Endoplasmic Reticulum Stress Inducer Thapsigargin Research Articles

CDNF induces the adaptive unfolded protein response and attenuates endoplasmic reticulum stress-induced cell death

The Cerebral Dopamine Neurotrophic Factor (CDNF) is a neurotrophic factor that has a protective effect in cell and animal models of several neurodegenerative diseases. The molecular mechanism of the protective effect of CDNF is unclear. Many neurodegenerative diseases have been related to a proteostasis dysregulation in the endoplasmic reticulum (ER). A failure of proteostasis produces ER stress, triggering the unfolded protein response (UPR) and, in the long-term, induces cell death. An adaptive UPR solves ER stress by attenuating protein synthesis, inducing chaperones expression, and degradation of misfolded proteins. Since CDNF is an ER resident protein, we investigated whether the role of CDNF is to regulate ER proteostasis. To this end, we determined the effect of CDNF in thapsigargin-induced ER stress in HEK293-T cells and cultured hippocampal neurons. Our results show that CDNF improved the viability of HEK293-T cells exposed to thapsigargin. CDNF increased levels of protective proteins of the early UPR, such as BiP, ATF4, ATF6, and XBP-1 in both HEK293-T cells and neurons. Conversely, expression of CDNF attenuated ER stress-induced apoptotic proteins, CHOP and cleaved caspase-3 in HEK293-T cells and neurons. A mutant CDNF lacking the ER retention sequence failed to protect against ER stress. In conclusion, CDNF regulates proteostasis in the ER by inducing the adaptive UPR response and inhibiting apoptotic pathways triggered by ER stress. We propose that neuroprotection induced by CDNF is mediated by regulating ER proteostasis.

P38 MAPK Facilitates Crosstalk Between Endoplasmic Reticulum Stress and IL-6 Release in the Intervertebral Disc.

Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2–5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1β and TNF-α. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 µM) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-α (5 and 10 ng/mL) did not activate ER stress, while IL-1β (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+]i flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD.

Crosstalk between transforming growth factor-β1 and endoplasmic reticulum stress regulates alpha-smooth muscle cell actin expression in podocytes

AimsTransforming growth factor-β1 (TGF-β1) plays a significant role in epithelial-mesenchymal transition (EMT). Furthermore, endoplasmic reticulum (ER) stress also can induce EMT. However, the relationship among TGF-β1, ER stress and EMT in podocytes is still unclear. Consequently, this study examines the crosstalk effect between TGF-β1 and ER stress on the regulation of EMT. Main methodsThe mRNA of EMT marker (α-smooth muscle actin: α-SMA) was evaluated by quantitative real-time PCR. In addition, the protein expressions of α-SMA and three ER stress biomarkers (glucose-regulated protein 78: GRP78; eukaryotic translation initiation factor 2α: eIF2α; CCAAT/enhancer-binding protein-homologous protein: CHOP) were evaluated by Western blot. Key findingsTGF-β1 increased the ER stress response biomarkers (GRP78, p-eIF2α/eIF2α and CHOP) and mRNA and protein levels of α-SMA in podocytes. Furthermore, ER stress inducer (thapsigargin) increased α-SMA protein expression. ER stress inhibitor (4-phenylbutyrate) attenuated the ER stress response and α-SMA protein expression under treatment with TGF-β1. Among the various TGF-β1 down-stream pathway inhibitors considered in the present study (SIS3: inhibitor of Smad2/3; U0126: inhibitor of MEK/ERK; SB203580: inhibitor of p38), SIS3 greatly attenuated the ER stress response biomarker (GRP78) under treatment with TGF-β1. SIS3, U0126 and SB203580 all partly attenuated α-SMA mRNA expression under TGF-β1 treatment. However, only SIS3 attenuated α-SMA protein expression. SignificanceThe present results confirm that ER stress induces α-SMA protein expression in podocytes. Furthermore, TGF-β1 mainly regulates ER stress and α-SMA protein expression through the Smad2/3 pathway. Therefore, ER stress and TGF-β1 may synergistically induce podocytes to undergo EMT.

High-Fat Diet/Palmitate–Induced ER Stress Promotes Protein O-GlcNAcylation in Retina and Retinal Muller Cells

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise due to the overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-type 2 diabetes, we evaluated the mechanism responsible for increased O-GlcNAcylation of retinal proteins following consumption of a diet high in saturated fat (HFD), as dysregulated O-GlcNAcylation contributes to the development of DR. In the retina of mice fed a HFD for 4 weeks, increased O-GlcNAcylation was observed concomitant with markers of endoplasmic reticulum (ER) stress. Similarly, in TR-MUL retinal cells in culture, addition of the saturated fatty acid palmitate or the ceramide analog Cer6 to culture medium increased protein O-GlcNAcylation and ER stress. One potential mechanism whereby ER stress increases O-GlcNAcylation is by upregulating flux through the hexosamine biosynthetic pathway (HBP) via increased expression of the rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT). In the retina of mice fed a HFD and in TR-MUL cells exposed to Cer6, mRNA and protein expression of GFAT2, but not GFAT1 were increased. Similarly, in TR-MUL cells treated with the ER stress inducer thapsigargin (TG) increased O-GlcNAcylation was observed concomitant with an increase in GFAT2. Alternatively, ER stress inhibitor prevented the effect. GFAT2 knockdown via siRNA attenuated O-GlcNAcylation induced by either TG or Cer6. The glial cell specific transcription factor OASIS (old astrocyte specifically-induced substance) plays a central role in coordinating the unfolded protein response. Unlike GFAT1, GFAT2 contains a putative binding site for OASIS. Both HFD and Cer6 increased OASIS expression, and OASIS was sufficient to promote GFAT2 expression and increase O-GlcNAcylation. Overall, the results support a model whereby HFD-induced ER stress acts to promote O-GlcNAcylation in retina through an OASIS/GFAT2-dependent mechanism. Disclosure W. Dai: None. A. Toro: None. S.K. Dierschke: None. M.D. Dennis: None.

GPNMB Induces BiP Expression by Enhancing Splicing of BiP Pre-mRNA during the Endoplasmic Reticulum Stress Response

Glycoprotein nonmetastatic melanoma protein B (GPNMB) has a neuroprotective effect against neuronal cell death caused by the accumulation of abnormal mutated proteins. It is known that the accumulation of pathological proteins induces endoplasmic-reticulum (ER) stress leading to cell damage. The aim of this study was to determine the role of GPNMB in the ER stress response. GPNMB was greatly up-regulated by thapsigargin-induced ER stress. Under the ER stress conditions, GPNMB relocated to the nucleus and specifically up-regulated expression of BiP at the mRNA level by promoting the BiP pre-mRNA splicing, not through the pathways initiated by the three major transducers of the unfolded protein response: IRE1, PERK, and ATF6. Furthermore, we found that the protein level of BiP and the infarction were increased and attenuated, respectively, in Gpnmb-transgenic mice after occlusion of the middle cerebral artery, in comparison with wild-type mice. Thus, our findings indicate that GPNMB enhances the BiP expression by promoting the splicing (thereby preventing cell death caused by ER stress) and could be a therapeutic target in ER stress-related disorders.

Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human β-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of β-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes.

Endoplasmic reticulum stress restrains hepatocyte growth factor expression in hepatic stellate cells and rat acute liver failure model

The aim of this study was to test whether endoplasmic reticulum (ER) stress suppresses hepatocyte growth factor (HGF) expression in hepatic stellate cells (HSCs) and whether ER stress plays a role in alleviating d-Galactosamine and Lipopolysaccharide (D-GalN/LPS)-induced acute liver failure (ALF) by regulating HGF expression. Rat HSCs line HSC-T6 were treated with the ER stress inducers tunicamycin or thapsigargin, and ER stress inhibitors sodium 4-phenylbutyrate (4-PBA) or salubrinal, respectively. Recombinant lentivirus containing eukaryotic translation initiation factor 2α (eIf2α), named LV-eIf2α-shRNA-GFP was produced to block eIf2α activated by ER stress. A rat ALF model was created by intraperitoneal injection of D-GalN/LPS, and 100 mg/kg 4-PBA was injected 6 h before injection as an inhibitor of ER stress. Levels of HGF, c-Met, vascular endothelial growth factor (VEGF), GRP78, eIf2α, phospho-eIF2α, ATF4 and CHOP in vitro and in vivo were measured. Our results demonstrated that ER stress stimulated VEGF but inhibited HGF expression in HSC-T6 cells. Both the stimulation of VEGF and the inhibition of HGF could be partly prevented by 4-PBA or Salubrinal. eIf2α expression was upregulated during ERS and interfering eIf2α expression proportionately down-regulated HGF expression. Inhibition of HGF and c-Met expression was also observed in the ALF rat. Treatment with 4-PBA prevented the reduction of HGF in ALF rats. ER stress regulates HGF expression in vitro and in vivo, which depends on eIf2α pathway. Reduction in liver damage of ALF by 4-PBA is associated with attenuation of ER stress and maintaining HGF production.

Effects of endoplasmic reticulum stress on apoptosis induction in radioresistant macrophages.

Macrophages are important in the host's immune defense against pathogens. However, recent evidence has demonstrated that macrophages are also involved in the development of disease, including cancer. Therefore, it is important to regulate apoptosis in tumor‑related macrophages for effective cancer treatment. In the present study, the effect of endoplasmic reticulum (ER) stress on apoptosis induction was examined in human monocytic cell‑derived macrophages. Radiation therapy in cancer results in irradiating macrophages as well as cancer cells in the tumor microenvironment. Since ER stress has been demonstrated to sensitize cancer cells to radiation, it was hypothesized that ER stress may induce a similar effect in macrophages. Therefore, the effect of combination treatment with ER stress inducers and ionizing radiation on macrophage apoptosis was examined. Treatment of macrophages with ER stress inducers thapsigargin and tunicamycin, enhanced unfolded protein responses, including phosphorylation of eukaryotic initiation factor 2‑α and increased expression of binding immunoglobulin protein. Furthermore, treatment with thapsigargin and tunicamycin induced apoptosis in macrophages compared with untreated cells, although ionizing radiation did not. The thapsigargin-induced apoptosis in macrophages was demonstrated to be caspase‑3‑dependent. Finally, combination treatment with thapsigargin and ionizing radiation, did not result in any significant change in macrophage apoptosis. The present study demonstrated that ER stress regulated apoptosis in radioresistant macrophages and that ionizing radiation had no added effect on ER stress‑induced apoptosis in macrophages.

Analysis of the potency of various low molecular weight chemical chaperones to prevent protein aggregation

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

Herpes Simplex Virus 1 UL41 Protein Suppresses the IRE1/XBP1 Signal Pathway of the Unfolded Protein Response via Its RNase Activity.

During viral infection, viruses hijack the host translation apparatus to produce large amounts of viral proteins, which leads to ER stress. To restore ER homeostasis, cells initiate the UPR to alleviate the effects of ER stress. The IRE1/XBP1 pathway is the most conserved UPR branch, and it activates ER-associated protein degradation (ERAD) to reduce the ER load. The IRE1/XBP1 branch is repressed during HSV-1 infection, but little is known about the underlying molecular mechanism. Our results show for the first time that UL41 suppresses the IRE1/XBP1 signal pathway by reducing the accumulation of XBP1 mRNA, and characterization of the underlying molecular mechanism provides new insight into the modulation of UPR by HSV-1.

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures.

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO2 NPs were used to compare the effects. Exposure to 32 μg/mL ZnO NPs (p < 0.05), but not TiO2 NPs (p > 0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO4 induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO2 NP exposure (p > 0.05). The presence of 250 nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p < 0.01), but did not significantly affect intracellular ROS or release of TNFα (p > 0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p > 0.05) except neutral red uptake assay (p < 0.01). We concluded ER stress is probably not associated with ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.

PERK and XBP1 differentially regulate CXCL10 and CCL2 production

Inflammation plays a key role in the pathogenesis of many retinal degenerative diseases related with photoreceptor dysfunction/degeneration. However the involvement of photoreceptor cells in inflammatory reactions is largely unknown as they are not considered as inflammatory cells. In this study, we assessed whether photoreceptor cells can produce CCL2 and CXCL10, two important players in inflammation during endoplasmic reticulum (ER) stress. After photoreceptor 661 W cells were treated with ER stress inducer thapsigargin (TG), induction of ER stress increased CXCL10 and CCL2 expression at both mRNA and protein levels, which was significantly blocked by an ER stress blocker 4-phenylbutyrate. ER stress contains three pathways: PERK, ATF6 and IRE1α. Knockdown of PERK attenuated TG-induced CXCL10 and CCL2 mRNA expression, associated with significant decreases in phosphorylation of NF-κB RelA and STAT3. In contrast to PERK, knockdown of XBP1, which is activated by IRE1α-mediated splicing, robustly enhanced TG-induced CXCL10 and CCL2 expression and phosphorylation of NF-κB RelA and STAT3. Blockade of NF-κB or STAT3 markedly diminished TG-induced CXCL10 and CCL2 expression. The specific roles of PERK and XBP1 in CXCL10 and CCL2 expression were further investigated by treating photoreceptor cells with advanced glycation end products (AGE) and high glucose (HG), two of the major contributors to diabetic complications. Similarly, AGE and HG induced CXCL10 and CCL2 expression in which PERK was a positive regulator while XBP1 was a negative regulator. These studies suggest that photoreceptors may be involved in retinal inflammation by expressing chemokines CXCL10 and CCL2. PERK and IRE1α/XBP1 in the unfolded protein response differentially regulate the expression of CXCL10 and CCL2 likely through modulation of ER stress-induced NF-κB RelA and STAT3 activation.

Evaluation of in vitro toxicity of polymeric micelles to human endothelial cells under different conditions

Polymeric micelles have been extensively studied in the area of antitumor therapy, and more recently explored as nanocarriers for atherosclerosis. These applications of polymeric micelles in biomedicine will increase their contact with human blood vessels. However, few studies have considered the interactions between polymeric micelles and endothelial cells, especially in a complex system. This study used human umbilical vein endothelial cells (HUVECs) as an in vitro model for endothelial cells to investigate the toxic effects of methoxy-poly(ethylene glycol)-poly(d,l-lactide) (MPEG-PLA) based micelles. In addition, an endoplasmic reticulum stress inducer thapsigargin (TG), and a pro-atherogenic stimulus palmitate (PA), were used to co-expose HUVECs to further mimic the responses of diseased endothelial cells to micelle exposure. Overall, up to 200 μg/mL micelles did not significantly induce cytotoxicity, reactive oxygen species (ROS), release of inflammatory mediators in terms of interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), or adhesion of THP-1 monocytes to HUVECs. TG and PA significantly induced cytotoxicity and THP-1 adhesion as well as modestly promoted the release of IL-6, but did not affect ROS or release of sVCAM-1 and IL-8. Co-exposure of micelles did not significantly enhance the effects of TG and PA to HUVECs, and ANOVA analysis indicated no interaction between concentrations of micelles and the presence of TG/PA. Taken together, these data indicated that micelles are not toxic to HUVECs under different conditions in vitro.

Palmitic acid aggravates inflammation of pancreatic acinar cells by enhancing unfolded protein response induced CCAAT-enhancer-binding protein β–CCAAT-enhancer-binding protein α activation

Hypertriglyceridemia is an independent risk factor for acute pancreatitis, in which the pathological mechanisms are not fully illustrated. Intracellular inflammatory response is a key pathological response in acute pancreatitis and endoplasmic reticulum stress has been suggested to induce inflammation and CCAAT-enhancer-binding protein expression. Therefore, the current study aims to elucidate the possible relationship between endoplasmic reticulum stress and inflammation in hypertriglyceridemia associated pancreatitis and the possible involvement of CCAAT-enhancer-binding protein. In cholecystokinin-8 stimulated rat primary acinar cells, incubation with palmitic acid caused the activation of endoplasmic reticulum stress and inflammatory responses. Pre-incubation with the chemical chaperone 4-phenylbutyric acid inhibited inflammatory responses induced by palmitic acid, whereas stimulation with the endoplasmic reticulum stress inducer thapsigargin alone induced inflammatory responses. Meanwhile we found that the transcription factors CCAAT-enhancer-binding protein α and CCAAT-enhancer-binding protein β were also induced in the palmitic acid-stimulated pancreatic acinar cells, and were similarly inhibited by 4-phenylbutyric acid pre-incubation and induced by thapsigargin stimulation alone, indicating that endoplasmic reticulum stress was responsible for CCAAT-enhancer-binding protein α and CCAAT-enhancer-binding protein β induction in the pancreatic acinar cells. Knockdown of CCAAT-enhancer-binding protein β by siRNA transfection inhibited inflammatory responses and CCAAT-enhancer-binding protein α induction but did not affect endoplasmic reticulum stress. Our study provides strong evidence that in response to palmitic acid stimulation, endoplasmic reticulum stress induces inflammatory responses in pancreatic acinar cells through induction of the CCAAT-enhancer-binding protein family, wherein CCAAT-enhancer-binding protein β activation is responsible for CCAAT-enhancer-binding protein α activation.

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway.

Endoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here, we investigated the effects and underlying mechanism of ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) on cardiac ER stress. Cell death, apoptosis and ER stress-related signalling pathways were measured in cultured neonatal rat cardiomyocytes, treated with the ER stress inducers tunicamycin, hydrogen peroxide and thapsigargin. Acute myocardial infarction was established using left coronary artery occlusion in mice, and infarct size was measured by triphenyltetrazolium chloride staining. Echocardiography was used to assess heart function and transmission electron microscopy for evaluating ER expansion. Ginkgolide K (GK) significantly decreased ER stress-induced cell death in both in vitro and in vivo models. In ischaemic injured mice, GK treatment reduced infarct size, rescued heart dysfunction and ameliorated ER dilation. Mechanistic studies revealed that the beneficial effects of GK occurred through enhancement of inositol-requiring enzyme 1α (IRE1α)/X box-binding protein-1 (XBP1) activity, which in turn led to increased ER-associated degradation-mediated clearance of misfolded proteins and autophagy. In addition, GK was also able to partly repress the pro-apoptotic action of regulated IRE1-dependent decay and JNK pathway. In conclusion, GK acts through selective activation of the IRE1α/XBP1 pathway to limit ER stress injury. GK is revealed as a promising therapeutic agent to ameliorate ER stress for treating cardiovascular diseases.

Astrocyte 세포와 C6 glioma 세포에서 ER stress 유도 물질 brefeldin A에 의한 CHOP 단백질의 발현 차이

Brefeldin A (BFA), a lactone antibiotic isolated from the fungus Eupenicillium brefeldianum, inhibits the transport of secreted and membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. BFA disrupts Golgi function, the accumulation of unfolded proteins in ER, and the in-duction of ER stress. Prolonged ER stress induces apoptosis at least in part through the transcription factor C/EBP (CCAAT/enhancer binding protein) homologous protein (CHOP),which is activated by the unfolded protein response (UPR). In this paper, we demonstrate that BFA-induced endoplasmic reticulum stress leads to different CHOP expression in primary astrocyte cells and C6 glioma cells. BFA induced lower CHOP expression levels in primary astrocyte cells than in C6 glioma cells; how-ever, other ER stress inducers (thapsigargin and tunicamycin) resulted in similar expression patterns in these two cell types. Interestingly, the three different ER stress inducers (BFA, thapsigargin, and tunicamycin) induced similar levels of CHOP mRNA expression in primary astrocyte cells. The ubiq-uitin-proteasome inhibitor MG132 also markedly up-regulated the BFA-mediated CHOP protein ex-pression in primary astrocyte cells. BFA also induced higher proteasome activity in primary astrocyte cells than in C6 glioma cells. Taken together, our results suggest that higher proteasomal activity might down-regulate BFA-induced CHOP expression in primary astrocyte cells.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn’s disease and type 2 diabetes1,2. ER stress induces the unfolded protein response (UPR), which involves activation of three transmembrane receptors, ATF6 (activating transcription factor 6), PERK (protein kinase RNA-like endoplasmic reticulum kinase) and IRE1α (inositol-requiring enzyme 1α)3 (Extended Data figure 1a). Once activated, IRE1α recruits TRAF2 (TNF receptor-associated factor 2) to the ER membrane to initiate inflammatory responses via the nuclear factor kappa B (NF-κB) pathway4. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), detect tissue damage or microbial infection. However, it is not clear which PRRs play a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NLR family of PRRs, are important mediators of ER stress-induced inflammation. The ER stress inducers thapsigargin and dithiothreitol (DTT) triggered production of the pro-inflammatory cytokine interleukin (IL)-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system (T4SS) effector protein VceC into host cells5, was TRAF2, NOD1/2 and RIP2-dependent and could be blunted by treatment with the ER-stress inhibitor tauroursodeoxycholate (TUDCA) or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signaling pathway provides a novel link between innate immunity and ER stress-induced inflammation.

Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis.

Neurofi brillary tangles (NFTs) of microtubule-associated protein tau are a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of AD. However, the exact role of ER stress in tau pathology has not yet been clearly elucidated. In present study, the possible relationship between tau pathology and ER stress was examined in terms of sorcin, which is a calcium binding protein and plays an important role in calcium homeostasis. Our previous yeast two hybrid study showed that sorcin is a novel tau interacting protein. Caspase-3-cleaved tau (T4C3) showed significantly increased tau-sorcin interaction compared to wild type tau (T4). Thapsigargin-induced ER stress and co-expression of constitutively active GSK3β (GSK3β-S9A) also exhibited significantly increased tau-sorcin interactions. T4C3-expressing cells showed potentiated thapsigargin-induced apoptosis and disruption of intracellular calcium homeostasis compared to T4-expressing cells. Overexpression of sorcin signifi cantly attenuated thapsigargin-induced apoptosis and disruption of calcium homeostasis. In contrary, siRNA-mediated knock-down of sorcin showed significantly increased thapsigargin-induced apoptosis and disruption of calcium homeostasis. These data strongly suggest that sequestration of sorcin by aberrant forms of tau compromises the function of sorcin, such as calcium homeostasis and cellular resistance by ER stress, which may consequently result in the contribution to the progression of AD.

Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma

Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1α and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1α and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1α transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1α and PERK, in PEL cells. However the IRE1α and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1α and PERK in PEL cells, transcriptional suppression of IRE1α by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress.