone secretion disruption in mice

2,2,4,4-Tetrabromodiphenyl ether (BDE-47) has received considerable attention because of its high level in biological samples and potential developmental toxicity. Whether BDE-47 ingestion affects ovarian hormone secretion and the detailed underlying mechanism have not been clearly elucidated. The present study aimed to evaluate the toxicity of BDE-47 on ovarian hormone secretion and explored the underlying mechanism. The results showed that exposure to BDE-47 caused ovarian lipid deposition and ovarian hormone disruption accompanied by oxidative stress (OS) and downregulation of hormone biosynthesis-related proteins in mice. Mechanistically, using ovarian granulosa cells (GCs) as a cellular model, it was shown that BDE-47 inhibited two ovarian hormone secretion-associated pathways: i) BDE-47 exposure induced OS via the Nrf2/HO-1 signaling pathway and further inhibited the expressions of ovarian hormone biosynthesis-related proteins, such as StAR, 3-βHSD, CYP11A1, and CYP17A1; ii) BDE-47 induced endoplasmic reticulum (ER) stress, mitochondrial abnormalities, and lipotoxicity, which in turn disrupted the hormone biosynthesis process and inhibited ovarian hormone secretion. Interestingly, autophagy could promote hormone secretion via downregulating the transcription levels of PPARγ and C/EBPα involved in lipid deposition. Moreover, the reactive oxygen species (ROS) scavenger NAC and ER stress inhibitor 4-PBA not only inhibited the decrease in mitochondrial membrane potential but also blocked apoptosis induced by BDE-47, indicating that two individual pathways mediated apoptosis in GCs: the ER stress-mediated signaling pathway and the ROS-mediated mitochondrial signaling pathway. Together, these findings indicate the possible health risks of BDE-47 pollution areas to women, particularly affecting their ovarian hormone secretion.

Citrinin-Induced Hepatotoxicity in Mice Is Regulated by the Ca2+/Endoplasmic Reticulum Stress Signaling Pathway.

Citrinin (CTN) is a mycotoxin found in crops and agricultural products and poses a serious threat to human and animal health. The aim of this study is to investigate the hepatotoxicity of CTN in mice and analyze its mechanisms from Ca2+-dependent endoplasmic reticulum (ER) stress perspective. We showed that CTN induced histopathological damage, caused ultrastructural changes in liver cells, and induced abnormal values of biochemical laboratory tests of some liver functions in mice. Treatment with CTN could induce nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation in mice, accompanied with losses of activities of superoxide dismutase (SOD) and catalase (CAT), levels of glutathione (GSH), and capacities of total antioxidant (T-AOC), resulting in oxidative stress in mice. Furthermore, CTN treatment significantly increased Ca2+ accumulation, upregulated protein expressions of ER stress-mediated apoptosis signal protein (glucose regulated protein 78 (GRP78/BIP), C/EBP-homologous protein (CHOP), Caspase-12, and Caspase-3), and induced hepatocyte apoptosis. These adverse effects were counteracted by 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. In summary, our results showed a possible underlying molecular mechanism for CTN that induced hepatocyte apoptosis in mice by the regulation of the Ca2+/ER stress signaling pathway.

Endoplasmic Reticulum Stress Provocation by Different Nanoparticles: An Innovative Approach to Manage the Cancer and Other Common Diseases.

A proper execution of basic cellular functions requires well-controlled homeostasis including correct protein folding. Endoplasmic reticulum (ER) implements such functions by protein reshaping and post-translational modifications. Different insults imposed on cells could lead to ER stress-mediated signaling pathways, collectively called the unfolded protein response (UPR). ER stress is also closely linked with oxidative stress, which is a common feature of diseases such as stroke, neurodegeneration, inflammation, metabolic diseases, and cancer. The level of ER stress is higher in cancer cells, indicating that such cells are already struggling to survive. Prolonged ER stress in cancer cells is like an Achilles’ heel, if aggravated by different agents including nanoparticles (NPs) may be exhausted off the pro-survival features and can be easily subjected to proapoptotic mode. Different types of NPs including silver, gold, silica, graphene, etc. have been used to augment the cytotoxicity by promoting ER stress-mediated cell death. The diverse physico-chemical properties of NPs play a great role in their biomedical applications. Some special NPs have been effectively used to address different types of cancers as these particles can be used as both toxicological or therapeutic agents. Several types of NPs, and anticancer drug nano-formulations have been engineered to target tumor cells to enhance their ER stress to promote their death. Therefore, mitigating ER stress in cancer cells in favor of cell death by ER-specific NPs is extremely important in future therapeutics and understanding the underlying mechanism of how cancer cells can respond to NP induced ER stress is a good choice for the development of novel therapeutics. Thus, in depth focus on NP-mediated ER stress will be helpful to boost up developing novel pro-drug candidates for triggering pro-death pathways in different cancers.

Effects of Cadmium on ZO-1 Tight Junction Integrity of the Blood Brain Barrier.

Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood–brain barrier (BBB); however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 μM cadmium chloride (CdCl2) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.

IRE1α Implications in Endoplasmic Reticulum Stress-Mediated Development and Pathogenesis of Autoimmune Diseases.

Inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) is the most prominent and evolutionarily conserved endoplasmic reticulum (ER) membrane protein. This transduces the signal of misfolded protein accumulation in the ER, named as ER stress, to the nucleus as “unfolded protein response (UPR).” The ER stress-mediated IRE1α signaling pathway arbitrates the yin and yang of cell life. IRE1α has been implicated in several physiological as well as pathological conditions, including immune disorders. Autoimmune diseases are caused by abnormal immune responses that develop due to genetic mutations and several environmental factors, including infections and chemicals. These factors dysregulate the cell immune reactions, such as cytokine secretion, antigen presentation, and autoantigen generation. However, the mechanisms involved, in which these factors induce the onset of autoimmune diseases, are remaining unknown. Considering that these environmental factors also induce the UPR, which is expected to have significant role in secretory cells and immune cells. The role of the major UPR molecule, IRE1α, in causing immune responses is well identified, but its role in inducing autoimmunity and the pathogenesis of autoimmune diseases has not been clearly elucidated. Hence, a better understanding of the role of IRE1α and its regulatory mechanisms in causing autoimmune diseases could help to identify and develop the appropriate therapeutic strategies. In this review, we mainly center the discussion on the molecular mechanisms of IRE1α in the pathophysiology of autoimmune diseases.

Regulation of gammaherpesvirus lytic replication by endoplasmic reticulum stress–induced transcription factors ATF4 and CHOP

The stress-induced unfolded protein response (UPR) in the endoplasmic reticulum (ER) involves various signaling cross-talks and controls cell fate. B-cell receptor (BCR) signaling, which can trigger UPR, induces gammaherpesvirus lytic replication and serves as a physiological mechanism for gammaherpesvirus reactivation in vivo However, how the UPR regulates BCR-mediated gammaherpesvirus infection is unknown. Here, we demonstrate that the ER stressors tunicamycin and thapsigargin inhibit BCR-mediated murine gammaherpesvirus 68 (MHV68) lytic replication by inducing expression of the UPR mediator Bip and blocking activation of Akt, ERK, and JNK. Both Bip and the downstream transcription factor ATF4 inhibited BCR-mediated MHV68 lytic gene expression, whereas UPR-induced C/EBP homologous protein (CHOP) was required for and promoted BCR-mediated MHV68 lytic replication by suppressing upstream Bip and ATF4 expression. Bip knockout was sufficient to rescue BCR-mediated MHV68 lytic gene expression in CHOP knockout cells, and this rescue was blocked by ectopic ATF4 expression. Furthermore, ATF4 directly inhibited promoter activity of the MHV68 lytic switch transactivator RTA. Altogether, we show that ER stress-induced CHOP inhibits Bip and ATF4 expression and that ATF4, in turn, plays a critical role in CHOP-mediated regulation of BCR-controlled MHV68 lytic replication. We conclude that ER stress-mediated UPR and BCR signaling pathways are interconnected and form a complex network to regulate the gammaherpesvirus infection cycle.

Association of changes in ER stress-mediated signaling pathway with lead-induced insulin resistance and apoptosis in rats and their prevention by A-type dimeric epigallocatechin-3-gallate

A-type dimeric epigallocatechin-3-gallate (A-type-EGCG-dimer, AEd), a new proanthocyanidins dimer from persimmon fruits, has been shown to have health benefit effects. However, A-type-EGCG-dimer affects gluose metabolism in the liver and the underlying mechanism is not clarified. The present study aims to examine the protective effects of A-type-EGCG-dimer on Pb-induced hepatic insulin resistance, endoplasmic reticulum (ER) stress and apoptosis in rats. Male wistar rats exposed to 0.05% w/v Pb acetate in the drinking water with or without A-type-EGCG-dimer coadministration (200 mg/kg body weight/day, intragastrically) for three months. We found that A-type-EGCG-dimer and pioglitazone supplementation significantly deceased glucose and insulin levels in plasma as compared with the Pb group. A-type-EGCG-dimer markedly prevents Pb-induced oxidative stress, ER stress and apoptosis in livers. A-type-EGCG-dimer and pioglitazone reduced the expression levels of the GRP78, PEPCK, G6Pase, p-PERK, p-IRE1, p-JNK, ATF4, CHOP and increased p-AKT in livers of the Pb group. Moreover, A-type-EGCG-dimer reduced ROS production and restored the activities of SOD and GPx in livers. A-type-EGCG-dimer decreased Bax, cytosolic cytochrome c and cleaved caspase-3 and increased Bcl-2 in livers of Pb-exposed rats. Our results suggest that A-type-EGCG-dimer might be a potential natural candidate for the prevention of hepatic insulin resistance and apoptosis induced by Pb.

Fibroblast growth factor 21 protects rat cardiomyocytes from endoplasmic reticulum stress by promoting the fibroblast growth factor receptor 1-extracellular signal‑regulated kinase 1/2 signaling pathway.

Fibroblast growth factor 21 (FGF21), as an endocrine factor, is secreted into circulation by injured cardiomyocytes. Endoplasmic reticulum (ER) stress-induced apoptosis has been proposed as an important pathophysiological mechanism for cardiomyocyte injury. However, whether the enhanced expression of FGF21 in cardiomyocytes is linked to ER stress, and the effect and underlying mechanism of FGF21 on ER stress-induced cardiomyocyte apoptosis remain unclear. In the present study, it was demonstrated that mild ER stress resulted in upregulated expression levels of FGF21 and its main receptors, as a response to cell compensation, at the induction of ≤5 µM tunicamycin (TM). However, excessive ER stress (TM ≥10 µM) activated the ER stress-mediated apoptosis signaling pathways, including PKR-like ER kinase (PERK)-eukaryotic translational initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/-enhancer-binding protein homologous protein (CHOP) and inositol-requiring kinase 1α (IRE1α)-c-Jun N-terminal kinases (JNK), as well as inhibited the expression of FGF21 and its primary receptors. In addition, FGF21 overexpression provided protection against ER stress-induced cardiomyocyte injury, as evidenced by increased cell viability and reduced apoptosis. These changes were associated with the inhibition of ER stress-mediated apoptosis signaling pathways, as well as increased phosphorylation of FGFR1 and ERK1/2. However, the protective effects of overexpressed FGF21 were abolished following treatment with FGFR1 and ERK1/2 inhibitors. Thus, mild ER stress may induce the expression of FGF21 and its primary receptors in cardiomyocytes. FGF21 inhibits ER stress-induced cardiomyocyte injury as least in part via the FGFR1-ERK1/2 signaling pathway.

Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology.

The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition. Conversely, the contribution of impaired ER homeostasis by severe/prolong ER stress-mediated UPR signaling pathways to several reproductive tissue pathologies including endometriosis, cancers, recurrent pregnancy loss and pregnancy complications associated with pre-term birth have been reported. This review focuses on ER stress and UPR signaling mechanisms, and their potential roles in female and male reproductive physiopathology involving in menstrual cycle changes, gametogenesis, preimplantation embryo development, implantation and placentation, labor, endometriosis, pregnancy complications and preterm birth as well as reproductive system tumorigenesis.

ER Stress-Mediated Signaling: Action Potential and Ca(2+) as Key Players.

The proper functioning of the endoplasmic reticulum (ER) is crucial for multiple cellular activities and survival. Disturbances in the normal ER functions lead to the accumulation and aggregation of unfolded proteins, which initiates an adaptive response, the unfolded protein response (UPR), in order to regain normal ER functions. Failure to activate the adaptive response initiates the process of programmed cell death or apoptosis. Apoptosis plays an important role in cell elimination, which is essential for embryogenesis, development, and tissue homeostasis. Impaired apoptosis can lead to the development of various pathological conditions, such as neurodegenerative and autoimmune diseases, cancer, or acquired immune deficiency syndrome (AIDS). Calcium (Ca2+) is one of the key regulators of cell survival and it can induce ER stress-mediated apoptosis in response to various conditions. Ca2+ regulates cell death both at the early and late stages of apoptosis. Severe Ca2+ dysregulation can promote cell death through apoptosis. Action potential, an electrical signal transmitted along the neurons and muscle fibers, is important for conveying information to, from, and within the brain. Upon the initiation of the action potential, increased levels of cytosolic Ca2+ (depolarization) lead to the activation of the ER stress response involved in the initiation of apoptosis. In this review, we discuss the involvement of Ca2+ and action potential in ER stress-mediated apoptosis.

Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation

Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the “adaptive” signaling cascade mediated by the XBP1 pathway and the “alarm” signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)–Luc cell-based assay in 1536-well format for monitoring IRE1’s RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

Losing p53 loosens up ER-stress.

