Elicit Endoplasmic Reticulum Stress Research Articles

ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Dyslipidemia-induced renal fibrosis related to ferroptosis and endoplasmic reticulum stress

Dyslipidemia may induce chronic kidney disease and trigger both ferroptosis and endoplasmic reticulum (ER) stress, but the instigating factors are incompletely understood. We tested the hypothesis that different models of dyslipidemia engage distinct kidney injury mechanisms. Wild-type (WT) or proprotein-convertase subtilisin/kexin type-9 (PCSK9)-gain-of-function (GOF) Ossabaw pigs were fed with a 6-month normal diet (ND) or high-fat diet (HFD) (n = 5–6 each). Renal function and fat deposition were studied in vivo using CT, and blood and kidney tissue studied ex-vivo for lipid profile, systemic and renal vein FFAs levels, and renal injury mechanisms including lipid peroxidation, ferroptosis, and ER stress. Compared with WT-ND pigs, both HFD and PCSK9-GOF elevated triglyceride levels, which were highest in WT-HFD, whereas total and LDL cholesterol levels rose only in PCSK9-GOF pigs, particularly in PCSK9-GOF/HFD. The HFD groups had worse kidney function than the ND groups. The WT-HFD kidneys retained more FFA than other groups, but all kidneys developed fibrosis. Furthermore, HFD-induced ferroptosis in WT-HFD indicated by increased free iron, lipid peroxidation, and decreased glutathione peroxidase-4 mRNA expression, while PCSK9-GOF induced ER stress with upregulated GRP94 and CHOP protein expression. In vitro, pig kidney epithelial cells treated with palmitic acid and oxidized LDL to mimic HFD and PCSK9-GOF showed similar trends to those observed in vivo. Taken together, HFD-induced hypertriglyceridemia promotes renal FFA retention and ferroptosis, whereas PCSK9-GOF–induced hypercholesterolemia elicits ER stress, both resulting in renal fibrosis. These observations suggest different targets for preventing and treating renal fibrosis in subjects with specific types of dyslipidemia.

A Native Drug-Free Macromolecular Therapeutic to Trigger Mutual Reinforcing of Endoplasmic Reticulum Stress and Mitochondrial Dysfunction for Cancer Treatment.

Drug-free macromolecular therapeutics are promising alternatives to traditional drugs. Nanomedicines with multiple organelles targeting can potentially increase the efficacy. Herein, a drug-free macromolecular therapeutic was designed to formulate endoplasmic reticulum (ER) and mitochondria dual-targeting nanoparticles (EMT-NPs), which can synergistically elicit ER stress and mitochondrial dysfunction. In vitro experiments indicated that EMT-NPs could effectively enter ER and mitochondria at an approximate ratio of 2 to 3. Subsequently, EMT-NPs could upregulate ER stress-related protein expression (IRE1α, CHOP), boosting calcium ion (Ca2+) efflux and activating the caspase-12 signaling cascade in cancer cells. In addition, EMT-NPs induced direct oxidative stress in mitochondria; some mitochondrial-related apoptotic events such as decreased mitochondrial membrane potential (MMP), upregulation of Bax, cytochrome c release, and caspase-3 activation were also observed for tumor cells upon incubation with EMT-NPs. Furthermore, the leaked Ca2+ from ER could induce mitochondrial Ca2+ overloading to further augment cancer cell apoptosis. In brief, mitochondrial and ER signaling networks collaborated well to promote cancer cell death. Extended photoacoustic and fluorescence imaging served well for the treatment of in vivo patient-derived xenografts cancer model. This drug-free macromolecular strategy with multiple subcellular targeting provides a potential paradigm for cancer theranostics in precision nanomedicine.

Neurotransmitter release progressively desynchronizes in induced human neurons during synapse maturation and aging.

Rapid release of neurotransmitters in synchrony with action potentials is considered a key hardwired property of synapses. Here, in glutamatergic synapses formed between induced human neurons, we show that action potential-dependent neurotransmitter release becomes progressively desynchronized as synapses mature and age. In this solely excitatory network, the emergence of NMDAR-mediated transmission elicits endoplasmic reticulum (ER) stress leading to downregulation of key presynaptic molecules, synaptotagmin-1 and cysteine string protein α, that synchronize neurotransmitter release. The emergence of asynchronous release with neuronal maturity and subsequent aging is maintained by the high-affinity Ca2+ sensor synaptotagmin-7 and suppressed by the introduction of GABAergic transmission into the network, inhibition of NMDARs, and ER stress. These results suggest that long-term disruption of excitation-inhibition balance affects the synchrony of excitatory neurotransmission in human synapses.

Exposure to deltamethrin at the NOAEL causes ER stress and disruption of hippocampal neurogenesis in adult mice

In addition to age and traumatic brain injury, environmental exposure to pesticides is a potential risk factor for neurodegenerative diseases and cognitive impairments in humans. Deltamethrin is a type II pyrethroid insecticide widely used in agriculture and homes for pest control. Previously, we reported that repeated exposure of mice to 3 mg/kg deltamethrin for 30 or 60 days caused a marked increase in the endoplasmic reticulum (ER) stress and reduced adult hippocampal neurogenesis that was accompanied by impaired learning and memory. However, it is unknown whether an acute exposure to low doses of deltamethrin elicits similar effects. Here, we sought to characterize the dose-related effects of deltamethrin on ER stress and hippocampal neurogenesis at different time points following acute exposure. Following oral administration of 0, 0.3, 1, or 3 mg/kg deltamethrin, doses below, at, and above the acute NOAEL, mice were euthanized at 24 h, 48 h, 7 d, or 14 d to assess the acute and intermediate-term effects of deltamethrin on neural progenitor cells (NPCs). Deltamethrin at both 1 and 3 mg/kg elicited ER stress response and activation of apoptotic signaling. Data revealed that a dose as low as 1 mg/kg of deltamethrin, considered the acute NOAEL, produced a significant reduction in BrdU+ and Ki-67+ neural stem cells in the subgranular zone of the dentate gyrus of the hippocampus as early as 48 h after exposure. Furthermore, mice treated with 1 and 3 mg/kg deltamethrin exhibited a decreased number of immature neurons, determined by counting DCX-positive cells 7 days after exposure. These data establish that 0.3 mg/kg should be considered a NOAEL and that the previously established acute NOAEL of 1 mg/kg shows significant effects on ER stress and apoptotic pathways accompanied by deficits in aspects of adult hippocampal neurogenesis.

Cancer-induced Changes In Endoplasmic Stress And Autophagy Responses After Mechanical Overload In ApcMin/+ Mice

Cancer cachexia is characterized by the severe weight and muscle loss. The endoplasmic reticulum (ER) stress and autophagy perpetuate skeletal muscle homeostasis. Tumor cells elicit ER stress and unfolded protein response (UPR) in skeletal muscle. The inhibition of ER stress also activates protein degradation and autophagy, which magnifies muscle mass declination. While UPR moderates mechanical stress, the adaptations in UPR and autophagy in cancer cachexia are unknown. PURPOSE: To determine whether tumor burden alters UPR and autophagy responses to mechanical overload in mice. METHODS: Age-matched male wild-type (WT, n = 5) and ApcMin/+ (Min, n = 5) mice were utilized. Synergist ablation (SA) was performed on the left leg, while the right leg served as an internal control. Seven days after mechanical overload, the plantaris muscles were removed. After tissue homogenization, routine Western blotting was conducted using 80 ~ 120 μg of the total protein. Independent t-test (WT vs. Min) and two-way ANOVA (genotype x treatment) were used for statistical analysis. The coefficient of determination (r^2) was used to examine a significant correlation. RESULTS: Min mice showed a decrease of BW by 9.8% compared to their peak BW. Similarly, sham control plantaris weight in Min mice was smaller than that of WT mice (18.7 ± 0.6 mg vs. 15.1 ± 0.3 mg for WT and Min, respectively). In both mice, mechanical overload increased the plantaris weights, but the response was small in Min mice (43.3 ± 5.8% vs. 21.3 ± 4.6%, p ≤ 0.05). Western blot analysis resulted in Min mice having a reduced phospho-p70S6K after mechanical overload than WT mice (4.2-fold vs. 1.9-fold, p ≤ 0.05). Phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α, 4.2-fold vs. 3.0-fold, p ≤ 0.05) and beclin (4.3-fold vs. 1.8-fold, p ≤ 0.05) levels were reduced in response to the stimulus in Min mice. A significant correlation was observed between p70S6K and eIF2α and between p70S6K and Beclin regardless of mouse genotype. The slopes of the regression curve in Min mice were smaller than WT mice (p ≤ 0.05). CONCLUSIONS: Reduced ER stress and autophagy responses to mechanical overload were associated with decreased mTORC1 signaling in cachectic mice. Supported by Louisiana BORSF (LEQSF(2017-20)-RD-A-22).

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.

Spermine-mediated tight sealing of the Magnaporthe oryzae appressorial pore-rice leaf surface interface.

Cellular adhesion mediates many important plant-microbe interactions. In the devastating blast fungus Magnaporthe oryzae1, powerful glycoprotein-rich mucilage adhesives2 cement melanized and pressurized dome-shaped infection cells-appressoria-to host rice leaf surfaces. Enormous internal turgor pressure is directed onto a penetration peg emerging from the unmelanized, thin-walled pore at the appressorial base1-4, forcing it through the leaf cuticle where it elongates invasive hyphae in underlying epidermal cells5. Mucilage sealing around the appressorial pore facilitates turgor build-up2, but the molecular underpinnings of mucilage secretion and appressorial adhesion are unknown. Here, we discovered an unanticipated and sole role for spermine in facilitating mucilage production by mitigating endoplasmic reticulum (ER) stress in the developing appressorium. Mutant strains lacking the spermine synthase-encoding gene SPS1 progressed through all stages of appressorial development, including penetration peg formation, but cuticle penetration was unsuccessful due to reduced appressorial adhesion, which led to solute leakage. Mechanistically, spermine neutralized off-target oxygen free radicals produced by NADPH oxidase-1 (Nox1)3,6 that otherwise elicited ER stress and the unfolded protein response, thereby critically reducing mucilage secretion. Our study reveals that spermine metabolism via redox buffering of the ER underpins appressorial adhesion and rice cell invasion and provides insights into a process that is fundamental to host plant infection.

The Diabetes Gene JAZF1 Is Essential for the Homeostatic Control of Ribosome Biogenesis and Function in Metabolic Stress

The ability of pancreatic β-cells to respond to increased demands for insulin during metabolic stress critically depends on proper ribosome homeostasis and function. Excessive and long-lasting stimulation of insulin secretion can elicit endoplasmic reticulum (ER) stress, unfolded protein response, and β-cell apoptosis. Here we show that the diabetes susceptibility gene JAZF1 is a key transcriptional regulator of ribosome biogenesis, global protein, and insulin translation. JAZF1 is excluded from the nucleus, and its expression levels are reduced upon metabolic stress and in diabetes. Genetic deletion of Jazf1 results in global impairment of protein synthesis that is mediated by defects in ribosomal protein synthesis, ribosomal RNA processing, and aminoacyl-synthetase expression, thereby inducing ER stress and increasing β-cell susceptibility to apoptosis. Importantly, JAZF1 function and its pleiotropic actions are impaired in islets of murine T2D and in human islets exposed to metabolic stress. Our study identifies JAZF1 as a central mediator of metabolic stress in β-cells.

Exenatide inhibits NF-κB and attenuates ER stress in diabetic cardiomyocyte models.

Exenatide is used to treat patients with type-2 diabetes and it also exerts cardioprotective effects. Here, we tested whether Exenatide attenuates hyperglycemia-related cardiomyocyte damage by inhibiting endoplasmic reticulum (ER) stress and the NF-κB signaling pathway. Our results demonstrated that hyperglycemia activates the NF-κB signaling pathway, eliciting ER stress. We also observed cardiomyocyte contractile dysfunction, inflammation, and cell apoptosis induced by hyperglycemia. Exenatide treatment inhibited inflammation, improved cardiomyocyte contractile function, and rescued cardiomyocyte viability. Notably, re-activation of the NF-κB signaling pathway abolished Exenatide’s protective effects on hyperglycemic cardiomyocytes. Taken together, our results demonstrate that Exenatide directly reduces hyperglycemia-induced cardiomyocyte damage by inhibiting ER stress and inactivating the NF-κB signaling pathway.

Stellate Ganglion Blockade repairs Intestinal Mucosal Barrier through suppression of Endoplasmic Reticulum Stress following Hemorrhagic Shock.

Background: Hemorrhagic shock-induced ischemia and hypoxia elicit endoplasmic reticulum stress (ERS) that leads to cell apoptosis, tissue structural damage and organ dysfunction and failure. Stellate ganglion blockade (SGB) has been demonstrated to improve intestinal barrier dysfunction induced by hemorrhagic shock. The present study sought to investigate whether the beneficial effect of SGB on the intestinal mucosal barrier function is via suppression of ERS.Materials and methods: A conscious rat model of hemorrhagic shock (40 ±2 mmHg for 1 hour, followed by resuscitation) was established. The parameters reflecting intestinal morphology and intestinal mucosal barrier function including wet-dry ratio (W/D), intestinal permeability, D-lactic acid (D-LA) and intestinal fatty acid binding protein (I-FABP) in plasma, and expressions of ATF6α, PERK, and IRE1α in intestinal tissues were then observed. Furthermore, the effects of either SGB or ERS inhibitor, 4-phenylbutyric acid (4-PBA), on these parameters in rats with hemorrhagic shock were assessed. The effect of ERS agonist tunicamycin (TM) on the rats subjected with both SGB and hemorrhagic shock was also determined.Results: Either SGB or administration of ERS inhibitor, 4-PBA, alleviated hemorrhagic shock-induced adverse effects such as intestinal mucosal barrier dysfunction and excessive autophagy, which were characterized by damaged intestinal tissue, enhanced intestinal permeability and D-LA and I-FABP levels in plasma, and increased expressions of ATF6α, PERK, IRE1α in intestinal tissue. In contrast, administration of ERS agonist, TM, suppressed the beneficial effects of SGB on intestinal tissue and function during hemorrhagic shock.Conclusion: The SGB repairs intestinal mucosal barrier through suppression of ERS following hemorrhagic shock.

Diosgenyl Saponin Inducing Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptotic Pathways in Liver Cancer Cells.

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.

MON-LB039 Developmental Programming: Prenatal Testosterone Excess Induces Endoplasmic Reticulum (ER) Stress in the Metabolic Tissues of the Female Sheep

Prenatal exposure to excess testosterone (T) programs peripheral insulin resistance (IR) and dyslipidemia along with tissue-specific changes in ectopic lipid accumulation, oxidative stress and IR in liver and muscle, and inflammation, oxidative stress, and reduced adipogenesis, differentiation, and adipocyte size but not IR in the visceral adipose tissue (VAT) of the female sheep. The reduced Insulin sensitivity in liver and muscle is likely the result of oxidative stress and lipotoxicity. Recently, homeostatic disturbance in endoplasmic reticulum (ER), a crucial site for protein and lipid metabolism, called ER stress has emerged as a key player in the development and maintenance of IR and adipocyte defects. Abnormal lipid metabolism and cellular protein damage due to oxidative stress or inflammation can elicit ER stress through double-stranded RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring protein 1 (IRE1), and activating transcription factor 6 (ATF6) and transcription factors C/EBP homologous protein (CHOP) and X box-binding protein 1 (XBP1). We therefore hypothesized that the tissue-specific ER stress may underlie the prenatal T programmed IR state and adipocyte defects in the female sheep. To test this premise and determine if ER stress are programmed by androgen or insulin (as T excess induces maternal hyperinsulinemia), liver, muscle and visceral adipose (VAT) tissues from control, prenatal T- (100mg T propionate twice a week from days 30-90 of gestation), prenatal T plus androgen antagonist, flutamide (15 mg/kg/day)-, and prenatal T plus insulin sensitizer, Rosiglitazone (0.11 mg/kg/day)-treated female sheep were studied at 24 months of age. The changes in expression of ER stress markers were assessed by real time RTPCR and data analyzed by ANOVA and Cohen’s effect size analysis. By effect size analysis, Prenatal T-treatment induced a large magnitude increase in only CHOP10 mRNA expression in the liver and muscle, a large magnitude decrease in XBP1 in the muscle and large magnitude increase in IRE1, PERK and CHOP10 In the VAT. Intervention with androgen antagonist and insulin sensitizer reversed prenatal T- induced decrease in XBP1 in the muscle but did not reverse the changes in other markers of ER stress. These preliminary findings support the hypothesis that prenatal T-treatment induces ER stress in the liver, muscle and VAT and appear to be programmed independent of androgen or insulin dependent pathways. Based on these data ER stress may potentially underlie the development or maintenance of IR status of liver and muscle and adipocyte defects of VAT in the prenatal T-treated sheep. These findings are of translational relevance to female offspring of hyperandrogenic pregnancies. Support: NIH P01 HD44232 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness.Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Degradation routes of trafficking-defective VLDLR mutants associated with Dysequilibrium syndrome

Low density lipoprotein receptor (LDLR) family members are involved in signaling in the developing brain. Previously we have reported that missense mutations in the Very Low Density Lipoprotein Receptor gene (VLDLR), causing Dysequilibrium syndrome (DES), disrupt ligand-binding, due to endoplasmic reticulum (ER) retention of the mutants. We explored the degradation routes of these VLDLR mutants in cultured cells. Our results indicate that VLDLR mutants are retained in the ER for prolonged periods which could be facilitated by association with the ER-resident chaperone calnexin. The mutants were prone to aggregation and capable of eliciting ER stress. The VLDLR mutants were found to be degraded predominantly by the proteasomal pathway, since ubiquitinated VLDLR was found to accumulate in response to proteasomal inhibition. Further, the mutants were found to interact with the ER degradation adaptor protein SEL1L. The degradation of VLDLR wild type and mutant were delayed in CRISPR/Cas9 edited SEL1L knock-out cells which was reversed by exogenous expression of SEL1L. In summary, ER retention of pathogenic VLDLR mutants involves binding to calnexin, elevated ER stress, and delayed degradation which is dependent on SEL1L. Since core LDLR family members share common structural domains, common mechanisms may be involved in their ER processing.

HBV suppresses thapsigargin-induced apoptosis via inhibiting CHOP expression in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) accounts for a proportion of cancer-associated mortalities worldwide. Hepatitis B virus (HBV) infection is a major cause of HCC in China. Thapsigargin (TG) is a potential antitumor prodrug, eliciting endoplasmic reticulum (ER) stress via the inhibition of the ER calcium pump, effectively inducing apoptosis. The present study therefore examined the role of HBV in TG-induced apoptosis using two HCC cell lines, HBV positive HepG2.2.15 and HBV negative HepG2. When these two cell lines were treated with TG, HepG2.2.15 was less susceptible to apoptosis than HepG2. This phenomenon was confirmed by an MTT assay and Annexin V-FITC/propidium iodide staining. Reverse transcription quantitative polymerase chain reaction and western blotting were used to detect the expression levels of genes in the ER stress pathway subsequent to treatment with TG. Notably, the mRNA and protein levels of the apoptosis factor DNA damage inducible transcript 3 (CHOP) increased significantly in the HepG2 cells compared with the HepG2.2.15 cells. Additionally, the HepG2.2.15 cells treated with interferon-α exhibited higher levels of CHOP compared with the untreated cells. The overexpression or knockdown of CHOP microRNA in HepG2.2.15 or HepG2 cells may reduce the difference in apoptosis status between the two cell lines. These results suggest that HBV may inhibit the apoptosis induced by ER stress. These findings may be useful in the development of selective therapies for patients with HBV-positive tumors.

ER-resident antioxidative GPx7 and GPx8 enzyme isoforms protect insulin-secreting INS-1E β-cells against lipotoxicity by improving the ER antioxidative capacity

Increased circulating levels of saturated fatty acids (FFAs) and glucose are considered to be major mediators of β-cell dysfunction and death in T2DM. Although it has been proposed that endoplasmic reticulum (ER) and oxidative stress play a crucial role in gluco/lipotoxicity, their interplay and relative contribution to β-cell dysfunction and apoptosis has not been fully elucidated. In addition it is still unclear how palmitate – the physiologically most abundant long-chain saturated FFA – elicits ER stress and which immediate signals commit β-cells to apoptosis. To study the underlying mechanisms of palmitate-mediated ER stress and β-cell toxicity, we exploited the observation that the recently described ER-resident GPx7 and GPx8 are not expressed in rat β-cells. Expression of GPx7 or GPx8 attenuated FFAs-mediated H2O2 generation, ER stress, and apoptosis induction. These results could be confirmed by a H2O2-specific inactivating ER catalase, indicating that accumulation of H2O2 in the ER lumen is critical in FFA-induced ER stress. Furthermore, neither the expression of GPx7 nor of GPx8 increased insulin content or facilitated disulfide bond formation in insulin-secreting INS-1E cells. Hence, reduction of H2O2 by ER-GPx isoforms is not rate-limiting in oxidative protein folding in rat β-cells. These data suggest that FFA-mediated ER stress is partially dependent on oxidative stress and selective expression of GPx7 or GPx8 improves the ER antioxidative capacity of rat β-cells without compromising insulin production and the oxidative protein folding machinery.

Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress

Despite substantial advances in the research exploring the pathogenesis of Type 1 Diabetes (T1D), the pathophysiological mechanisms involved remain unknown. We hypothesized in this study that interferon alpha (IFNα) participates in the early stages of T1D development by triggering endoplasmic reticulum (ER) stress. To verify our hypothesis, human islets and human EndoC-βH1 cells were exposed to IFNα and tested for ER stress markers, glucose stimulated insulin secretion (GSIS) and insulin content. IFNα treatment induced upregulation of ER stress markers including Binding immunoglobulin Protein, phospho-eukaryotic translation initiation factor 2α, spliced- X-box binding protein-1, C/EBP homologous protein and activating transcription factor 4. Intriguingly, IFNα treatment did not impair GSIS but significantly decreased insulin production in both human islets and EndoC-βH1 cells. Furthermore, IFNα decreased the expression of both proinsulin convertase 1 and proinsulin convertase 2, suggesting an altered functional state of the beta cells characterized by a slower proinsulin-insulin conversion. Pretreatment of both human islets and EndoC-βH1 cells with chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid completely prevented IFNα effects, indicating an ER stress-mediated impairment of insulin production. We demonstrated for the first time that IFNα elicits ER stress in human beta cells providing a novel mechanistic role for this virus-induced cytokine in the development of T1D. Compounds targeting molecular processes altered in ER-stressed beta cells could represent a potential therapeutic strategy to prevent IFNα-induced beta cell dysfunction in the early onset of T1D.

Hypoxia-Induced Iron Accumulation in Oligodendrocytes Mediates Apoptosis by Eliciting Endoplasmic Reticulum Stress.

This study was aimed at evaluating the role of increased iron accumulation in oligodendrocytes and its role in their apoptosis in the periventricular white matter damage (PWMD) following a hypoxic injury to the neonatal brain. In response to hypoxia, in the PWM, there was increased expression of proteins involved in iron acquisition, such as iron regulatory proteins (IRP1, IRP2) and transferrin receptor in oligodendrocytes. Consistent with this, following a hypoxic exposure, there was increased accumulation of iron in primary cultured oligodendrocytes. The increased concentration of iron within hypoxic oligodendrocytes was found to elicit ryanodine receptor (RyR) expression, and the expression of endoplasmic reticulum (ER) stress markers such as binding-immunoglobulin protein (BiP) and inositol-requiring enzyme (IRE)-1α. Associated with ER stress, there was reduced adenosine triphosphate (ATP) levels within hypoxic oligodendrocytes. However, treatment with deferoxamine reduced the increased expression of RyR, BiP, and IRE-1α and increased ATP levels in hypoxic oligodendrocytes. Parallel to ER stress there was enhanced reactive oxygen species production within mitochondria of hypoxic oligodendrocytes, which was attenuated when these cells were treated with deferoxamine. At the ultrastructural level, hypoxic oligodendrocytes frequently showed dilated ER and disrupted mitochondria, which became less evident in those treated with deferoxamine. Associated with these subcellular changes, the apoptosis of hypoxic oligodendrocytes was evident with an increase in p53 and caspase-3 expression, which was attenuated when these cells were treated with deferoxamine. Thus, the present study emphasizes that the excess iron accumulated within oligodendrocytes in hypoxic PWM could result in their death by eliciting ER stress and mitochondrial disruption.