The endoplasmic reticulum (ER) is the central intracellular organelle responsible for proper folding, localization, and post-translational modifications of proteins in eukaryotic cells. During conditions of cellular stress, incorrectly folded proteins can accumulate in the ER and trigger the unfolded protein response (UPR), which involves the activation of signal transduction pathways designed to restore correct protein folding and recovery from ER stress [1]. In rapidly dividing tumor cells, UPR can become constitutively activated, presumably because higher levels of proliferation require increased protein synthesis [1, 2]. In addition, the tumor micro-environment is often host to additional adverse conditions including nutrient and oxygen deprivation, leading to further perturbations of ER function. Therefore, it may come as no surprise that UPR has been shown to play a crucial cytoprotective role in tumor cells [2]. We have recently unveiled an unexpected role for the tumor suppressor p53 in regulating ER function via the IRE1α/XBP1 pathway [3]. IRE1α is an ER stress sensor that activates the UPR to restore ER function and maintain cellular homeostasis. It was observed that wild-type p53 suppresses IRE1α expression and activation, while the loss of active p53 potentiates the IRE1α/XBP1 pathway, enhancing ER function [3]. This finding suggests a survival benefit for cancer cells that can inactivate p53. Because cancer cells with high protein load on the ER would need enhanced ER function to cope with constant ER stress, losing p53 function would assist tumors to grow in adverse conditions by increasing IRE1α/XBP1 pathway activation. In addition, we found that a p53 target, CDIP1, acts as a proapototic signal mediator in conditions of ER stress [4]. The CDIP1/BAP31 complex was shown to transduce ER stress-mediated apoptotic signaling to the mitochondria and activate the intrinsic apoptosis pathway, suggesting that loss of p53 function may contribute to the survival of cancer cells by enhancing ER function as well as hindering ER stress-mediated apoptosis signaling. The loss of p53 function in relation to ER stress is likely to have important consequences for the sustenance of cancer cell proliferation. Previous studies have demonstrated that the activation of p53 suppresses mTOR activity and consequently inhibits protein translation [5, 6]. The elimination of wild-type p53 function may therefore provide an opportunity for cancer cells to further promote unregulated proliferation, via elevated protein synthesis and a subsequent increase in ER function. We have revealed that wild-type p53 directly interacts with synoviolin (SYVN1) and can thereby stimulate SYVN1-dependent degradation of IRE1α. The loss of p53 disrupts SYVN1-mediated IRE1α downregulation, producing an ER stress-resistant phenotype by increased IRE1α/XBP1 pathway activity [3]. However, although the loss of p53 function facilitates enhanced ER function to manage high ER stress, this also generates an overreliance on IRE1α in cancer cells with p53 mutations. Indeed, we have found that pharmacological inhibition of IRE1α strongly suppresses protein secretion and potently induces cell death in such p53-deficient cancer cells [3]. Importantly, the IRE1α inhibitor exhibited significant inhibitory effects against p53-deficient human tumors in vivo compared to those with wild-type p53. These results illuminate an important proof-of-concept for targeting tumors with mutant p53 via inhibition of the IRE1α/XBP1 pathway. Malignant transformation occurs through a combination of oncogenic pathway activation and the loss of tumor suppression. Understanding the consequences and identifying vulnerabilities caused by such changes can provide promising opportunities to selectively target cancer cells. Mutation in the p53 gene is the most frequent genetic alteration found in human cancer and inactivation of p53 is known as one of the strongest driving factors in cancer development and chemoresistance [7]. The new finding that loss of p53 activates the IRE1α/XBP1 pathway leading to enhanced ER function broadens our understanding of how cancer cells with mutant p53 survive, and also highlights a possibility for the development of novel therapeutic strategies.

Attenuation of endoplasmic reticulum stress as a treatment strategy against ischemia/reperfusion injury.

Attenuation of endoplasmic reticulum stress as a treatment strategy against ischemia/reperfusion injury.

Dipeptidyl petidase-IV inhibitor (gemigliptin) inhibits tunicamycin-induced endoplasmic reticulum stress, apoptosis and inflammation in H9c2 cardiomyocytes

The direct effects of dipeptidyl peptidase-IV (DPP-IV) inhibitors on endoplasmic reticulum (ER) stress-induced apoptosis and inflammation in cardiomyocytes have not been elucidated. H9c2 cell viability, which was reduced by tunicamycin, was increased after DPP-IV inhibitor gemigliptin treatment. Gemigliptin significantly decreased the tunicamycin-mediated increase in glucose regulated protein 78 (GRP78) expression and ER stress-mediated signaling molecules such as protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C-EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α)/c-Jun N-terminal kinase (JNK)-p38. Furthermore, gemigliptin effectively induced Akt phosphorylation in a dose-dependent manner. Using flow cytometry and Hoechst staining, we showed that treatment with Akt inhibitor significantly blocked the anti-apoptotic effects mediated by gemigliptin. The reduction in tunicamycin-induced GRP78 level and PERK/CHOP pathway activity by gemigliptin was reversed after treatment with Akt inhibitor. In conclusion, gemigliptin effectively inhibited ER stress-induced apoptosis and inflammation in cardiomyocytes via Akt/PERK/CHOP and IRE1α/JNK-p38 pathways, suggesting its direct protective role in cardiovascular diseases.

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.

The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Effect of quercetin on apoptosis of PANC-1 cells

PurposeTo investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells.MethodsVarious anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 µg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin.ResultsTreatment with quercetin resulted in the increased accumulation of intracellular Ca2+ ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction.ConclusionThese data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents.