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Sample records for inhibits endoplasmic reticulum

  1. Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of resistance blood vessel structure and function.

    PubMed

    Carlisle, Rachel E; Werner, Kaitlyn E; Yum, Victoria; Lu, Chao; Tat, Victor; Memon, Muzammil; No, Yejin; Ask, Kjetil; Dickhout, Jeffrey G

    2016-08-01

    Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.

  2. Chronic inhibition of cGMP-specific phosphodiesterase 5 suppresses endoplasmic reticulum stress in heart failure

    PubMed Central

    Gong, Wei; Duan, Quanlu; Cai, Zhejun; Chen, Chen; Ni, Li; Yan, Mengwen; Wang, Xingxu; Cianflone, Katherine; Wang, Dao Wen

    2013-01-01

    BACKGROUND AND PURPOSE Inhibition of the cGMP-specific phosphodiesterase 5 (PDE5) exerts profound beneficial effects on failing hearts. However, the mechanisms underlying the therapeutic effects of PDE5 inhibition on heart failure are unclear. The purpose of this study was to investigate whether PDE5 inhibition decreases endoplasmic reticulum (ER) stress, a key event in heart failure. EXPERIMENTAL APPROACH Heart failure was induced by isoprenaline s.c. injection in Sprague–Dawley rats and transverse aortic constriction (TAC) in mice. PDE5 was inhibited with sildenafil. Heart function was detected by invasive pressure–volume analysis and echocardiography. ER stress markers were analysed by Western blotting. Apoptosis was measured by flow cytometric analysis. KEY RESULTS PDE5 inhibition markedly attenuated isoprenaline-induced and TAC-induced cardiac hypertrophy and dysfunction, and reduced ER stress and apoptosis. Further, PDE5 inhibition with sildenafil largely prevented ER stress and reduced apoptosis in isoprenaline- or thapsigargin-treated cardiomyocytes. PKG inhibition markedly prevented the protective effects of sildenafil in vivo and in vitro. To further understand the mechanism of the effect of PDE5 inhibition on ER stress, we demonstrated that PDE5 inhibitor increased sarco-(endo)-plasmic reticulum Ca2+-ATPase activity via phosphorylation of phospholamban at Ser16. This may contribute to the attenuation of ER stress induced by PDE5 inhibition. CONCLUSION AND IMPLICATIONS These results suggest that PDE5 inhibition can attenuate ER stress and improve cardiac function in vivo and in vitro. Suppression of ER stress by inhibiting PDE5 may contribute to the therapeutic effects on heart failure. PMID:24032459

  3. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress

    PubMed Central

    Yang, Rui; Teng, Xu; Li, Hui; Xue, Hong-Mei; Guo, Qi; Xiao, Lin; Wu, Yu-Ming

    2016-01-01

    In this study, the vitamin D3 plus nicotine (VDN) model of rats was used to prove that H2S alleviates vascular calcification (VC) and phenotype transformation of vascular smooth muscle cells (VSMC). Besides, H2S can also inhibit endoplasmic reticulum stress (ERS) of calcified aortic tissues. The effect of H2S on alleviating VC and phenotype transformation of VSMC can be blocked by TM, while PBA also alleviated VC and phenotype transformation of VSMC that was similar to the effect of H2S. These results suggest that H2S may alleviate rat aorta VC by inhibiting ERS, providing new target and perspective for prevention and treatment of VC. PMID:27022436

  4. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Yang, Rui; Teng, Xu; Li, Hui; Xue, Hong-Mei; Guo, Qi; Xiao, Lin; Wu, Yu-Ming

    2016-01-01

    In this study, the vitamin D3 plus nicotine (VDN) model of rats was used to prove that H2S alleviates vascular calcification (VC) and phenotype transformation of vascular smooth muscle cells (VSMC). Besides, H2S can also inhibit endoplasmic reticulum stress (ERS) of calcified aortic tissues. The effect of H2S on alleviating VC and phenotype transformation of VSMC can be blocked by TM, while PBA also alleviated VC and phenotype transformation of VSMC that was similar to the effect of H2S. These results suggest that H2S may alleviate rat aorta VC by inhibiting ERS, providing new target and perspective for prevention and treatment of VC.

  5. Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

    PubMed Central

    Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

    2017-01-01

    This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress. PMID:28071725

  6. STUDIES ON THE ENDOPLASMIC RETICULUM

    PubMed Central

    Palade, George E.

    1955-01-01

    A survey of a large number of different cell types has indicated the presence of a network of membrane-bound cavities (the endoplasmic reticulum) in the cytoplasm of all cell types examined, with the exception of the mature erythrocyte. In its simplest form, encountered in seminal epithelia and in leucocytes, the reticulum consists mainly of interconnected strings of vesicles and appears to be randomly disposed in three dimensions. Local differentiations occur within the endoplasmic reticulum of all the cell types studied. The membrane limiting the cavities of the endoplasmic reticulum appears to be continuous with the cell membrane and the nuclear membranes. PMID:13278367

  7. Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress

    PubMed Central

    Liu, Mi; Xue, Mei; Wang, Xiao-Reng; Tao, Tian-Qi; Xu, Fei-Fei; Liu, Xiu-Hua; Shi, Da-Zhuo

    2015-01-01

    Background Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings

  8. Chronic Intermittent Hypobaric Hypoxia Improves Cardiac Function through Inhibition of Endoplasmic Reticulum Stress.

    PubMed

    Yuan, Fang; Zhang, Li; Li, Yan-Qing; Teng, Xu; Tian, Si-Yu; Wang, Xiao-Ran; Zhang, Yi

    2017-08-11

    We investigated the role of endoplasmic reticulum stress (ERS) in chronic intermittent hypobaric hypoxia (CIHH)-induced cardiac protection. Adult male Sprague-Dawley rats were exposed to CIHH treatment simulating 5000 m altitude for 28 days, 6 hours per day. The heart was isolated and perfused with Langendorff apparatus and subjected to 30-min ischemia followed by 60-min reperfusion. Cardiac function, infarct size, and lactate dehydrogenase (LDH) activity were assessed. Expression of ERS molecular chaperones (GRP78, CHOP and caspase-12) was assayed by western blot analysis. CIHH treatment improved the recovery of left ventricular function and decreased cardiac infarct size and activity of LDH after I/R compared to control rats. Furthermore, CIHH treatment inhibited over-expression of ERS-related factors including GRP78, CHOP and caspase-12. CIHH-induced cardioprotection and inhibition of ERS were eliminated by application of dithiothreitol, an ERS inducer, and chelerythrine, a protein kinase C (PKC) inhibitor. In conclusion CIHH treatment exerts cardiac protection against I/R injury through inhibition of ERS via PKC signaling pathway.

  9. Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2008-10-15

    To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention "receptor" since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

  10. Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria

    PubMed Central

    Mohammed-Ali, Zahraa; Lu, Chao; Marway, Mandeep K.; Carlisle, Rachel E.; Ask, Kjetil; Lukic, Dusan; Krepinsky, Joan C.; Dickhout, Jeffrey G.

    2017-01-01

    Endoplasmic reticulum (ER) stress is implicated in chronic kidney disease (CKD) development in patients and in animal models. Here we show that ER stress inhibition through 4-phenylbutyric acid (4-PBA) administration decreases blood pressure, albuminuria, and tubular casts in an angiotensin II/deoxycorticosterone acetate/salt murine model of CKD. Lower albuminuria in 4-PBA-treated mice was associated with higher levels of cubilin protein in renal tissue membrane fractions. 4-PBA decreased renal interstitial fibrosis, renal CD3+ T-cell and macrophage infiltration, mRNA expression of TGFβ1, Wnt signaling molecules, and ER stress-induced pro-inflammatory genes. CHOP deficient mice that underwent this model of CKD developed hypertension comparable to wild type mice, but had less albuminuria and tubular casts. CHOP deficiency resulted in higher nephrin levels and decreased glomerulosclerosis compared to wild type mice; this effect was accompanied by lower macrophage infiltration and fibrosis. Our findings portray ER stress inhibition as a means to alleviate hypertensive CKD by preserving glomerular barrier integrity and tubular function. These results demonstrate ER stress modulation as a novel target for preserving renal function in hypertensive CKD. PMID:28148966

  11. Inhibition of telomerase causes vulnerability to endoplasmic reticulum stress-induced neuronal cell death.

    PubMed

    Hosoi, Toru; Nakatsu, Kanako; Shimamoto, Akira; Tahara, Hidetoshi; Ozawa, Koichiro

    2016-08-26

    Endoplasmic reticulum (ER) stress is implicated in several diseases, such as cancer and neurodegenerative diseases. In the present study, we investigated the possible involvement of telomerase in ER stress-induced cell death. ER stress-induced cell death was ameliorated in telomerase reverse transcriptase (TERT) over-expressing MCF7 cells (MCF7-TERT cell). Telomerase specific inhibitor, BIBR1532, reversed the inhibitory effect of TERT on ER stress-induced cell death in MCF7-TERT cells. These findings suggest that BIBR1532 may specifically inhibit telomerase activity, thereby inducing cell death in ER stress-exposed cells. TERT was expressed in the SH-SY5Y neuroblastoma cell line. To analyze the possible involvement of telomerase in ER stress-induced neuronal cell death, we treated SH-SY5Y neuroblastoma cells with BIBR1532 and analyzed ER stress-induced cell death. We found that BIBR1532 significantly enhanced the ER stress-induced neuronal cell death. These findings suggest that inhibition of telomerase activity may enhance vulnerability to neuronal cell death caused by ER stress.

  12. Endoplasmic Reticulum Stress and Obesity.

    PubMed

    Yilmaz, Erkan

    2017-01-01

    In recent years, the world has seen an alarming increase in obesity and closely associated with insulin resistance which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) play in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably other causes for obesity-related insulin resistance and inflammation. One of these appears to be endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.

  13. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity.

    PubMed

    Yamauchi, Mika; Tsuruma, Kazuhiro; Imai, Shunsuke; Nakanishi, Tomohiro; Umigai, Naofumi; Shimazawa, Masamitsu; Hara, Hideaki

    2011-01-10

    Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨ(m)), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H(2)O(2)) exposure. Crocetin at a concentration of 3μM showed the inhibitory effect of 50-60% against tunicamycin- and H(2)O(2)-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48h after light exposure. Crocetin at 100mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage.

  14. Inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA1) by rutin derivatives.

    PubMed

    Viskupicova, Jana; Majekova, Magdalena; Horakova, Lubica

    2015-04-01

    The effect of lipophilic rutin derivatives (acylated with fatty acid chain length of 16-22) on sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA1 isoform) compared to the parent molecule rutin was evaluated. Rutin derivatives caused concentration dependent decrease of SERCA1 activity (IC50 ~ 23-64 µM) and significant conformational alterations in the transmembrane region of the enzyme. Upon treatment by peroxynitrite, rutin derivatives exerted a hormetic effect, i.e. prevented enzyme activity decrease at low concentrations, while additionally inhibited at high concentrations. Concerning the posttranslational modifications of SERCA1, rutin esters: (i) induced a significant loss of free sulfhydryl groups, (ii) protected the enzyme from protein carbonyl formation, and (iii) prevented SERCA from tyrosine nitration (except R20:4 and R22:1). In silico study revealed a strong affinity of the derivative R20:4 to the transmembrane region of SERCA1, stabilized via hydrogen bonds with Glu90, Glu771, Thr778 and Thr848 residues. Interaction of rutin derivatives with Glu771, a residue involved in Ca(2+) binding, is likely to be responsible for the inhibitory effect of the esters.

  15. Inhibition of Cardiomyocytes Hypertrophy by Resveratrol Is Associated with Amelioration of Endoplasmic Reticulum Stress.

    PubMed

    Lin, Yan; Zhu, Jingbin; Zhang, Xiaojie; Wang, Jun; Xiao, Wei; Li, Bo; Jin, Li; Lian, Jie; Zhou, Li; Liu, Jicheng

    2016-01-01

    Resveratrol (Res), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. This study was undertaken to investigate whether Res can protect the heart suffering from hypertrophy injuries induced by isoproterenol (ISO), and whether the protective effect is mediated by endoplasmic reticulum (ER) stress. Cardiomyocytes were randomly assigned to the control group, ISO group (100 nM ISO for 48 h), Res + ISO group (50 μM Res and 100 nM ISO for 48 h) and Res group (50 μM Res for 48h only). Hypertrophy was estimated by measuring the cell surface area and the atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using Hoechst 33258 staining and transmission electron microscopy. Protein expression of ER stress and apoptosis factors was analyzed using Western Blot analysis. Res effectively suppress the cardiomyocytes hypertrophy and apoptosis induced by ISO, characterized by the reduction of the myocardial cell surface area, the ANP gene expression, the LDH and MDA leakage amount and the rate of cell apoptosis, while decrease of the protein expression of GRP78, GRP94 and CHOP, and reverse the expression of Bcl-2 and Bax. In summary, Res treatment effectively suppressed myocardial hypertrophy and apoptosis at least partially via inhibiting ER stress. © 2016 The Author(s) Published by S. Karger AG, Basel.

  16. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    PubMed Central

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-01-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro. PMID:28145529

  17. Valsartan protects HK-2 cells from contrast media-induced apoptosis by inhibiting endoplasmic reticulum stress.

    PubMed

    Peng, Ping-An; Wang, Le; Ma, Qian; Xin, Yi; Zhang, Ou; Han, Hong-Ya; Liu, Xiao-Li; Ji, Qing-Wei; Zhou, Yu-Jie; Zhao, Ying-Xin

    2015-12-01

    Contrast-induced acute kidney injury (CI-AKI) is associated with increasing in-hospital and long-term adverse clinical outcomes in high-risk patients undergoing percutaneous coronary intervention (PCI). Contrast media (CM)-induced renal tubular cell apoptosis is reported to participate in this process by activating endoplasmic reticulum (ER) stress. An angiotensin II type 1 receptor (AT1R) antagonist can alleviate ER stress-induced renal apoptosis in streptozotocin (STZ)-induced diabetic mice and can reduce CM-induced renal apoptosis by reducing oxidative stress and reversing the enhancement of bax mRNA and the reduction of bcl-2 mRNA, but the effect of the AT1R blocker on ER stress in the pathogenesis of CI-AKI is still unknown. In this study, we explored the effect of valsartan on meglumine diatrizoate-induced human renal tubular cell apoptosis by measuring changes in ER stress-related biomarkers. The results showed that meglumine diatrizoate caused significant cell apoptosis by up-regulating the expression of ER stress markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase 12, in a time- and dose-dependent manner, which could be alleviated by preincubation with valsartan. In conclusion, valsartan had a potential nephroprotective effect on meglumine diatrizoate-induced renal cell apoptosis by inhibiting ER stress.

  18. Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress.

    PubMed

    Li, Jian-Shuang; Wang, Wen-Jun; Sun, Yu; Zhang, Yu-Hao; Zheng, Ling

    2015-05-01

    Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10-30 μg ml(-1)) significantly decreased palmitic acid induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD treatment.

  19. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    NASA Astrophysics Data System (ADS)

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-02-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro.

  20. Aberrant Neuronal Differentiation and Inhibition of Dendrite Outgrowth Resulting from Endoplasmic Reticulum Stress

    PubMed Central

    Kawada, Koichi; Iekumo, Takaaki; Saito, Ryo; Kaneko, Masayuki; Mimori, Seisuke; Nomura, Yasuyuki; Okuma, Yasunobu

    2014-01-01

    Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis-related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin-induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII-tubulin in 8-day-differentiated cells. However, the neurites of dendrite marker microtubule-associated protein-2 (MAP-2)-positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP-2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII-tubulin and MAP-2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression. PMID:24723324

  1. Schisandra chinensis extract ameliorates nonalcoholic fatty liver via inhibition of endoplasmic reticulum stress.

    PubMed

    Jang, Min-Kyung; Nam, Jeong Soo; Kim, Ji Ha; Yun, Ye-Rang; Han, Chang Woo; Kim, Byung Joo; Jeong, Han-Sol; Ha, Ki-Tae; Jung, Myeong Ho

    2016-06-05

    Schisandra chinensis (Turcz.) Baill. (SC) is a traditional Chinese herbal medicine with diverse pharmacological activities for treatment of various human diseases. Endoplasmic reticulum (ER) stress is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effects of methanol extract of Schisandra chinensis (SC extract) against ER stress-induced NAFLD in vitro and in vivo. The protective effects of SC extract were examined in tunicamycin- or palmitate-treated HepG2 cells in vitro, and in tunicamycin-injected mice or high fed diet (HFD) obese mice in vivo. Expression of ER stress markers including glucose regulated protein 78 (GRP78), C/EBP homolog protein (CHOP), and X-box-binding protein-1 (XBP-1), and triglyceride accumulation were measured in HepG2 cells and in the liver of mice. SC extract significantly inhibited expression of tunicamycin-induced ER stress markers in tunicamycin-treated HepG2 cells and in the liver of tunicamycin-injected mice, and it also inhibited tunicamycin-induced triglyceride accumulation. Similar observations were made under physiological ER stress conditions such as in palmitate-treated HepG2 cells and in the liver of HFD obese mice. In addition, SC extract repressed the expression of inflammatory genes and lipogenic genes in palmitate-treated HepG2 cells. Schisandrin, an abundant bioactive lignan in SC extract, inhibited the expression of ER stress markers in tunicamycin-or palmitate-treated HepG2 cells, whereas Gomisin J did not affect ER stress markers. SC attenuates ER stress and prevents development of NAFLD. SC may be useful as a pharmacological agent for protection against ER stress-induced human diseases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Sustained endoplasmic reticulum stress inhibits hepatocyte proliferation via downregulation of c-Met expression.

    PubMed

    He, Yihuai; Long, Jun; Zhong, Weiwei; Fu, Yu; Li, Ying; Lin, Shide

    2014-04-01

    The molecular mechanisms of impaired liver regeneration in several liver diseases remain poorly understood. Endoplasmic reticulum (ER) stress has been observed in a variety of liver diseases. The aims of this study were to explore the impacts of ER stress on hepatocyte growth factor (HGF)-induced proliferation and c-Met expression in human hepatocyte L02 cells. Human hepatocyte L02 cells were incubated with thapsigargin (TG) to induce ER stress. 4-Phenylbutyric acid (PBA) was used to rescue ER stress. Activation of glucose-regulated protein 78, phosphorylation of PKR-like ER kinase and eukaryotic translation initiation factor-2α, and the expression of c-Met were determined by western blotting. The expression of c-Met mRNA was observed by reverse transcription polymerase chain reaction. L02 cell proliferation was determined by the MTS assay. L02 cell proliferation was significantly impaired in TG-treated L02 cells from 24 to 48 h, while PBA partly restored the proliferation of L02 cells. In addition, TG treatment significantly decreased the sensitivity of L02 cells to HGF-induced proliferation. PBA partly resumed the sensitivity of L02 cells to HGF-induced proliferation. The expression of c-Met protein in L02 cells was downregulated from 6 h after TG treatment, and PBA partly restored c-Met expression inhibited by TG. The expression of c-Met mRNA was also significantly downregulated from 24 to 48 h after TG treatment. Our results strongly suggest that sustained ER stress inhibits hepatocyte proliferation via downregulation of both c-Met mRNA and protein expression in human hepatocyte L02 cells.

  3. Cortistatin inhibits calcification of vascular smooth muscle cells by depressing osteoblastic differentiation and endoplasmic reticulum stress.

    PubMed

    Liu, Yue; Lin, Fang; Fu, Yu; Chen, Wenjia; Liu, Wenxiu; Chi, Jinyu; Zhang, Xiaohui; Yin, Xinhua

    2016-11-01

    Accumulating evidence has indicated that vascular smooth muscular cells (VSMCs) play an important role in the development of vascular calcification (VC). Cortistatin (CST), a novel bio-active peptide, has been shown to exert multiple protective effects on the cardiovascular system. However, the role and possible mechanism of CST in VC remain unclear. Therefore, we used β-glycerophosphoric acid (β-GP) to induce calcification in rat and human VSMCs to determine the effects of CST on osteoblastic differentiation and VSMC mineralization in vitro. Compared with the control, β-GP significantly increased alkaline phosphatase (ALP) activity and calcium content in cultured rat and human VSMCs, as well as multicellular node formation and calcium deposition, as confirmed by von Kossa and Alizarin Red S staining assays. After incubating rat and human VSMCs with β-GP in the presence of different doses of CST (10(-8) or 10(-7) mol/L), CST clearly reversed the β-GP-induced increases in ALP activity and calcium content and formation of pathological calcified nodes of VSMCs in a dose-independent manner. Moreover, 10(-8) and 10(-7) mol/L CST inhibited the phenotypic transformation of VSMCs into osteoblastic cells by decreasing the osteocalcin protein levels, increasing the SM-α-actin protein levels, and reducing endoplasmic reticulum stress by decreasing the protein expression of glucose-regulated protein 94 and CCAAT/enhancer-binding protein homologous protein. In conclusion, CST directly inhibited β-GP-induced calcification of VSMCs in vitro, probably by suppressing ERS and phenotypic transformation of VSMCs into osteoblastic cells. These results indicate that CST represents a potential target for the prevention and treatment of VC.

  4. Withaferin A Induces Proteasome Inhibition, Endoplasmic Reticulum Stress, the Heat Shock Response and Acquisition of Thermotolerance

    PubMed Central

    Heikkila, John J.

    2012-01-01

    In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge. PMID:23226310

  5. Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity.

    PubMed

    McLellan, Catherine A; Whitesell, Luke; King, Oliver D; Lancaster, Alex K; Mazitschek, Ralph; Lindquist, Susan

    2012-09-21

    In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.

  6. Atorvastatin attenuates atherosclerotic plaque destabilization by inhibiting endoplasmic reticulum stress in hyperhomocysteinemic mice.

    PubMed

    Jia, Fang; Wu, Chunfang; Chen, Zhenyue; Lu, Guoping; Sun, Jianhui

    2016-04-01

    Endoplasmic reticulum (ER) stress has been suggested to play a role in the progression of plaque vulnerability and the occurrence of acute complications of coronary atherosclerosis. Atorvastatin is known to exert pleiotropic effects on the cardiovascular system. The present study aimed to examine the stabilizing effects of atorvastatin on vulnerable plaques within hyperhomocysteinemic apolipoprotein E‑deficient (ApoE‑/‑) mice, and to investigate the potential mechanisms underlying ER stress in ApoE‑/‑ mice and macrophages. In the present study, ApoE‑/‑ mice were administrated methionine or atorvastatin, and were sacrificed after 2 months. Necrotic core size, collagen content and inflammatory cytokine infiltration were subsequently measured in the aortic lesions, in order to investigate plaque stability. Treatment with atorvastatin decreased the number and size of necrotic cores, increased collagen content, and downregulated tumor necrosis factor (TNF)‑α and matrix metalloproteinase (MMP)‑9 mRNA expression, as compared with the methionine group. Immunohistochemical analysis indicated that atorvastatin administration prevented ER stress activation in aortic lesions of hyperhomocysteinemic mice. Furthermore, macrophages were challenged with homocysteine (Hcy) in the presence or absence of atorvastatin and thapsigargin (an ER stress inducer). Atorvastatin suppressed Hcy‑induced ER stress, and downregulated TNF‑α and MMP‑9 mRNA expression in the macrophages. Conversely, thapsigargin attenuated the inhibitory effects of atorvastatin against Hcy‑induced TNF‑α and MMP‑9 expression. These results indicated that hyperhomocysteinemia may promote atherosclerotic plaque development and instability. In addition, atorvastatin was able to improve atherosclerotic plaque stability in hyperhomocysteinemic mice by inhibiting ER stress.

  7. Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells.

    PubMed

    Quan, Xiaojuan; Wang, Juyan; Liang, Chunlian; Zheng, Huadong; Zhang, Lin

    2015-08-07

    The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels of which may accelerate T2D. Endoplasmic reticulum stress (ERS) has been linked to insulin resistance in multiple tissues, but the role of melatonin on ERS and insulin resistance in skeletal muscle has not yet been investigated. In this study, the results showed that tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the phosphorylation of protein kinase R-like ER protein kinase (PERK) time-dependently in C2C12 cells. Consistently, ERS gene markers, including binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) expression and the splicing of X box binding protein 1 (XBP-1), were activated by tunicamycin time-dependently. Interestingly, melatonin pretreatment reversed the elevated PERK phosphorylation, as well as the activation of Bip expression and XBP-1 splicing, and prevented the inhibitory effect of tunicamycin on Akt phosphorylation. In addition, the insulin-provoked glucose transport was reduced by tunicamycin, and then promoted by melatonin pretreatment. A strong phosphorylation of inositol-requiring enzyme 1 (IRE-1), c-JUN NH2-terminal kinase (JNK), and insulin receptor substrate 1 (IRS-1) serine, and simultaneously, a dramatic decrease of IRS-1 tyrosine phosphorylation were observed in the presence of tunicamycin, leading to a blockade of insulin signaling, which was reversed by melatonin pretreatment. Furthermore, luzindole pretreatment acted inversely with melatonin action on glucose uptake and insulin signaling. Therefore, these results demonstrated that melatonin pretreatment inhibited the activated role of tunicamycin on ERS and insulin resistance through melatonin receptor-mediated IRE-1/JNK/IRS-1 insulin signaling in skeletal muscle cells.

  8. Oroxin A inhibits breast cancer cell growth by inducing robust endoplasmic reticulum stress and senescence.

    PubMed

    He, Jun; Du, Longsheng; Bao, Meimei; Zhang, Bin; Qian, Haixin; Zhou, Quansheng; Cao, Zhifei

    2016-03-01

    Breast cancer is a major cause of cancer death among women. Although various anticancer drugs have been used in clinics, drugs that are effective against advanced and metastatic breast cancer are still lacking and in great demand. In this study, we found that oroxin A, an active component isolated from the herb Oroxylum indicum (L.) Kurz, effectively inhibited the growth of human breast cancer cells MDA-MB-231 and MCF7 by inducing endoplasmic reticulum (ER) stress-mediated senescence. Oroxin A caused breast cancer cell cycle arrest at the G2/M stage, and reorganization of microtubules and actin cytoskeleton accompanied by a decrease in cellular mitosis. ER-specific probe ER-Tracker Red and confocal microscope imaging showed that ER-Tracker Red-positive cells increased in an oroxin A dosage-dependent manner. In addition, oroxin A increased cell population with high β-Gal activity and SAHF-positive staining; these data suggest that oroxin A induces breast cancer cell ER stress and senescence. Mechanistic studies showed that oroxin A led to a significant increase in intracellular reactive oxygen species levels, promoted expression of ER stress markers ATF4 and GRP78, and increased the phosphorylation of a key stress-response signaling protein p38, resulting in an ER stress-mediated senescence. Taken together, our data indicate that oroxin A exerts its antibreast cancer effects by inducing ER stress-mediated senescence, activating the key stress p38 signaling pathway, and increasing key ER stress genes ATF4 and GRP78 expression levels.

  9. Amelioration of bleomycin-induced pulmonary fibrosis by chlorogenic acid through endoplasmic reticulum stress inhibition.

    PubMed

    Wang, Yi-Chun; Dong, Jing; Nie, Jing; Zhu, Ji-Xiang; Wang, Hui; Chen, Qiong; Chen, Jun-Yi; Xia, Jia-Mei; Shuai, Wei

    2017-07-04

    To investigate the inhibitory effects of chlorogenic acid on pulmonary fibrosis and the internal mechanisms in vivo and in vitro. 30 male BALB/C mice were randomized into 5 groups: control group, pulmonary fibrosis model group, low, middle and high dose of chlorogenic acid groups. Mice in pulmonary fibrosis model group were administered 5.0 mg/kg bleomycin with intracheal instillation and mice in 3 chlorogenic acid groups were treated with chlorogenic acid every day for 28 days after bleomycin administration. Lung tissue histology was observed using HE staining. Primary pulmonary fibroblasts were isolated and cultured. The expressions of fibrosis related factors (α-SMA and collagen I), as well as ER stress markers (CHOP and GRP78) were determined by both real-time PCR assay and Western blotting, while the expressions of other ER stress signaling pathway factors PERK, IRE-1, ATF-6 and protein levels of caspase-12, caspase-9, caspase-3, PARP were determined by Western blotting. RLE-6TN cell line induced by TGF-β1 was also used to verify the amelioration effects in vitro study. In both in vivo and in vitro studies, TUNEL staining was used to evaluate cell apoptosis. Expressions of collagen I, α-SMA, GRP78, and CHOP were significantly inhibited by chlorogenic acid in dose-dependent manner. Similarly, decreasing levels of cleaved caspase-12, caspase-9, caspase-3 and increasing level of uncleaved PARP were observed in chlorogenic acid groups compared with those in the fibrosis group both in vivo and in vitro. Chlorogenic acid could also significantly down-regulate the level of phosphorylation of PERK and cleaved ATF-6 in vivo study. Moreover, MTT assay demonstrated chlorogenic acid could enhance proliferation of RLE-6TN cells induced by TGFβ1 in vitro. And the apoptosis assays indicated that chlorogenic acid could significantly inhibit cell apoptosis both in vivo and in vitro studies. Chlorogenic acid could inhibit the pulmonary fibrosis through endoplasmic

  10. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

    PubMed

    Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

    2015-12-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

  11. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  12. Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis.

    PubMed

    San-Miguel, Beatriz; Crespo, Irene; Sánchez, Diana I; González-Fernández, Bárbara; Ortiz de Urbina, Juan J; Tuñón, María J; González-Gallego, Javier

    2015-09-01

    This study aimed to investigate whether inhibition of autophagy and endoplasmic reticulum (ER stress) associates with the antifibrogenic effect of melatonin in mice treated with carbon tetrachloride (CCl4 ). Mice received CCl4 5 μL/g body wt i.p. twice a week for 4 wk or 6 wk. Melatonin was given at 5 or 10 mg/kg/day i.p, beginning 2 wk after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA)-positive cells. CCl4 induced an autophagic response measured as the presence of autophagic vesicles, protein 1 light chain 3 (LC3) staining, conversion of LC3-I to autophagosome-associated LC3-II, changes in expression of beclin-1, UV radiation resistance-associated gene (UVRAG), ubiquitin-like autophagy-related (Atg5), Atg12, Atg16L1, sequestosome 1 (p62/SQSTM1), and lysosome-associated membrane protein (LAMP)-2, and increased phosphorylation of the mammalian target of rapamycin (mTOR). There was an increase in the expression of the ER stress chaperones CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin-heavy-chain-binding protein (BiP/GRP78), and 94-kDa glucose-regulated protein (GRP94), and in the mRNA levels of pancreatic ER kinase (PERK), activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), and spliced X-box-binding protein-1 (XBP1). Phospho-IRE1, ATF6, and phospho-PERK protein concentration also increased significantly. Immunohistochemical staining of α-SMA indicated an abrogation of hepatic stellate cells activation by melatonin. Furthermore, treatment with the indole resulted in significant inhibition of the autophagic flux and the unfolded protein response. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in fibrogenesis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

    PubMed

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C

    2014-08-15

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. ©2014 American Association for Cancer Research.

  14. Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow.

    PubMed

    Chung, Jihwa; Kim, Kyoung Hwa; Lee, Seok Cheol; An, Shung Hyun; Kwon, Kihwan

    2015-10-01

    Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.

  15. Salubrinal Alleviates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Endoplasmic Reticulum Stress Pathway

    PubMed Central

    Rani, Shilpa; Sreenivasaiah, Pradeep Kumar; Cho, Chunghee; Kim, Do Han

    2017-01-01

    Pathological hypertrophy of the heart is closely associated with endoplasmic reticulum stress (ERS), leading to maladaptations such as myocardial fibrosis, induction of apoptosis, and cardiac dysfunctions. Salubrinal is a known selective inhibitor of protein phosphatase 1 (PP1) complex involving dephosphorylation of phospho-eukaryotic translation initiation factor 2 subunit (p-eIF2)-α, the key signaling process in the ERS pathway. In this study, the effects of salubrinal were examined on cardiac hypertrophy using the mouse model of transverse aortic constriction (TAC) and cell model of neonatal rat ventricular myocytes (NRVMs). Treatment of TAC-induced mice with salubrinal (0.5 mg·kg−1·day−1) alleviated cardiac hypertrophy and tissue fibrosis. Salubrinal also alleviated hypertrophic growth in endothelin 1 (ET1)-treated NRVMs. Therefore, the present results suggest that salubrinal may be a potentially efficacious drug for treating pathological cardiac remodeling. PMID:28152298

  16. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  17. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism*

    PubMed Central

    Dickey, Deborah M.; Edmund, Aaron B.; Otto, Neil M.; Chaffee, Thomas S.; Robinson, Jerid W.; Potter, Lincoln R.

    2016-01-01

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound 125I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a “constitutively phosphorylated” enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. PMID:26980729

  18. Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.

    PubMed

    Dong, Yunzhou; Zhang, Miao; Wang, Shuangxi; Liang, Bin; Zhao, Zhengxing; Liu, Chao; Wu, Mingyuan; Choi, Hyoung Chul; Lyons, Timothy J; Zou, Ming-Hui

    2010-06-01

    The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo. ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet. Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet. We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.

  19. Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress.

    PubMed

    Su, Lijuan; Chen, Xiaoyi; Wu, Jun; Lin, Biyun; Zhang, Haitao; Lan, Liubo; Luo, Hui

    2013-12-01

    Prolonged endoplasmic reticulum (ER) stress may activate apoptotic pathways in cancer cells. It is suggested that ER stress has the potential of enhancing tumor death in cancer therapy. Galangin, a flavonol derived from Alpinia officinarum Hance, has been shown to suppress the proliferation of hepatocellular carcinoma cells (HCC). The aim of this study was to determine whether galangin was able to induce ER stress in HepG2, Hep3B and PLC/PRF/5 cells. The proliferation of HCC was tested by MTT method. Intracellular Ca(2+) levels were measured with Fluo3-AM.The proteins levels of GRP94, GRP78 and CHOP were detected by Western blot. To further understand the anti-HCC mechanism of galangin, mitogen-activated protein kinases (MAPKs) were detected. The results showed that galangin treatment induced ER stress was evidenced by increased protein levels of GRP94, GRP78 and CHOP, as well as increased free cytosolic Ca(2+) concentration. ER stress inhibitor 4-PBA and CHOP siRNA blocked significantly galangin-induced ER in all three cell lines. Further experiments showed that MAPKs involved in ER stress induced by galangin. In summary, galangin is identified as a stimulator of ER stress to suppress the proliferation of HCC, and may be used as a potential anti-cancer agent.

  20. Inhibition of endoplasmic reticulum stress improves coronary artery function in the spontaneously hypertensive rats

    PubMed Central

    Choi, Soo-Kyoung; Lim, Mihwa; Byeon, Seon-Hee; Lee, Young-Ho

    2016-01-01

    Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of cardiovascular complications. However, the role and mechanisms of ER stress in hypertension remain unclear. Thus, we hypothesized that enhanced ER stress contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). Sixteen-week old male SHRs and Wistar Kyoto Rats (WKYs) were used in this study. The SHRs were treated with ER stress inhibitor (Tauroursodeoxycholic acid; TUDCA, 100 mg/kg/day) for two weeks. There was a decrease in systolic blood pressure in SHR treated with TUDCA. The pressure-induced myogenic tone was significantly increased, whereas endothelium-dependent relaxation was significantly attenuated in SHR compared with WHY. Interestingly, treatment of ER stress inhibitor normalized myogenic responses and endothelium-dependent relaxation in SHR. These data were associated with an increase in expression or phosphorylation of ER stress markers (Bip, ATF6, CHOP, IRE1, XBP1, PERK, and eIF2α) in SHRs, which were reduced by TUDCA treatment. Furthermore, phosphorylation of MLC20 was increased in SHRs, which was reduced by the treatment of TUDCA. Therefore, our results suggest that ER stress could be a potential target for hypertension. PMID:27550383

  1. Reduction of endoplasmic reticulum stress inhibits neointima formation after vascular injury.

    PubMed

    Ishimura, Shutaro; Furuhashi, Masato; Mita, Tomohiro; Fuseya, Takahiro; Watanabe, Yuki; Hoshina, Kyoko; Kokubu, Nobuaki; Inoue, Katsumi; Yoshida, Hideaki; Miura, Tetsuji

    2014-11-06

    Endoplasmic reticulum (ER) stress and inappropriate adaptation through the unfolded protein response (UPR) are predominant features of pathological processes. However, little is known about the link between ER stress and endovascular injury. We investigated the involvement of ER stress in neointima hyperplasia after vascular injury. The femoral arteries of 7-8-week-old male mice were subjected to wire-induced vascular injury. After 4 weeks, immunohistological analysis showed that ER stress markers were upregulated in the hyperplastic neointima. Neointima formation was increased by 54.8% in X-box binding protein-1 (XBP1) heterozygous mice, a model of compromised UPR. Knockdown of Xbp1 in human coronary artery smooth muscle cells (CASMC) in vitro promoted cell proliferation and migration. Furthermore, treatment with ER stress reducers, 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), decreased the intima-to-media ratio after wire injury by 50.0% and 72.8%, respectively. Chronic stimulation of CASMC with PDGF-BB activated the UPR, and treatment with 4-PBA and TUDCA significantly suppressed the PDGF-BB-induced ER stress markers in CASMC and the proliferation and migration of CASMC. In conclusion, increased ER stress contributes to neointima formation after vascular injury, while UPR signaling downstream of XBP1 plays a suppressive role. Suppression of ER stress would be a novel strategy against post-angioplasty vascular restenosis.

  2. Inhibition of endoplasmic reticulum stress and atherosclerosis by 2-aminopurine in apolipoprotein e-deficient mice.

    PubMed

    Zhou, Lichun; Yang, Dezhi; Wu, Dong Fang; Guo, Zhong Mao; Okoro, Emmanuel; Yang, Hong

    2013-01-01

    We previously reported that the apolipoprotein (apo) B48-carrying lipoproteins obtained from apoE knockout (apoE (-/-) ) mice, so called E(-)/B48 lipoproteins, transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2 α (eIF-2 α ). Furthermore, the eIF-2 α phosphorylation inhibitor, 2-aminopurine (2-AP), attenuated E(-)/B48 lipoprotein-induced foam cell formation. The present report studied the effect of 2-AP on atherosclerosis in apoE (-/-) mice. Our results showed that the level of food intake, bodyweight, plasma cholesterol, and triglycerides was comparable in apoE (-/-) mice treated with or without 2-AP. However, the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-AP-treated apoE (-/-) mice were reduced by about 55% and 39%, respectively, compared to samples from untreated control apoE (-/-) mice. In addition, the 2-AP-treated apoE (-/-) mice showed a significant decrease in glucose-regulated protein 78 (GRP78) and phosphorylated eIF-2 α in their aortic samples as compared to levels in untreated control apoE (-/-) mice. These observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoE (-/-) mice and that therapeutic strategies can be developed for using eIF-2 α phosphorylation inhibitors, such as 2-AP, to prevent or treat atherosclerosis.

  3. STUDIES ON THE ENDOPLASMIC RETICULUM

    PubMed Central

    Porter, Keith R.; Yamada, Eichi

    1960-01-01

    Pigment epithelial cells of the frog's retina have been examined by methods of electron microscopy with special attention focused on the fine structure of the endoplasmic reticulum and the myeloid bodies. These cells, as reported previously, send apical prolongations into the spaces between the rod outer segments, and within these extensions, pigment migrates in response to light stimulation. The cytoplasm of these cells is filled with a compact lattice of membrane-limited tubules, the surfaces of which are smooth or particle-free. In this respect, the endoplasmic reticulum here resembles that encountered in cells which produce lipid-rich secretions. The myeloid bodies comprise paired membranes arranged in stacks shaped like biconvex lenses. At their margins the membranes are continuous with elements of the ER and in consequence of this the myeloid body is referred to as a differentiation of the reticulum. The paired membranes resemble in their thickness and spacings those which make up the outer segments; they are therefore regarded as intracellular photoreceptors of possible significance in the activation of pigment migration and other physiologic functions of these cells. The fuscin granules are enclosed in membranes which are also continuous with those of the ER. The granules seem to move independently of the prolongations in which they are contained. The report also describes the fine structure of the terminal bar apparatus, the fibrous layer intervening between the epithelium and the choroid blood vessels, and comments on the functions of the organelles depicted. PMID:13737277

  4. Heat shock inhibits. alpha. -amylase synthesis in barley aleurone without inhibiting the activity of endoplasmic reticulum marker enzymes

    SciTech Connect

    Sticher, L.; Biswas, A.K.; Bush, D.S.; Jones, R.L. )

    1990-02-01

    The effects of heat shock on the synthesis of {alpha}-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25{degree}C to 40{degree}C for 3 hours, inhibits the accumulation of {alpha}-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca{sup 2+}. When ER is isolated from heat-shocked aleurone layers, less newly synthesized {alpha}-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca{sup 2+} transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

  5. Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols.

    PubMed

    Révész, Katalin; Tütto, Anna; Margittai, Eva; Bánhegyi, Gábor; Magyar, Judit E; Mandl, József; Csala, Miklós

    2007-01-01

    Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.

  6. Sulindac sulfide inhibits sarcoendoplasmic reticulum Ca2+ ATPase, induces endoplasmic reticulum stress response, and exerts toxicity in glioma cells: relevant similarities to and important differences from celecoxib.

    PubMed

    White, M C; Johnson, G G; Zhang, W; Hobrath, J V; Piazza, G A; Grimaldi, M

    2013-03-01

    Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca(2+) homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca(2+) from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR. Copyright © 2012 Wiley Periodicals, Inc.

  7. Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

    PubMed

    Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D

    2008-09-01

    Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

  8. Inhibition of Endoplasmic Reticulum Stress and Atherosclerosis by 2-Aminopurine in Apolipoprotein E-Deficient Mice

    PubMed Central

    Zhou, Lichun; Yang, Dezhi; Wu, Dong Fang; Guo, Zhong Mao; Yang, Hong

    2013-01-01

    We previously reported that the apolipoprotein (apo) B48-carrying lipoproteins obtained from apoE knockout (apoE−/−) mice, so called E−/B48 lipoproteins, transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2α (eIF-2α). Furthermore, the eIF-2α phosphorylation inhibitor, 2-aminopurine (2-AP), attenuated E−/B48 lipoprotein-induced foam cell formation. The present report studied the effect of 2-AP on atherosclerosis in apoE−/− mice. Our results showed that the level of food intake, bodyweight, plasma cholesterol, and triglycerides was comparable in apoE−/− mice treated with or without 2-AP. However, the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-AP-treated apoE−/− mice were reduced by about 55% and 39%, respectively, compared to samples from untreated control apoE−/− mice. In addition, the 2-AP-treated apoE−/− mice showed a significant decrease in glucose-regulated protein 78 (GRP78) and phosphorylated eIF-2α in their aortic samples as compared to levels in untreated control apoE−/− mice. These observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoE−/− mice and that therapeutic strategies can be developed for using eIF-2α phosphorylation inhibitors, such as 2-AP, to prevent or treat atherosclerosis. PMID:23984090

  9. L-carnitine attenuates H2O2-induced neuron apoptosis via inhibition of endoplasmic reticulum stress.

    PubMed

    Ye, Junli; Han, Yantao; Chen, Xuehong; Xie, Jing; Liu, Xiaojin; Qiao, Shunhong; Wang, Chunbo

    2014-12-01

    Both oxidative stress and endoplasmic reticulum stress (ER stress) have been linked to pathogenesis of neurodegenerative diseases. Our previous study has shown that L-carnitine may function as an antioxidant to inhibit H2O2-induced oxidative stress in neuroblastoma SH-SY5Y cells. To further explore the neuroprotection of L-carnitine, here we study the effects of L-carnitine on the ER stress response in H2O2-induced SH-SY5Y cell injury. Our results showed that L-carnitine pretreatment could increase cell viability; inhibit apoptosis and ROS accumulation caused by H2O2 or tunicamycin (TM). L-carnitine suppress the endoplasmic reticulum dilation and activation of ER stress-associated proteins including glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), JNK, Bax and Bim induced by H2O2 or TM. In addition, H2O2-induced cell apoptosis and activation of ER stress can also be attenuated by antioxidant N-acetylcysteine (NAC), CHOP siRNA and the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, our results demonstrated that H2O2 could trigger both oxidative stress and ER stress in SH-SY5Y cells, and ER stress participated in SH-SY5Y apoptosis mediated by H2O2-induced oxidative stress. CHOP/Bim or JNK/Bim-dependent ER stress signaling pathways maybe related to the neuroprotective effects of L-carnitine against H2O2-induced apoptosis and oxidative injury.

  10. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos-2 cells to cannabinoid receptor agonist WIN55,212-2.

    PubMed

    Zhang, Guodong; Bi, Haiyong; Gao, Ji; Lu, Xing; Zheng, Yanping

    2016-07-01

    WIN55,212-2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti-tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212-2 on osteosarcoma cell line Saos-2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212-2. Our results showed that the cell proliferation of Saos-2 was inhibited by WIN55,212-2 in a dose-dependent and time-dependent manner. WIN55,212-2-induced Saos-2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212-2 can induce endoplasmic reticulum stress and autophagy in Saos-2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212-2 in Saos-2 cells. These findings indicated that WIN55,212-2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    PubMed Central

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-01-01

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl4)-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl4-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl4-treated group relative to the control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl4-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl4, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. PMID:25827057

  12. 4‑Phenylbutyrate protects rat skin flaps against ischemia‑reperfusion injury and apoptosis by inhibiting endoplasmic reticulum stress.

    PubMed

    Yue, Zhen-Shuang; Zeng, Lin-Ru; Quan, Ren-Fu; Tang, Yang-Hua; Zheng, Wen-Jie; Qu, Gang; Xu, Can-Da; Zhu, Fang-Bing; Huang, Zhong-Ming

    2016-02-01

    4‑phenylbutyrate (4‑PBA) is a low molecular weight fatty acid, which has been demonstrated to regulate endoplasmic reticulum (ER) stress. ER stress‑induced cell apoptosis has an important role in skin flap ischemia; however, a pharmacological approach for treating ischemia‑induced ER dysfunction has yet to be reported. In the present study, the effects of 4‑PBA‑induced ER stress inhibition on ischemia‑reperfusion injury were investigated in the skin flap of rats, and transcriptional regulation was examined. 4‑PBA attenuated ischemia‑reperfusion injury and inhibited cell apoptosis in the skin flap. Furthermore, 4‑PBA reversed the increased expression levels of two ER stress markers: CCAAT/enhancer-binding protein‑homologous protein and glucose‑regulated protein 78. These results suggested that 4‑PBA was able to protect rat skin flaps against ischemia‑reperfusion injury and apoptosis by inhibiting ER stress marker expression and ER stress‑mediated apoptosis. The beneficial effects of 4‑PBA may prove useful in the treatment of skin flap ischemia‑reperfusion injury.

  13. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    SciTech Connect

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  14. Nafamostat mesilate attenuates transient focal ischemia/reperfusion-induced brain injury via the inhibition of endoplasmic reticulum stress.

    PubMed

    Kwon, Sun Kwan; Ahn, Moonsang; Song, Hee-Jung; Kang, Shin Kwang; Jung, Saet-Byel; Harsha, Nagar; Jee, Sungju; Moon, Jae Young; Suh, Kwang-Sun; Lee, Sang Do; Jeon, Byeong Hwa; Kim, Dong Woon; Kim, Cuk-Seong

    2015-11-19

    Nafamostat mesilate (NM), a serine protease inhibitor, has a broad range of clinical applications that include use as an anticoagulant during hemodialysis in cerebral hemorrhage patients, as a hemoperfusion anticoagulant for patients with intravascular coagulation, hemorrhagic lesions, and hemorrhagic tendencies, and for the improvement of acute pancreatitis. However, the effects of NM on acute cerebral ischemia have yet to be investigated. Thus, the present study utilized a rat model in which transient middle cerebral artery occlusion (MCAO) was used to induce ischemic injury to investigate the effects of NM on infarct volume and histological and biological changes. NM (1mg/kg) was intravenously administered prior to and after the MCAO procedure. Compared to control rats, the administration of NM significantly decreased infarct size and the extent of brain edema after the induction of focal ischemia via MCAO. Additionally, NM treatment attenuated MCAO-induced neuronal degeneration and activation of microglia and astrocytes. NM treatment also inhibited the MCAO-induced expression levels of glucose-regulated protein 78 (GRP78), CATT/EBP homologous protein (CHOP), and p-eukaryotic initiation factor 2α (eIF2α), which are endoplasmic reticulum (ER) stress markers, in the cerebral cortex. The present findings demonstrate that NM exerts neuroprotective effects in the brain following focal ischemia via, at least in part, the inhibition of ER stress.

  15. Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress

    PubMed Central

    Wang, Ting; Chen, Kaixian; Zhu, Weiliang; Wang, Heyao

    2015-01-01

    Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes. PMID:26147439

  16. Fibroblast growth factor 21 reverses suppression of adiponectin expression via inhibiting endoplasmic reticulum stress in adipose tissue of obese mice.

    PubMed

    Guo, Qinyue; Xu, Lin; Liu, Jiali; Li, Huixia; Sun, Hongzhi; Wu, Shufang; Zhou, Bo

    2017-02-01

    Fibroblast growth factor 21 (FGF21) has recently emerged as a novel endocrine hormone involved in the regulation of glucose and lipid metabolism. However, the exact mechanisms whereby FGF21 mediates insulin sensitivity remain not fully understood. In the present study, FGF21was administrated in high-fat diet-induced obese mice and tunicamycin-induced 3T3-L1 adipocytes, and metabolic parameters, endoplasmic reticulum (ER) stress indicators, and insulin signaling molecular were assessed by Western blotting. The administration of FGF21 in obese mice reduced body weight, blood glucose and serum insulin, and increased insulin sensitivity, resulting in alleviation of insulin resistance. Meanwhile, FGF21 treatment reversed suppression of adiponectin expression and restored insulin signaling via inhibiting ER stress in adipose tissue of obese mice. Additionally, suppression of ER stress via the ER stress inhibitor tauroursodeoxycholic acid increased adiponectin expression and improved insulin resistance in obese mice and in tunicamycin-induced adipocytes. In conclusion, our results showed that the administration of FGF21 reversed suppression of adiponectin expression and restored insulin signaling via inhibiting ER stress under the condition of insulin resistance, demonstrating the causative role of ER stress in downregulating adiponectin levels.

  17. Inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by flavonoids: a quantitative structure-activity relationship study.

    PubMed

    Ogunbayo, Oluseye A; Harris, Robert M; Waring, Rosemary H; Kirk, Christopher J; Michelangeli, Francesco

    2008-12-01

    Flavonoids are commonly found in fruit and vegetables and have been shown to reach concentrations of several micromolars in human blood plasma. Flavonoids are also believed to have cancer chemoprotective properties. One hypothesis is that flavonoids are able to initiate apoptosis, especially in cancer cells, via a Ca(2+)-dependent mitochondrial pathway. This pathway can be activated through an exaggerated elevation of cytosolic [Ca(2+)], and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA) play an essential role in ameliorating such changes. In this study, we demonstrate that flavonoids (especially flavones) can inhibit the activity of Ca(2+)-ATPases isoforms SERCA1A and SERCA2B in the micromolar concentration range. Of the 25 flavonoids tested, 3,6-dihydroxyflavone (IC(50), 4.6 microM) and 3,3',4',5,7-pentahydroxyflavone (quercetin) (IC(50), 8.9 microM) were the most potent inhibitors. We show that polyhydroxylation of the flavones are important for inhibition, with hydroxylation at position 3 (for SERCA1A) and position 6 (for SERCA2B) being particularly relevant.

  18. YiQiFuMai Powder Injection Ameliorates Cerebral Ischemia by Inhibiting Endoplasmic Reticulum Stress-Mediated Neuronal Apoptosis

    PubMed Central

    Hu, Yang

    2016-01-01

    YiQiFuMai (YQFM) powder injection as a modern preparation derived from Sheng Mai San, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, its neuroprotective effect and underlying mechanism in cerebral ischemia remain to be explored. The present study was designed to investigate the neuroprotective effect of YQFM on endoplasmic reticulum (ER) stress-mediated neuronal apoptosis in the permanent middle cerebral artery occlusion- (MCAO-) injured mice and the oxygen-glucose deprivation- (OGD-) induced pheochromocytoma (PC12) cells. The results showed that single administration of YQFM (1.342 g/kg, i.p.) could reduce the brain infarction and improve the neurological deficits and the cerebral blood flow (CBF) after MCAO for 24 h in mice. Moreover, incubation with YQFM (100, 200, and 400 μg/mL) could increase the cell viability, decrease the caspase-3 activity, and inhibit the cell apoptosis in OGD-induced PC12 cells for 12 h. In addition, YQFM treatment could significantly modulate cleaved caspase-3 and Bcl-2 expressions and inhibit the expressions of ER stress-related marker proteins and signaling pathways in vivo and in vitro. In conclusion, our findings provide the first evidence that YQFM ameliorates cerebral ischemic injury linked with modulating ER stress-related signaling pathways, which provided some new insights for its prevention and treatment of cerebral ischemia diseases. PMID:27087890

  19. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

    PubMed Central

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  20. Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum.

    PubMed

    Abe, M; Nishida, I; Minemura, M; Qadota, H; Seyama, Y; Watanabe, T; Ohya, Y

    2001-07-20

    1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

  1. Effect of endoplasmic reticulum stress inhibition on hyperoxaluria-induced oxidative stress: influence on cellular ROS sources.

    PubMed

    Bhardwaj, Rishi; Tandon, Chanderdeep; Dhawan, Devinder K; Kaur, Tanzeer

    2017-08-24

    Hyperoxaluria-induced calcium oxalate crystallisation is associated with the generation of reactive oxygen species (ROS) via mitochondria and NADPH oxidase. Endoplasmic reticulum (ER) has emerged as an organelle which could influence mitochondrial functioning and ROS generation. Plugging an upstream pathway of mitochondrial and NADPH oxidase-induced ROS generation may have better prophylaxis. Therefore, we propose to investigate the linkage of hyperoxaluria-induced ROS generation with ER stress by inhibiting the later with 4-Phenylbutyric acid (4-PBA). Male wistar rats were divided into three groups: a normal control group, an ethylene glycol with ammonium chloride-induced hyperoxaluric group (EA) and a third group which has hyperoxaluric animals given 4-PBA at a dose of 300 mg/kg. After 9 days of treatment, animals were sacrificed and renal tissues were analysed for histopathological examination, ROS, mitochondrial dysfunction, ER stress markers, inflammatory markers and NADPH oxidase subunits expression. Hyperoxaluric rats exhibited a significant increase in the levels of ROS, subsequently up-regulated levels of ER stress markers, inflammatory indicators, NADPH oxidase subunits and compromised mitochondrial functioning. However, ER stress amelioration appreciably curtailed the alterations caused by hyperoxaluric abuse. Therefore, suggesting the major role of ER in hyperoxaluric manifestations thereby providing an opportunity to target ER stress for future therapeutic interventions.

  2. Akebia trifoliate (Thunb.) Koidz Seed Extract Inhibits the Proliferation of Human Hepatocellular Carcinoma Cell Lines via Inducing Endoplasmic Reticulum Stress

    PubMed Central

    Lu, Wen-Li; Ren, Hong-Yan; Liang, Cao; Zhang, Yuan-Yuan; Xu, Ji; Pan, Zhi-Qiang; Liu, Xiao-Mei; Wu, Zhong-Hua; Fang, Zhao-Qin

    2014-01-01

    Akebia Fructus has long been used for hepatocellular carcinoma (HCC) in China, while the molecular mechanism remains obscure. Our recent work found that Akebia trifoliate (Thunb.) Koidz seed extract (ATSE) suppressed proliferation and induced endoplasmic reticulum (ER) stress in SMMC-7721. The present study aimed to throw more light on the mechanism. ER stress occurred after ATSE treatment in HepG2, HuH7, and SMMC-7721 cells, manifested as ER expansion, and SMMC-7721 was the most sensitive kind in terms of morphology. Cell viability assay showed that ATSE significantly inhibited cells proliferation. Flow cytometry analysis indicated that ATSE leads to an upward tendency of G0/G1 phase and a reduced trend of the continuous peak after G2/M phase in HepG2; ATSE promoted apoptosis in HuH7 and a notable reduction in G0/G1 phase; ATSE does not quite influence cell cycles of SMMC-7721. Western blot analysis showed an increased trend of the chosen ER stress-related proteins after different treatments but nonsignificantly; only HYOU1 and GRP78 were decreased notably by ATSE in HuH7. Affymetrix array indicated that lots of ER stress-related genes' expressions were significantly altered, and downward is the main trend. These results suggest that ATSE have anticancer potency in HCC cells via partly inducing ER stress. PMID:25389441

  3. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice.

    PubMed

    Park, Sin-Hye; Gong, Ju-Hyun; Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.

  4. RAGE deficiency alleviates aortic valve calcification in ApoE(-/-) mice via the inhibition of endoplasmic reticulum stress.

    PubMed

    Wang, Bo; Cai, Zhejun; Liu, Baoqing; Liu, Zongtao; Zhou, Xianming; Dong, Nianguo; Li, Fei

    2017-03-01

    Receptor for advanced glycation end products (RAGE) and endoplasmic reticulum (ER) stress have been shown to be involved in calcific aortic valve disease (CAVD). However, the association between RAGE and ER stress remains unknown in the pathogenesis of CAVD. The current study aims to test the hypothesis that RAGE deficiency alleviates aortic valve calcification via the inhibition of ER stress. Up-regulation of RAGE and ER stress markers in calcified human aortic valves were confirmed by immunoblotting. Aortic valve calcification was evaluated in atherosclerotic prone ApoE(-/-) mice or in mice with dual deficiencies of ApoE and RAGE (ApoE(-/-)RAGE(-/-)) fed with high cholesterol diet for 24weeks. Echocardiography and histological examination show that genetic deficiency of RAGE attenuates aortic valve calcification in ApoE(-/-) mice. Meanwhile, RAGE deficiency inhibited the osteogenic signaling and ER stress activation as well as suppressed macrophage infiltration in vivo. Cultured human aortic valve interstitial cells (AVICs) were treated with high molecular group box 1 protein (HMGB1) as in vitro model. We found that HMGB1 induced osteoblastic differentiation and calcification through RAGE/ER stress. Furthermore, Sox9 up-regulation and intranuclear translocation mediated the pro-osteogenic effect of HMGB1 on AVICs. RAGE or ER stress knockdown reduced the up-regulation of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in human AVICs exposed to HMGB1.These novel findings demonstrate that RAGE deficiency protects against aortic valve calcification in high cholesterol diet-fed ApoE(-/-) mice via inhibition of ER stress. HMGB1 induces AVIC osteoblastic differentiation and calcification through RAGE/ER stress/Sox9 pathway. Copyright © 2016. Published by Elsevier B.V.

  5. Fish oil supplementation inhibits endoplasmic reticulum stress and improves insulin resistance: involvement of AMP-activated protein kinase.

    PubMed

    Yang, Wenqi; Chen, Xu; Chen, Ming; Li, Yanping; Li, Qing; Jiang, Xinwei; Yang, Yan; Ling, Wenhua

    2017-04-19

    The beneficial effects of fish oil consumption on glucose metabolism have been generally reported. However, the mechanism underlying the fish oil-induced protective effects against insulin resistance remains unclear. Endoplasmic reticulum (ER) stress is recognized as an important contributor to insulin resistance. The aim of this study is to evaluate whether fish oil supplementation reduces ER stress and ameliorates insulin resistance in diet-induced obese mice, and to investigate the molecular mechanism of fish oil-induced benefits on ER stress. C57BL/6J mice were fed one of the following diets for 12 weeks: the low-fat diet (LFD), the high-fat diet (HFD) or the fish oil-supplemented high-fat diet (FOD). Fish oil supplementation led to lower blood glucose, better glucose tolerance and improved insulin sensitivity in high-fat diet-induced obese mice. Importantly, fish oil administration inhibited high-fat feeding-induced ER stress and reduced adipose tissue dysfunction. The fish oil-induced improvements were accompanied by the elevation of phosphorylated AMP-activated protein kinase (AMPK) expression in white adipose tissue. Correspondingly, the results of in vitro experiments showed that docosahexaenoic acid (DHA), the main n-3 polyunsaturated fatty acid (PUFA) in the fish oil used in the study, led to a dose-dependent increase in AMPK phosphorylation and suppressed palmitic acid (PA)-triggered ER stress in differentiated 3T3-L1 adipocytes. Furthermore, AMPK inhibitor (compound C) treatment largely blocked the effects of DHA to inhibit PA-induced ER stress. Our data indicate that n-3 PUFAs suppress ER stress in adipocytes through AMPK activation, and may thereby exert protective effects against high-fat feeding-induced adipose tissue dysfunction and insulin resistance.

  6. Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

    PubMed

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arévalo, Juan Carlos; Castellano, José; Fernández-Sanz, Celia; Galenkamp, Koen M O; Planells-Ferrer, Laura; Moubarak, Rana S; Llecha-Cano, Núria; Reix, Stéphanie; García-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X

    2016-01-15

    Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

  7. Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells*

    PubMed Central

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arévalo, Juan Carlos; Castellano, José; Fernández-Sanz, Celia; Galenkamp, Koen M. O.; Planells-Ferrer, Laura; Moubarak, Rana S.; Llecha-Cano, Núria; Reix, Stéphanie; García-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X.

    2016-01-01

    Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG. PMID:26582200

  8. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

    Hasel, K W; Glass, J R; Godbout, M; Sutcliffe, J G

    1991-01-01

    Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. Images PMID:1710767

  9. Studies on the Endoplasmic Reticulum

    PubMed Central

    Porter, Keith R.; Machado, Raul D.

    1960-01-01

    Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO4. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO4-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell. PMID:14434278

  10. Endoplasmic reticulum stress implicated in chronic traumatic encephalopathy.

    PubMed

    Lucke-Wold, Brandon P; Turner, Ryan C; Logsdon, Aric F; Nguyen, Linda; Bailes, Julian E; Lee, John M; Robson, Matthew J; Omalu, Bennet I; Huber, Jason D; Rosen, Charles L

    2016-03-01

    Chronic traumatic encephalopathy is a progressive neurodegenerative disease characterized by neurofibrillary tau tangles following repetitive neurotrauma. The underlying mechanism linking traumatic brain injury to chronic traumatic encephalopathy has not been elucidated. The authors investigate the role of endoplasmic reticulum stress as a link between acute neurotrauma and chronic neurodegeneration. The authors used pharmacological, biochemical, and behavioral tools to assess the role of endoplasmic reticulum stress in linking acute repetitive traumatic brain injury to the development of chronic neurodegeneration. Data from the authors' clinically relevant and validated rodent blast model were compared with those obtained from postmortem human chronic traumatic encephalopathy specimens from a National Football League player and World Wrestling Entertainment wrestler. The results demonstrated strong correlation of endoplasmic reticulum stress activation with subsequent tau hyperphosphorylation. Various endoplasmic reticulum stress markers were increased in human chronic traumatic encephalopathy specimens, and the endoplasmic reticulum stress response was associated with an increase in the tau kinase, glycogen synthase kinase-3β. Docosahexaenoic acid, an endoplasmic reticulum stress inhibitor, improved cognitive performance in the rat model 3 weeks after repetitive blast exposure. The data showed that docosahexaenoic acid administration substantially reduced tau hyperphosphorylation (t = 4.111, p < 0.05), improved cognition (t = 6.532, p < 0.001), and inhibited C/EBP homology protein activation (t = 5.631, p < 0.01). Additionally the data showed, for the first time, that endoplasmic reticulum stress is involved in the pathophysiology of chronic traumatic encephalopathy. Docosahexaenoic acid therefore warrants further investigation as a potential therapeutic agent for the prevention of chronic traumatic encephalopathy.

  11. Chronic inhibition of endoplasmic reticulum stress and inflammation prevents ischaemia-induced vascular pathology in type II diabetic mice.

    PubMed

    Amin, Ali; Choi, Soo-kyoung; Galan, Maria; Kassan, Modar; Partyka, Megan; Kadowitz, Philip; Henrion, Daniel; Trebak, Mohamed; Belmadani, Souad; Matrougui, Khalid

    2012-06-01

    Endoplasmic reticulum (ER) stress and inflammation are important mechanisms that underlie many of the serious consequences of type II diabetes. However, the role of ER stress and inflammation in impaired ischaemia-induced neovascularization in type II diabetes is unknown. We studied ischaemia-induced neovascularization in the hind-limb of 4-week-old db - /db- mice and their controls treated with or without the ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) and interleukin-1 receptor antagonist (anakinra, 0.5 µg/mouse per day) for 4 weeks. Blood pressure was similar in all groups of mice. Blood glucose, insulin levels, and body weight were reduced in db - /db- mice treated with TUDCA. Increased cholesterol and reduced adiponectin in db - /db- mice were restored by TUDCA and anakinra treatment. ER stress and inflammation in the ischaemic hind-limb in db - /db- mice were attenuated by TUDCA and anakinra treatment. Ischaemia-induced neovascularization and blood flow recovery were significantly reduced in db - /db- mice compared to control. Interestingly, neovascularization and blood flow recovery were restored in db - /db- mice treated with TUDCA or anakinra compared to non-treated db - /db- mice. TUDCA and anakinra enhanced eNOS-cGMP, VEGFR2, and reduced ERK1/2 MAP-kinase signalling, while endothelial progenitor cell number was similar in all groups of mice. Our findings demonstrate that the inhibition of ER stress and inflammation prevents impaired ischaemia-induced neovascularization in type II diabetic mice. Thus, ER stress and inflammation could be potential targets for a novel therapeutic approach to prevent impaired ischaemia-induced vascular pathology in type II diabetes.

  12. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts.

    PubMed

    Zhang, Aiping; Hildreth, Richard L; Colberg-Poley, Anamaris M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

  13. alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress.

    PubMed

    Katsoulieris, Elias; Mabley, Jon G; Samai, Mohamed; Green, Irene C; Chatterjee, Prabal K

    2009-11-25

    Unsaturated fatty acids may counteract the lipotoxicity associated with saturated fatty acids. Palmitic acid induced endoplasmic reticulum (ER) stress and caused apoptotic and necrotic cell death in the renal proximal tubular cell line, NRK-52E. We investigated whether alpha-linolenic acid, an unsaturated fatty acid, protected against ER stress and cell death induced by palmitic acid or by other non-nutrient ER stress generators. Incubation of NRK-52E cells for 24h with palmitic acid produced a significant increase in apoptosis and necrosis. Palmitic acid also increased levels of three indicators of ER stress - the phosphorylated form of the eukaryotic initiation factor 2alpha (eIF2alpha), C/EBP homologous protein (CHOP), and glucose regulated protein 78 (GRP78). alpha-Linolenic acid dramatically reduced cell death and levels of all three indicators of ER stress brought about by palmitic acid. Tunicamycin, which induces ER stress by glycosylation of proteins, produced similar effects to those obtained using palmitic acid; its effects were partially reversed by alpha-linolenic acid. Salubrinal (a phosphatase inhibitor) causes increased levels of the phosphorylated form of eIF2alpha - this effect was partially reversed by alpha-linolenic acid. Palmitoleate, a monosaturated fatty acid, had similar effects to those of alpha-linolenic acid. These results suggest that part of the mechanism of protection of the kidney by unsaturated fatty acids is through inhibition of ER stress, eIF2alpha phosphorylation and consequential reduction of CHOP protein expression and apoptotic renal cell death.

  14. Combined inhibition of Hsp90 and heme oxygenase-1 induces apoptosis and endoplasmic reticulum stress in melanoma.

    PubMed

    Barbagallo, Ignazio; Parenti, Rosalba; Zappalà, Agata; Vanella, Luca; Tibullo, Daniele; Pepe, Francesco; Onni, Toniangelo; Li Volti, Giovanni

    2015-10-01

    Heat shock proteins are ubiquitous molecular chaperones involved in post-translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment since it may act as a key regulator of various oncogene products and cell-signaling molecules. Heme oxygenase-1 (HO-1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO-1 activation may play a role in tumor development and progression. In the present study we investigated the chemotherapic effects of combining an Hsp90 inhibitor (NMS E973) and an HO-1 inhibitor (SnMP) on A375 melanoma cells. NMS E973 treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1α, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS E973 treatment resulted in a significant HO-1 overexpression, which in turn serves as a possible chemoresistance molecular mechanism. Interestingly, the combination of NMS E973 and SnMP produced an increase of ROS and reduced cell viability compared to NMS E973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by bifunctional apoptosis regulator (BFAR) mRNA expression and lower phosphorylation of Akt when compared to NMS E973 alone. In conclusion, these data suggest that HO-1 inhibition potentiates NMS E973 toxicity and may be exploited as a strategy for melanoma treatment.

  15. IL-15 expression increased in response to treadmill running and inhibited endoplasmic reticulum stress in skeletal muscle in rats.

    PubMed

    Yang, Hong-Tao; Luo, Li-Jie; Chen, Wen-Jia; Zhao, Lei; Tang, Chao-Shu; Qi, Yong-Fen; Zhang, Jing

    2015-02-01

    Interleukin 15 (IL-15) has recently been proposed as a circulating myokine involved in glucose uptake and utilization in skeletal muscle. However, the role and mechanism of IL-15 in exercise improving insulin resistance (IR) is unclear. Here, we investigated the alteration in expression of IL-15 and IL-15 receptor α (IL-15Rα) in skeletal muscle during treadmill running in rats with IR induced by a high-fat diet (HFD) and elucidated the mechanism of the anti-IR effects of IL-15. At 20 weeks of HFD, rats showed severe IR, with increased levels of fasting blood sugar and plasma insulin, impaired glucose tolerance, and reduced glucose transport activity. IL-15 immunoreactivity and mRNA level in gastrocnemius muscle were decreased markedly as compared with controls. IL-15Rα protein and mRNA levels in both soleus and gastrocnemius muscle were significantly decreased, which might attenuate the signaling or secretion of IL-15 in muscle. Eight-week treadmill running completely ameliorated HFD-induced IR and reversed the downregulated level of IL-15 and IL-15Rα in skeletal muscle of HFD-fed rats. To investigate whether IL-15 exerts its anti-IR effects directly in muscle, we pre-incubated muscle strips with the endoplasmic reticulum stress (ERS) inducer dithiothreitol (DTT) or tunicamycin (Tm); IL-15 treatment markedly decreased the protein expression of the ERS markers 78-kDa glucose-regulated protein, 94-kDa glucose-regulated protein and C/EBP homologous protein and inhibited ERS induced by DTT or Tm. Therefore, treadmill running promoted skeletal IL-15 and IL-15Rα expression in HFD-induced IR in rats. The inhibitory effect of IL-15 on ERS may be involved in improved insulin sensitivity with exercise training.

  16. Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1

    PubMed Central

    Zhang, Ping; Chen, Li-Xun; Wang, Li; Xie, Ming; Wang, Chun-Yan; Tang, Xiao-Qing

    2014-01-01

    Background Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. Objective We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. Principal Findings We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. Conclusion/Significance These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity. PMID:24587076

  17. The retention of prion protein in the endoplasmic reticulum prevents N2A cells from proteasome inhibition-induced cytotoxicity.

    PubMed

    Fang, Shuping; Wang, Ruixue; Liu, Honghao; Zhuang, Weiliang; Wang, Zhen; Zhang, Jianjun; Pei, Lili; Liu, Yumei; Su, Yunpeng

    2017-09-16

    Prion disease is a fatal neurodegenerative disease that may result from the conversion of normal cellular prion protein (PrP(C)) to the pathogenic scrapie PrP isoform (PrP(Sc)), however, how proliferation of prion leads to neuronal apoptosis is still not clear. In this study, to explore the role of the endoplasmic reticulum (ER) in prion diseases, we engineered the KDEL ER-retention motif to the C-terminus of PrP(C) and studied its effect on N2A cell toxicity. The KDEL retention signal led to the accumulation of PrP in the ER, and KDEL signal could effectively deplete PrP from the cell surface and trap PrP in the ER/Cis-Golgi compartment. PrP(C) molecules were delayed in their transit along the early pathway of the secretory compartment, however, they did not aggregate, and were not resistant to Proteinase K (PK) or become detergent-insoluble. Moreover, we found that the ER was not the site where PrP became detergent-insoluble and acquired PK resistance. In addition, an MTT assay indicated cells expressing PrP(C)/N2A were sensitive to proteasome inhibition, but not N2A cells expressing PrP(KDEL). Our findings suggest that the ER is not a compartment in which wild type PrP(C) is able to initiate aggregation, protease resistance or other scapie-like properties of PrP. Copyright © 2017. Published by Elsevier Inc.

  18. Cold-inducible RBM3 inhibits PERK phosphorylation through cooperation with NF90 to protect cells from endoplasmic reticulum stress.

    PubMed

    Zhu, Xinzhou; Zelmer, Andrea; Kapfhammer, Josef P; Wellmann, Sven

    2016-02-01

    The cold-inducible RNA-binding motif protein 3 (RBM3) is involved in the protection of neurons in hypoxic-ischemic and neurodegenerative disorders. RBM3 belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down. To investigate the molecular mechanisms underlying RBM3 action, we subjected hippocampal organotypic slice cultures from RBM3 knockout mice to various stressors and found exuberant signaling of the endoplasmic reticulum (ER) stress pathway PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-CCAAT/enhancer-binding protein homologous protein (CHOP) as compared with wild-type mice. Further, blocking RBM3 expression in human embryonic kidney HEK293 cells by specific small interfering RNAs increased phosphorylation of PERK and eIF2α, whereas overexpression of RBM3 prevented PERK-eIF2α-CHOP signaling during ER stress induced by thapsigargin or tunicamycin. RBM3 did not affect expression of the ER stress sensor immunoglobulin binding protein/GRP78. However, based on affinity purification coupled with mass spectrometry, coimmunoprecipitation, and proximity ligation assay, we revealed that nuclear factor 90 (NF90) is a novel protein interactor of PERK and that this interaction is essential for RBM3-mediated regulation of PERK activity, which requires an RNA-dependent interaction. In conclusion, our data provide evidence for a central role of RBM3 in preventing cell death by inhibiting the PERK-eIF2α-CHOP ER stress pathway through cooperation with NF90.

  19. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.

    PubMed

    Dixon, Scott J; Patel, Darpan N; Welsch, Matthew; Skouta, Rachid; Lee, Eric D; Hayano, Miki; Thomas, Ajit G; Gleason, Caroline E; Tatonetti, Nicholas P; Slusher, Barbara S; Stockwell, Brent R

    2014-05-20

    Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc (-) is implicated in numerous pathologies. Pharmacological agents that inhibit system xc (-) activity with high potency have long been sought, but have remained elusive. In this study, we report that the small molecule erastin is a potent, selective inhibitor of system xc (-). RNA sequencing revealed that inhibition of cystine-glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc (-) inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc (-) function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis.DOI: http://dx.doi.org/10.7554/eLife.02523.001.

  20. Inhibition of apolipoprotein B synthesis stimulates endoplasmic reticulum autophagy that prevents steatosis

    PubMed Central

    Conlon, Donna M.; Thomas, Tiffany; Fedotova, Tatyana; Di Paolo, Gilbert; Chan, Robin B.; Gibeley, Sarah; Liu, Jing; Ginsberg, Henry N.

    2016-01-01

    Inhibition of VLDL secretion reduces plasma levels of atherogenic apolipoprotein B (apoB) lipoproteins but can also cause hepatic steatosis. Approaches targeting apoB synthesis, which lies upstream of VLDL secretion, have potential to effectively reduce dyslipidemia but can also lead to hepatic accumulation of unsecreted triglycerides (TG). Here, we found that treating mice with apoB antisense oligonucleotides (ASOs) for 6 weeks decreased VLDL secretion and plasma cholesterol without causing steatosis. The absence of steatosis was linked to an increase in ER stress in the first 3 weeks of ASO treatment, followed by development of ER autophagy at the end of 6 weeks of treatment. The latter resulted in increased fatty acid (FA) oxidation that was inhibited by both chloroquine and 3-methyl adenine, consistent with trafficking of ER TG through the autophagic pathway before oxidation. These findings support the concept that inhibition of apoB synthesis traps lipids that have been transferred to the ER by microsomal TG transfer protein (MTP), inducing ER stress. ER stress then triggers ER autophagy and subsequent lysosomal lipolysis of TG, followed by mitochondrial oxidation of released FA, leading to prevention of steatosis. The identification of this pathway indicates that inhibition of VLDL secretion remains a viable target for therapies aiming to reduce circulating levels of atherogenic apoB lipoproteins. PMID:27599291

  1. Inhibition of endoplasmic reticulum stress signaling pathway: A new mechanism of statins to suppress the development of abdominal aortic aneurysm

    PubMed Central

    Li, Yuanyuan; Lu, Gangsheng; Sun, Dating; Zuo, Houjuan; Wang, Dao Wen; Yan, Jiangtao

    2017-01-01

    Background Abdominal aortic aneurysm (AAA) is a potentially lethal disease with extremely poor survival rates once the aneurysm ruptures. Statins may exert beneficial effects on the progression of AAA. However, the underlying mechanism is still not known. The purpose of the present study is to investigate whether statin could inhibit AAA formation by inhibiting the endoplasmic reticulum (ER) stress signal pathway. Methods A clinically relevant AAA model was induced in Apolipoprotein E-deficient (ApoE−/−) mice, which were infused with angiotensin II (Ang II) for 28 days. These mice were randomly divided into following 4 groups: saline infusion alone; Ang II infusion alone; Ang II infusion plus Atorvastatin (20mg/kg/d); and Ang II infusion plus Atorvastatin (30mg/kg/d). Besides, another AAA model was induced in C57 mice with extraluminal CaCl2, which were divided into 3 groups: sham group, CaCl2-induced AAA group, and CaCl2-induced AAA plus atorvastatin (20mg/kg/d) group. Then, aortic tissue was excised for further examinations, respectively. In vitro studies, Ang II with or without simvastatin treatment were applied to the vascular smooth muscle cells (VSMCS) and Raw 264.7 cells. The ER stress signal pathway, apoptosis and inflammatory response were evaluated by in vivo and in vitro assays. Results We found that higher dose of atorvastatin can effectively suppress the development and progression of AAA induced by Ang II or CaCl2. Mechanistically, the activation of ER stress and inflammatory response were found involved in Ang II-induced AAA formation. The atorvastatin infusion significantly reduced ER stress signaling proteins, the number of apoptotic cells, and the activation of Caspase12 and Bax in the Ang II-induced ApoE−/− mice, compared with mice treated by Ang II alone. Furthermore, proinflammatory cytokines such as IL-6, IL-8, IL-1β were all remarkably inhibited after atorvastatin treatment. In vitro, the inhibitory effect of simvastatin on the ER

  2. Inhibition of endoplasmic reticulum stress signaling pathway: A new mechanism of statins to suppress the development of abdominal aortic aneurysm.

    PubMed

    Li, Yuanyuan; Lu, Gangsheng; Sun, Dating; Zuo, Houjuan; Wang, Dao Wen; Yan, Jiangtao

    2017-01-01

    Abdominal aortic aneurysm (AAA) is a potentially lethal disease with extremely poor survival rates once the aneurysm ruptures. Statins may exert beneficial effects on the progression of AAA. However, the underlying mechanism is still not known. The purpose of the present study is to investigate whether statin could inhibit AAA formation by inhibiting the endoplasmic reticulum (ER) stress signal pathway. A clinically relevant AAA model was induced in Apolipoprotein E-deficient (ApoE-/-) mice, which were infused with angiotensin II (Ang II) for 28 days. These mice were randomly divided into following 4 groups: saline infusion alone; Ang II infusion alone; Ang II infusion plus Atorvastatin (20mg/kg/d); and Ang II infusion plus Atorvastatin (30mg/kg/d). Besides, another AAA model was induced in C57 mice with extraluminal CaCl2, which were divided into 3 groups: sham group, CaCl2-induced AAA group, and CaCl2-induced AAA plus atorvastatin (20mg/kg/d) group. Then, aortic tissue was excised for further examinations, respectively. In vitro studies, Ang II with or without simvastatin treatment were applied to the vascular smooth muscle cells (VSMCS) and Raw 264.7 cells. The ER stress signal pathway, apoptosis and inflammatory response were evaluated by in vivo and in vitro assays. We found that higher dose of atorvastatin can effectively suppress the development and progression of AAA induced by Ang II or CaCl2. Mechanistically, the activation of ER stress and inflammatory response were found involved in Ang II-induced AAA formation. The atorvastatin infusion significantly reduced ER stress signaling proteins, the number of apoptotic cells, and the activation of Caspase12 and Bax in the Ang II-induced ApoE-/- mice, compared with mice treated by Ang II alone. Furthermore, proinflammatory cytokines such as IL-6, IL-8, IL-1β were all remarkably inhibited after atorvastatin treatment. In vitro, the inhibitory effect of simvastatin on the ER stress signal pathway could be

  3. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy

    PubMed Central

    Montague, Karli; Malik, Bilal; Gray, Anna L.; La Spada, Albert R.; Hanna, Michael G.; Szabadkai, Gyorgy

    2014-01-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington’s disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases. PMID:24898351

  4. Endoplasmic reticulum aminopeptidases: biochemistry, physiology and pathology.

    PubMed

    Hattori, Akira; Tsujimoto, Masafumi

    2013-09-01

    The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized.

  5. Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity, and perturb Ca2+ homeostasis in human coronary artery endothelial cells

    PubMed Central

    Cook, Naomi L.; Viola, Helena M.; Sharov, Victor S.; Hool, Livia C.; Schöneich, Christian; Davies, Michael J.

    2013-01-01

    The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a critical role in Ca2+ homeostasis via sequestration of this ion into the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in ageing tissues and cardiovascular disease. We have shown previously that the myeloperoxidase- (MPO) derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca2+ levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to pre-formed or enzymatically-generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrently with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca2+, to HOSCN or HOCl, resulted in a time- and concentration-dependent increase in intracellular Ca2+ under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca2+ pumps/channels, completely attenuated the increase in intracellular Ca2+ consistent with a critical role for SERCA in maintaining endothelial cell Ca2+ homeostasis. Angiotensin II pre-treatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca2+ levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers due to their higher SCN− levels. PMID:22214747

  6. Inhibition of the mitochondrial calcium uniporter inhibits Aβ-induced apoptosis by reducing reactive oxygen species-mediated endoplasmic reticulum stress in cultured microglia.

    PubMed

    Xie, Nanchang; Wu, Chuanjie; Wang, Cui; Cheng, Xuan; Zhang, Lu; Zhang, Haifeng; Lian, Yajun

    2017-09-19

    Amyloid-beta (Aβ) has been shown to induce microglial apoptosis, which is itself sensitive to disturbed mitochondrial calcium (Ca(2+)) homeostasis. The mitochondrial calcium uniporter (MCU) plays an important regulatory role in mitochondrial Ca(2+) homeostasis, but its role in Aβ-induced microglia apoptosis is unknown. In this study, we found increased mitochondrial Ca(2+) concentration in Aβ-treated primary microglia and BV-2 cells; also, the MCU inhibitor Ru360 significantly attenuated Aβ-induced microglial apoptosis, whereas the MCU activator spermine augmented it. In addition, Ru360 significantly attenuated Aβ-induced mitochondrial reactive oxygen species (ROS) production, as well as endoplasmic reticulum (ER) stress characterized by glucose-regulated protein 78 (GRP78) and C/-EBP homologous protein (CHOP) expression. Spermine, however, exerted the opposite effects on mitochondrial ROS production and ER stress. We also found that mitochondria-targeted antioxidant (Mito-TEMPO) treatment decreased GRP78 and CHOP expression in Aβ-treated microglia. Moreover, blocking endogenous CHOP expression using a CHOP small interfering RNA (siRNA) attenuated Aβ-induced cell death. Altogether, our data suggested that 1) inhibition of MCU exerts a neuroprotective effect on Aβ-induced microglia apoptosis, and 2) that the underlying mechanism may be related to reducing mitochondrial ROS-mediated ER stress. Copyright © 2017. Published by Elsevier B.V.

  7. Cancer: Untethering Mitochondria from the Endoplasmic Reticulum?

    PubMed Central

    Herrera-Cruz, Maria Sol; Simmen, Thomas

    2017-01-01

    Following the discovery of the mitochondria-associated membrane (MAM) as a hub for lipid metabolism in 1990 and its description as one of the first examples for membrane contact sites at the turn of the century, the past decade has seen the emergence of this structure as a potential regulator of cancer growth and metabolism. The mechanistic basis for this hypothesis is that the MAM accommodates flux of Ca2+ from the endoplasmic reticulum (ER) to mitochondria. This flux then determines mitochondrial ATP production, known to be low in many tumors as part of the Warburg effect. However, low mitochondrial Ca2+ flux also reduces the propensity of tumor cells to undergo apoptosis, another cancer hallmark. Numerous regulators of this flux have been recently identified as MAM proteins. Not surprisingly, many fall into the groups of tumor suppressors and oncogenes. Given the important role that the MAM could play in cancer, it is expected that proteins mediating its formation are particularly implicated in tumorigenesis. Examples for such proteins are mitofusin-2 and phosphofurin acidic cluster sorting protein 2 that likely act as tumor suppressors. This review discusses how these proteins that mediate or regulate ER–mitochondria tethering are (or are not) promoting or inhibiting tumorigenesis. The emerging picture of MAMs in cancer seems to indicate that in addition to the downregulation of mitochondrial Ca2+ import, MAM defects are but one way how cancer cells control mitochondria metabolism and apoptosis. PMID:28603693

  8. Transport and transporters in the endoplasmic reticulum.

    PubMed

    Csala, Miklós; Marcolongo, Paola; Lizák, Beáta; Senesi, Silvia; Margittai, Eva; Fulceri, Rosella; Magyar, Judit E; Benedetti, Angelo; Bánhegyi, Gábor

    2007-06-01

    Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.

  9. Endoplasmic Reticulum Stress and Associated ROS

    PubMed Central

    Zeeshan, Hafiz Maher Ali; Lee, Geum Hwa; Kim, Hyung-Ryong; Chae, Han-Jung

    2016-01-01

    The endoplasmic reticulum (ER) is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS). Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI)-endoplasmic reticulum oxidoreductin (ERO)-1, glutathione (GSH)/glutathione disuphide (GSSG), NADPH oxidase 4 (Nox4), NADPH-P450 reductase (NPR), and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases. PMID:26950115

  10. Dietary Cocoa Powder Improves Hyperlipidemia and Reduces Atherosclerosis in apoE Deficient Mice through the Inhibition of Hepatic Endoplasmic Reticulum Stress

    PubMed Central

    Guan, Hua; Lin, Yan; Bai, Liang; An, Yingfeng; Shang, Jianan; Wang, Zhao; Zhao, Sihai; Fan, Jianglin

    2016-01-01

    Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress. PMID:26980943

  11. Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway.

    PubMed

    Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won

    2016-07-01

    Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

  12. Mesencephalic astrocyte-derived neurotrophic factor inhibits oxygen-glucose deprivation-induced cell damage and inflammation by suppressing endoplasmic reticulum stress in rat primary astrocytes.

    PubMed

    Zhao, Hua; Liu, Yi; Cheng, Lei; Liu, Ben; Zhang, Wen; Guo, Ying-Jun; Nie, Lin

    2013-11-01

    Astrocyte inflammation plays important roles both in physiological and pathological processes in the central nervous system (CNS). Ischemic injury in the CNS causes damage to astrocytes and the release of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β, and interleukin-6. This current study investigates whether mesencephalic astrocyte-derived neurotrophic factor (MANF) inhibits oxygen-glucose deprivation (OGD)-induced cell damage and inflammatory cytokine secretion by suppressing endoplasmic reticulum stress in rat primary astrocytes. We found that MANF alleviated OGD-induced astrocyte damage and rescued the cell viability, and the upregulation of GRP78 (endoplasmic reticulum (ER) stress marker) and NF-κB p65 (one of the central mediators of proinflammatory pathways) induced by OGD were significantly reduced by preincubation of MANF. In addition, the increases of secretion and mRNA expression levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in astrocytes induced by OGD were significantly suppressed by MANF. These findings demonstrate that MANF shows the potential to alleviate cell damage and inflammation in rat primary astrocytes by suppressing ER stress, indicating that MANF plays an important role in astrocyte inflammation and functioning and may suggest a promising strategy for neuroprotection in the CNS.

  13. Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia–reperfusion

    PubMed Central

    Mosbah, I Ben; Alfany-Fernández, I; Martel, C; Zaouali, M A; Bintanel-Morcillo, M; Rimola, A; Rodés, J; Brenner, C; Roselló-Catafau, J; Peralta, C

    2010-01-01

    During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R. PMID:21364657

  14. Endoplasmic reticulum stress inhibition limits the progression of chronic kidney disease in the Dahl salt-sensitive rat.

    PubMed

    Yum, Victoria; Carlisle, Rachel E; Lu, Chao; Brimble, Elise; Chahal, Jasmine; Upagupta, Chandak; Ask, Kjetil; Dickhout, Jeffrey G

    2017-01-01

    Proteinuria is one of the primary risk factors for the progression of chronic kidney disease (CKD) and has been implicated in the induction of endoplasmic reticulum (ER) stress. We hypothesized that the suppression of ER stress with a low molecular weight chemical chaperone, 4-phenylbutyric acid (4-PBA), would reduce the severity of CKD and proteinuria in the Dahl salt-sensitive (SS) hypertensive rat. To induce hypertension and CKD, 12-wk-old male rats were placed on a high-salt (HS) diet for 4 wk with or without 4-PBA treatment. We assessed blood pressure and markers of CKD, including proteinuria, albuminuria, and renal pathology. Furthermore, we determined if HS feeding resulted in an impaired myogenic response, subsequent to ER stress. 4-PBA treatment reduced salt-induced hypertension, proteinuria, and albuminuria and preserved myogenic constriction. Furthermore, renal pathology was reduced with 4-PBA treatment, as indicated by lowered expression of profibrotic markers and fewer intratubular protein casts. In addition, ER stress in the glomerulus was reduced, and the integrity of the glomerular filtration barrier was preserved. These results suggest that 4-PBA treatment protects against proteinuria in the SS rat by preserving the myogenic response and by preventing ER stress, which led to a breakdown in the glomerular filtration barrier. As such, alleviating ER stress serves as a viable therapeutic strategy to preserve kidney function and to delay the progression of CKD in the animal model under study. Copyright © 2017 the American Physiological Society.

  15. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

    SciTech Connect

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

  16. Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy.

    PubMed

    Huang, He; Kang, Rongyan; Wang, Ji; Luo, Guangxiang; Yang, Wei; Zhao, Zhendong

    2013-02-01

    Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.

  17. Obesity and endoplasmic reticulum (ER) stresses

    PubMed Central

    Tripathi, Yamini B.; Pandey, Vivek

    2012-01-01

    In obesity, the adipose cells behave as inflammatory source and result to low grade inflammation. This systemic inflammation along with oxidative stress is a silent killer and damages other vital organs also. High metabolic process, induced due to high nutritional intake, results to endoplasmic reticulum (ER) stress and mitochondrial stress. This review describes the triggering factor and basic mechanism behind the obesity mediated these stresses in relation to inflammation. Efforts have been made to describe the effect-response cycle between adipocytes and non-adipocyte cells with reference to metabolic syndrome (MS). PMID:22891067

  18. Protein Translocation across the Rough Endoplasmic Reticulum

    PubMed Central

    Mandon, Elisabet C.; Trueman, Steven F.; Gilmore, Reid

    2013-01-01

    The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration. PMID:23251026

  19. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

    PubMed

    Montague, Karli; Malik, Bilal; Gray, Anna L; La Spada, Albert R; Hanna, Michael G; Szabadkai, Gyorgy; Greensmith, Linda

    2014-07-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain.

  20. Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress.

    PubMed

    Li, Huang; Zhang, Xiang-Yu; Wu, Tuo-Jiang; Cheng, Wei; Liu, Xin; Jiang, Ting-Ting; Wen, Juan; Li, Jie; Ma, Qiao-Ling; Hua, Zi-Chun

    2013-06-21

    Compressive mechanical stress-induced cartilage thinning has been characterized as a key step in the progression of temporomandibular joint diseases, such as osteoarthritis. However, the regulatory mechanisms underlying this loss have not been thoroughly studied. Here, we used an established animal model for loading compressive mechanical stress to induce cartilage thinning in vivo. The mechanically stressed mandibular chondrocytes were then isolated to screen potential candidates using a proteomics approach. A total of 28 proteins were identified that were directly or indirectly associated with endoplasmic reticulum stress, including protein disulfide-isomerase, calreticulin, translationally controlled tumor protein, and peptidyl-prolyl cis/trans-isomerase protein. The altered expression of these candidates was validated at both the mRNA and protein levels. The induction of endoplasmic reticulum stress by mechanical stress loading was confirmed by the activation of endoplasmic reticulum stress markers, the elevation of the cytoplasmic Ca(2+) level, and the expansion of endoplasmic reticulum membranes. More importantly, the use of a selective inhibitor to block endoplasmic reticulum stress in vivo reduced the apoptosis observed at the early stages of mechanical stress loading and inhibited the proliferation observed at the later stages of mechanical stress loading. Accordingly, the use of the inhibitor significantly restored cartilage thinning. Taken together, these results demonstrated that endoplasmic reticulum stress is significantly activated in mechanical stress-induced mandibular cartilage thinning and, more importantly, that endoplasmic reticulum stress inhibition alleviates this loss, suggesting a novel pharmaceutical strategy for the treatment of mechanical stress-induced temporomandibular joint diseases.

  1. Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum.

    PubMed

    Tang, Yan-Dong; Na, Lei; Zhu, Chun-Hui; Shen, Nan; Yang, Fei; Fu, Xian-Qiu; Wang, Yu-Hong; Fu, Li-Hua; Wang, Jia-Yi; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun; Zhou, Jian-Hua; Li, Cheng-Yao

    2014-11-01

    Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction

  2. Phenylbutyrate prevents disruption of blood-spinal cord barrier by inhibiting endoplasmic reticulum stress after spinal cord injury

    PubMed Central

    Zhou, Yulong; Ye, Libing; Zheng, Binbin; Zhu, Sipin; Shi, Hongxue; Zhang, Hongyu; Wang, Zhouguang; Wei, Xiaojie; Chen, Daqing; Li, Xiaokun; Xu, Huazi; Xiao, Jian

    2016-01-01

    This study aims to investigate the role of endocytoplasmic reticulum (ER) stress induced by spinal cord injury (SCI) in blood-spinal cord barrier (BSCB) disruption and the effect of phenylbutyrate (PBA) on BSCB disruption after SCI. After a moderate contusion injury at the T9 level of spinal cord with a vascular clip, PBA was immediately administered into injured rat via intraperitoneal injection (100 mg/kg) and then further treated once a day for 2 weeks for behavior test. Spinal cord was collected at 1 day post-injury for evaluation of the effects of ER stress and PBA on BSCB disruption after SCI. PBA significantly attenuated BSCB permeability and degradation of tight junction molecules such as P120, β-catenin, Occludin and Claudin5 at 1 day after injury and improved functional recovery in the rat model of trauma. The BSCB protective effect of PBA is related to the inhibition of ER stress induced by SCI. In addition, PBA significantly inhibited the increase of ER stress markers and prevents loss of tight junction and adherens junction proteins in TG-treated human brain microvascular endothelial cells (HBMEC). Taken together, our data demonstrate that therapeutic strategies targeting ER stress may be suitable for the therapy of preserving BSCB integrity after SCI. PBA may be a new candidate as a therapeutic agent for protecting SCI by a compromised BSCB. PMID:27186310

  3. The endoplasmic reticulum-resident chaperone heat shock protein 47 protects the Golgi apparatus from the effects of O-glycosylation inhibition.

    PubMed

    Miyata, Shingo; Mizuno, Tatsunori; Koyama, Yoshihisa; Katayama, Taiichi; Tohyama, Masaya

    2013-01-01

    The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.

  4. Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

    PubMed Central

    Hoo, Ruby L. C.; Shu, Lingling; Cheng, Kenneth K. Y.; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

    2017-01-01

    Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases. PMID:28094778

  5. The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

    PubMed Central

    Miyata, Shingo; Mizuno, Tatsunori; Koyama, Yoshihisa; Katayama, Taiichi; Tohyama, Masaya

    2013-01-01

    The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria. PMID:23922785

  6. Bilobalide protection of normal human melanocytes from hydrogen peroxide-induced oxidative damage via promotion of antioxidase expression and inhibition of endoplasmic reticulum stress.

    PubMed

    Lu, L; Wang, S; Fu, L; Liu, D; Zhu, Y; Xu, A

    2016-01-01

    H2 O2 accumulating in the epidermis contributes to the melanocyte damage characteristic of vitiligo. Removal of H2 O2 by antioxidants is thus considered beneficial for patients with vitiligo. To investigate the protective effects and underlying mechanism of bilobalide, the main terpenoid constituent of Ginkgo biloba extract, against H2 O2 -induced oxidative damage in normal human melanocytes. Effects of bilobalide on melanocytes were investigated by measuring cell viability, heat shock protein (Hsp)70 release and levels of intracellular reactive oxygen species (ROS). Pretreatment of melanocytes with bilobalide for 24 h significantly inhibited H2 O2 -induced apoptosis, Hsp70 release and intracellular ROS increase in a dose-dependent manner. Bilobalide pretreatment increased the level of catalase (CAT) and glutathione peroxidase (GPx)1 mRNA in melanocytes. Although bilobalide moderately increased the phosphorylation of eukaryotic initiation factor 2, α subunit (eIF2α), suggesting its role in stimulating unfolded protein response signalling pathways, it also blocked the induction of anti-C/EBP homologous protein expression by H2 O2 . This study indicates for the first time that bilobalide protects human melanocytes from oxidative damage by inhibiting H2 O2 -induced apoptosis and suppressing autoimmune response to melanocytes through reducing Hsp70 release. Instead of working as an ROS scavenger, bilobalide promotes the expression of antioxidases including CAT and GPx1, and inhibits H2 O2 -induced endoplasmic reticulum stress to protect melanocytes. © 2015 British Association of Dermatologists.

  7. Inhibition of Endoplasmic Reticulum Stress is Involved in the Neuroprotective Effect of bFGF in the 6-OHDA-Induced Parkinson’s Disease Model

    PubMed Central

    Cai, Pingtao; Ye, Jingjing; Zhu, Jingjing; Liu, Dan; Chen, Daqing; Wei, Xiaojie; Johnson, Noah R.; Wang, Zhouguang; Zhang, Hongyu; Cao, Guodong; Xiao, Jian; Ye, Junming; Lin, Li

    2016-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder with complicated pathophysiologic mechanisms. Endoplasmic reticulum (ER) stress appears to play a critical role in the progression of PD. We demonstrated that basic fibroblast growth factor (bFGF), as a neurotropic factor, inhibited ER stress-induced neuronal cell apoptosis and that 6-hydroxydopamine (6-OHDA)-induced ER stress was involved in the progression of PD in rats. bFGF administration improved motor function recovery, increased tyrosine hydroxylase (TH)-positive neuron survival, and upregulated the levels of neurotransmitters in PD rats. The 6-OHDA-induced ER stress response proteins were inhibited by bFGF treatment. Meanwhile, bFGF also increased expression of TH. The administration of bFGF activated the downstream signals PI3K/Akt and Erk1/2 in vivo and in vitro. Inhibition of the PI3K/Akt and Erk1/2 pathways by specific inhibitors partially reduced the protective effect of bFGF. This study provides new insight towards bFGF translational drug development for PD involving the regulation of ER stress. PMID:27493838

  8. Inhibiting endoplasmic reticulum stress by lithium chloride contributes to the integrity of blood-spinal cord barrier and functional recovery after spinal cord injury

    PubMed Central

    He, Zili; Zhou, Yulong; Wang, Qingqing; Li, Jiawei; Zheng, Zengming; Chen, Jian; Zhang, Hongyu; Wang, Zhouguang; Xu, Huazi; Xiao, Jian

    2017-01-01

    Endoplasmic reticulum (ER) stress play important roles in the spinal cord injury (SCI), which including blood-spinal cord barrier (BSCB) disruption. Lithium chloride (LiCl) is a clinical drug for bipolar mood disorders and contributes to neuroprotection. This study aims to investigate the effects of LiCl on BSCB disruption and the ER stress pathway induced by spinal cord injury. We examined the integrity of the BSCB with Evans Blue dye and macrophages extravasation, measured the microvessels loss, the junction proteins degeneration, the activation ER stress, and the locomotor function recovery. Our data indicated that LiCl treatment could attenuates BSCB disruption and improved the recovery of functional locomotion in rats SCI model, reduced the structure damage and number loss of microvessels, increased the expressions of junction proteins, including p120, β-catenin, occludin, and claudin-5, via reversed the upregulated ER stress associated proteins. In addition, LiCl significantly inhibited the increase of ER stress markers and prevents loss of junction proteins in thapsigargin (TG)-treated human brain microvascular endothelial cells (HBMEC). These findings suggest that LiCl treatment alleviates BSCB disruption and promote the neurological function recovery after SCI, partly through inhibiting the activation of ER stress. PMID:28386329

  9. Squamous Cell Carcinoma Antigen 1 Promotes Caspase-8-Mediated Apoptosis in Response to Endoplasmic Reticulum Stress While Inhibiting Necrosis Induced by Lysosomal Injury▿

    PubMed Central

    Ullman, Erica; Pan, Ji-An; Zong, Wei-Xing

    2011-01-01

    Squamous cell carcinoma antigen 1 (SCCA1) is a member of the serine protease inhibitor (serpin) family of proteins, whose target proteases include the cathepsins. Initially identified as a serological marker for advanced squamous cell carcinomas of the cervix, SCCA1 has also been found to be associated with other cancer types of epithelial or endodermal origins such as lung cancer, head and neck cancer, melanoma, and hepatocellular carcinoma. While the biological function of SCCA1 remains largely unclear, it is believed to limit cellular damage resulting from lysosomal cathepsin release. Here, we show that SCCA1 acts as a molecular switch that inhibits cell death induced by lysosomal injury resulting from DNA alkylating agents and hypotonic shock, whereas it promotes a caspase-8-mediated apoptosis in response to endoplasmic reticulum (ER) stress. In response to ER stress, SCCA1 blocks both lysosomal and proteasomal protein degradation pathways and enhances the interaction between sequestosome 1/p62 and caspase-8, which leads to the aggregation of intracellular caspase-8 and its subsequent cleavage and activation. Hence, on one hand, SCCA1 inhibits cell death induced by lysosomal injury while, on the other hand, it sensitizes cells to ER stress by activating caspase-8 independently of the death receptor apoptotic pathway. PMID:21576355

  10. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders.

    PubMed

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-02-11

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.

  11. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

    PubMed Central

    Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

  12. Protein quality control at the endoplasmic reticulum.

    PubMed

    McCaffrey, Kathleen; Braakman, Ineke

    2016-10-15

    The ER (endoplasmic reticulum) is the protein folding 'factory' of the secretory pathway. Virtually all proteins destined for the plasma membrane, the extracellular space or other secretory compartments undergo folding and maturation within the ER. The ER hosts a unique PQC (protein quality control) system that allows specialized modifications such as glycosylation and disulfide bond formation essential for the correct folding and function of many secretory proteins. It is also the major checkpoint for misfolded or aggregation-prone proteins that may be toxic to the cell or extracellular environment. A failure of this system, due to aging or other factors, has therefore been implicated in a number of serious human diseases. In this article, we discuss several key features of ER PQC that maintain the health of the cellular secretome. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  13. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    PubMed Central

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  14. Endoplasmic-Reticulum Calcium Depletion and Disease

    PubMed Central

    Mekahli, Djalila; Bultynck, Geert; Parys, Jan B.; De Smedt, Humbert; Missiaen, Ludwig

    2011-01-01

    The endoplasmic reticulum (ER) as an intracellular Ca2+ store not only sets up cytosolic Ca2+ signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca2+ depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca2+ may no longer sustain essential cell functions. On the other hand, loss of luminal Ca2+ causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca2+ depletion. PMID:21441595

  15. Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

    PubMed Central

    Lev, Sima

    2012-01-01

    The transport of lipids from their synthesis site at the endoplasmic reticulum (ER) to different target membranes could be mediated by both vesicular and nonvesicular transport mechanisms. Nonvesicular lipid transport appears to be the major transport route of certain lipid species, and could be mediated by either spontaneous lipid transport or by lipid-transfer proteins (LTPs). Although nonvesicular lipid transport has been extensively studied for more than four decades, its underlying mechanism, advantage and regulation, have not been fully explored. In particular, the function of LTPs and their involvement in intracellular lipid movement remain largely controversial. In this article, we describe the pathways by which lipids are synthesized at the ER and delivered to different cellular membranes, and discuss the role of LTPs in lipid transport both in vitro and in intact cells. PMID:23028121

  16. Structural organization of the endoplasmic reticulum

    PubMed Central

    Voeltz, Gia K.; Rolls, Melissa M.; Rapoport, Tom A.

    2002-01-01

    The endoplasmic reticulum (ER) is a continuous membrane system but consists of various domains that perform different functions. Structurally distinct domains of this organelle include the nuclear envelope (NE), the rough and smooth ER, and the regions that contact other organelles. The establishment of these domains and the targeting of proteins to them are understood to varying degrees. Despite its complexity, the ER is a dynamic structure. In mitosis it must be divided between daughter cells and domains must be re-established, and even in interphase it is constantly rearranged as tubules extend along the cytoskeleton. Throughout these rearrangements the ER maintains its basic structure. How this is accomplished remains mysterious, but some insight has been gained from in vitro systems. PMID:12370207

  17. Endoplasmic Reticulum Stress Response in Arabidopsis Roots

    PubMed Central

    Cho, Yueh; Kanehara, Kazue

    2017-01-01

    Roots are the frontier of plant body to perceive underground environmental change. Endoplasmic reticulum (ER) stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on the ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of immunoglobulin-binding protein 3 (BiP3), a representative marker for the response. Roots promptly responded to the TM-induced ER stress through the induction of similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity. PMID:28298914

  18. [Endoplasmic reticulum stress response in osteogenesis].

    PubMed

    Saito, Atsushi; Imaizumi, Kazunori

    2013-11-01

    Various cellular conditions such as synthesis of abundant proteins, expressions of mutant proteins and oxidative stress lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. This type of stress is called ER stress. The excessive ER stress causes cellular damages followed by apoptosis. When ER stress occurs, cells are activated ER stress response (unfolded protein response) to avoid cellular damages. Recently, it has been clear that ER stress response plays crucial roles not only in cell survival after ER stress but also in regulating various cellular functions and tissue formations. In particular, ER stress and ER stress response regulate protein quality control, secretory protein production, and smooth secretion of proteins in the cells such as osteoblasts which synthesize and secrete enormous matrix proteins.

  19. Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

    Saheki, Yasunori; De Camilli, Pietro

    2017-06-20

    The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca(2+) dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

  20. Endoplasmic reticulum stress and intestinal inflammation.

    PubMed

    Kaser, A; Blumberg, R S

    2010-01-01

    The intestinal epithelial cell (IEC) is increasingly recognized to play a prominent role as an important intermediary between the commensal microbiota and the intestinal immune system. Moreover, it is now recognized that intestinal inflammation in inflammatory bowel disease (IBD) may arise primarily from IEC dysfunction due to unresolved endoplasmic reticulum (ER) stress as a consequence of genetic disruption of X box binding protein-1 function. In addition to primary (genetic) abnormalities of the unfolded protein response, a variety of secondary (inflammation and environmental) factors are also likely to be important regulators of ER stress. ER stress pathways are also well known to regulate (and be regulated by) autophagy pathways. Therefore, the host's ability to manage ER stress is likely to be a major pathway in the pathogenesis of intestinal inflammation that arises primarily from the IEC. Herein we discuss ER stress in the IEC as both an originator and perpetuator of intestinal inflammation in IBD.

  1. Endoplasmic Reticulum Stress and Ethanol Neurotoxicity.

    PubMed

    Yang, Fanmuyi; Luo, Jia

    2015-10-14

    Ethanol abuse affects virtually all organ systems and the central nervous system (CNS) is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD) which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER) regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR) which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity.

  2. Inhibition of Glycogen Synthase Kinase 3β Ameliorates D-GalN/LPS-Induced Liver Injury by Reducing Endoplasmic Reticulum Stress-Triggered Apoptosis

    PubMed Central

    Zhang, Haiyan; Wen, Tao; Piao, Zhengfu; Zhou, Li; Zheng, Sujun; Zhang, Jing; Chen, Yu; Han, Yuanping; Duan, Zhongping; Ma, Yingji

    2012-01-01

    Background Glycogen synthase kinase 3β(GSK3β) is a ubiquitous serine-threonine protein kinase that participates in numerous cellular processes and disease pathophysiology. We aimed to determine therapeutic potential of GSK3β inhibition and its mechanism in a well-characterized model of lipopolysaccharide (LPS)-induced model of acute liver failure (ALF). Methodology In a murine ALF model induced by D-GalN(700 mg/kg)/LPS(10 µg/kg), we analyzed GSK3β mechanisms using a specific chemical inhibitor, SB216763, and detected the role of endoplasmic reticulum stress (ERS). Mice were administered SB216763 at 2 h before or after D-GalN/LPS injection, respectively, and then sacrificed 6 h after D-GalN/LPS treatment to evaluate its prophylactic and therapeutic function. The lethality rate, liver damage, ERS, cytokine expression, MAP kinase, hepatocyte apoptosis and expression of TLR 4 were evaluated, respectively. Whether the inhibition of GSK3β activation protected hepatocyte from ERS-induced apoptosis was investigated in vitro. Principal Findings GSK3β became quickly activated (dephosphorylated) upon D-GalN/LPS exposure. Administration of SB216763 not only ameliorated liver injury, as evidenced by reduced transaminase levels, and well-preserved liver architecture, but also decreased lethality. Moreover, GSK3β inhibition resulted in down-regulation of pro-apoptotic proteins C/EBP–homologous protein(CHOP) and caspase-12, which are related to ERS. To further demonstrate the role of ERS, we found that GSK3β inhibition protected hepatocyte from ERS-induced cell death. GSK3β inhibition down-regulated the MAPK pathways, reduced expression of inflammatory cytokines and decreased expression of TLR4. Conclusions Our findings demonstrate the key function of GSK3β signaling in the pathophysiology of ALF, especially in regulating the ERS, and provide a rationale for targeting GSK3β as a potential therapeutic strategy to ameliorate ALF. PMID:23028846

  3. Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells

    PubMed Central

    Xie, Yuxi; He, Yonggui; Cai, Zhiliang; Cai, Jianhang; Xi, Mengyao; Zhang, Yidong; Xi, Jinkun

    2016-01-01

    This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3β) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3β phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3β phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H2O2-induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3β mutant (GSK-3β-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3β inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3β inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3β inactivation. PMID:27904664

  4. Development of Endoplasmic Reticulum Stress during Experimental Oxalate Nephrolithiasis.

    PubMed

    Motin, Yu G; Lepilov, A V; Bgatova, N P; Zharikov, A Yu; Motina, N V; Lapii, G A; Lushnikova, E L; Nepomnyashchikh, L M

    2016-01-01

    Morphological and ultrastructural study of the kidney was performed in rats with oxalate nephrolithiasis. Specific features of endoplasmic reticulum stress were evaluated during nephrolithiasis and treatment with α-tocopherol. We observed the signs of endoplasmic reticulum stress with activation of proapoptotic pathways and injury to the cell lining in nephron tubules and collecting ducts. Ultrastructural changes were found in the organelles, nuclei, and cell membranes of epitheliocytes. A relationship was revealed between endoplasmic reticulum stress and oxidative damage, which developed at the early state of lithogenesis.

  5. Pre-ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress-dependent autophagy via PERK and IRE1 signalings.

    PubMed

    Feng, Dayun; Wang, Bao; Wang, Lei; Abraham, Neeta; Tao, Kai; Huang, Lu; Shi, Wei; Dong, Yushu; Qu, Yan

    2017-04-01

    Melatonin has demonstrated a potential protective effect in central nervous system. Thus, it is interesting to determine whether pre-ischemia melatonin administration could protect against cerebral ischemia/reperfusion (IR)-related injury and the underlying molecular mechanisms. In this study, we revealed that IR injury significantly activated endoplasmic reticulum (ER) stress and autophagy in a middle cerebral artery occlusion mouse model. Pre-ischemia melatonin treatment was able to attenuate IR-induced ER stress and autophagy. In addition, with tandem RFP-GFP-LC3 adeno-associated virus, we demonstrated pre-ischemic melatonin significantly alleviated IR-induced autophagic flux. Furthermore, we showed that IR induced neuronal apoptosis through ER stress related signalings. Moreover, IR-induced autophagy was significantly blocked by ER stress inhibitor (4-PBA), as well as ER-related signaling inhibitors (PERK inhibitor, GSK; IRE1 inhibitor, 3,5-dibromosalicylaldehyde). Finally, we revealed that melatonin significantly alleviated cerebral infarction, brain edema, neuronal apoptosis, and neurological deficiency, which were remarkably abolished by tunicamycin (ER stress activator) and rapamycin (autophagy activator), respectively. In summary, our study provides strong evidence that pre-ischemia melatonin administration significantly protects against cerebral IR injury through inhibiting ER stress-dependent autophagy. Our findings shed light on the novel preventive and therapeutic strategy of daily administration of melatonin, especially among the population with high risk of cerebral ischemic stroke. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Gypenoside L inhibits autophagic flux and induces cell death in human esophageal cancer cells through endoplasm reticulum stress-mediated Ca2+ release

    PubMed Central

    Li, Yan; Xu, Hong; Kang, Qiangrong; Fan, Long; Hu, Xiaopeng; Jin, Zhe; Zeng, Yong; Kong, Xiaoli; Zhang, Jian; Wu, Xuli; Wu, Haiqiang; Liu, Lizhong; Xiao, Xiaohua; Wang, Yifei; He, Zhendan

    2016-01-01

    Esophageal cancer is one of the leading cause of cancer mortality in the world. Due to the increased drug and radiation tolerance, it is urgent to develop novel anticancer agent that triggers nonapoptotic cell death to compensate for apoptosis resistance. In this study, we show that treatment with gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, induced nonapoptotic, lysosome-associated cell death in human esophageal cancer cells. Gyp-L-induced cell death was associated with lysosomal swelling and autophagic flux inhibition. Mechanistic investigations revealed that through increasing the levels of intracellular reactive oxygen species (ROS), Gyp-L triggered protein ubiquitination and endoplasm reticulum (ER) stress response, leading to Ca2+ release from ER inositol trisphosphate receptor (IP3R)-operated stores and finally cell death. Interestingly, there existed a reciprocal positive-regulatory loop between Ca2+ release and ER stress in response to Gyp-L. In addition, protein synthesis was critical for Gyp-L-mediated ER stress and cell death. Taken together, this work suggested a novel therapeutic option by Gyp-L through the induction of an unconventional ROS-ER-Ca2+-mediated cell death in human esophageal cancer. PMID:27329722

  7. Inhibition of GADD34, the stress-inducible regulatory subunit of the endoplasmic reticulum stress response, does not enhance functional recovery after spinal cord injury.

    PubMed

    Ohri, Sujata Saraswat; Mullins, Ashley; Hetman, Michal; Whittemore, Scott R

    2014-01-01

    Activation of the endoplasmic reticulum stress response (ERSR) is a hallmark of various pathological diseases and/or traumatic injuries. Restoration of ER homeostasis can contribute to improvement in the functional outcome of these diseases. Using genetic and pharmacological inhibition of the PERK-CHOP arm of the ERSR, we recently demonstrated improvements in hindlimb locomotion after spinal cord injury (SCI) and implicated oligodendrocyte survival as a potential mechanism. Here, we investigated the contribution of stress-inducible PPP1R15A/GADD34, an ERSR signaling effector downstream of CHOP that dephosphorylates eIF2α, in the pathogenesis of SCI. We show that although genetic ablation of GADD34 protects oligodendrocyte precursor cells (OPCs) against ER stress-mediated cell death in vitro and results in differential ERSR attenuation in vivo after SCI, there is no improvement in hindlimb locomotor function. Guanabenz, a FDA approved antihypertensive drug, was recently shown to reduce the burden of misfolded proteins in the ER by directly targeting GADD34. Guanabenz protected OPCs from ER stress-mediated cell death in vitro and attenuated the ERSR in vivo after SCI. However, guanabenz administration failed to rescue the locomotor deficits after SCI. These data suggest that deletion of GADD34 alone is not sufficient to improve functional recovery after SCI.

  8. Inhibition of Cellular Protein Secretion by Norwalk Virus Nonstructural Protein p22 Requires a Mimic of an Endoplasmic Reticulum Export Signal

    PubMed Central

    Sharp, Tyler M.; Guix, Susana; Katayama, Kazuhiko; Crawford, Sue E.; Estes, Mary K.

    2010-01-01

    Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development. PMID:20976190

  9. Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4.

    PubMed

    Warfield, Kelly L; Plummer, Emily M; Sayce, Andrew C; Alonzi, Dominic S; Tang, William; Tyrrell, Beatrice E; Hill, Michelle L; Caputo, Alessandro T; Killingbeck, Sarah S; Beatty, P Robert; Harris, Eva; Iwaki, Ren; Kinami, Kyoko; Ide, Daisuke; Kiappes, J L; Kato, Atsushi; Buck, Michael D; King, Kevin; Eddy, William; Khaliq, Mansoora; Sampath, Aruna; Treston, Anthony M; Dwek, Raymond A; Enterlein, Sven G; Miller, Joanna L; Zitzmann, Nicole; Ramstedt, Urban; Shresta, Sujan

    2016-05-01

    The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease.

  10. Protective effect of S-allyl-L-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.

    PubMed

    Imai, Toru; Kosuge, Yasuhiro; Endo-Umeda, Kaori; Miyagishi, Hiroko; Ishige, Kumiko; Makishima, Makoto; Ito, Yoshihisa

    2014-02-01

    Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca(2+) and leads to activation of calpain, a Ca(2+)-dependent cysteine protease. We have shown previously that S-allyl-L-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, L-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 μM), which prevents interaction of Ca(2+) with the Ca(2+)-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca(2+)-binding site.

  11. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  12. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  13. Wogonin induces apoptosis and endoplasmic reticulum stress in HL-60 leukemia cells through inhibition of the PI3K-AKT signaling pathway.

    PubMed

    Hu, Chengjun; Xu, Maozhong; Qin, Rujuan; Chen, Weifeng; Xu, Xin

    2015-06-01

    Wogonin is a flavonoid isolated from Scutellaria baicalensis root and has multiple pharmacological effects, including anticancer effects. Recent studies have shown that wogonin induces cell cycle arrest and reverses multi-drug resistance in the human K562 leukemia cell line. However, its pharmacological function in the apoptosis of leukemia cells remains unknown. Therefore, we hypothesized that wogonin can induce apoptosis in the HL-60 leukemia cell line. In the present study, the HL-60 cells were treated with different doses of wogonin (0-150 µM). Wogonin inhibited the viability of HL-60 cells in a dose-dependent and time-dependent manner. Flow cytometry and analyses of caspase and PARP-1 activation and the Bax/Bcl-2 ratio, demonstrated that the cytotoxic effect of wogonin on HL-60 cells was mediated by caspase-dependent and mitochondrial-dependent apoptosis. Wogonin also induced the expression of certain members of the endoplasmic reticulum (ER) stress pathway (CHOP, GRP94 and GRP78) and the activation of multiple branches of ER stress transducers (IRE1α, PERK-eIF2α and ATF6) in the HL-60 cells. In addition, wogonin reduced the phosphorylation of PI3K and AKT in the HL-60 cells. Furthermore, constitutive activation of AKT induced by adenoviral vectors inhibited the pro-apoptotic effects and ER stress induced by wogonin in the HL-60 cells. In summary, our results indicated that wogonin induced apoptosis and ER stress in HL-60 cells, which was mediated by the inhibition of the PI3K-AKT signaling pathway.

  14. Intermedin1-53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway.

    PubMed

    Chang, Jin-Rui; Duan, Xiao-Hui; Zhang, Bao-Hong; Teng, Xu; Zhou, Ye-Bo; Liu, Yue; Yu, Yan-Rong; Zhu, Yi; Tang, Chao-Shu; Qi, Yong-Fen

    2013-10-01

    We previously reported that endoplasmic reticulum (ER) stress-mediated apoptosis participated in vascular calcification. Importantly, a novel paracrine/autocrine peptide intermedin1-53 (IMD1-53) in the vasculature inhibited vascular calcification in rats. But the mechanisms needed to be fully elucidated. Vascular smooth muscle cells (VSMCs) calcification was induced by CaCl2 and β-glycerophosphate. Tunicamycin (Tm) or dithiothreitol (DTT) was used to induce ER stress. We found that IMD1-53 (10(-7)mol/L) treatment significantly alleviated the protein expression of ER stress hallmarks activating transcription factor 4 (ATF4), ATF6, glucose-regulated protein 78 (GRP78) and GRP94 induced by Tm or DTT. ER stress occurred in early and late calcification of VSMCs but was inhibited by IMD1-53. These inhibitory effects of IMD1-53 were abolished by treatment with the protein kinase A (PKA) inhibitor H89. Pretreatment with IMD1-53 decreased the number of apoptotic VSMCs and downregulated protein expression of cleaved caspase 12 and C/EBP homologous protein (CHOP) in calcified VSMCs. Concurrently, IMD1-53 restored the loss of VSMC lineage markers and ameliorated calcium deposition and alkaline phosphatase activity in calcified VSMCs as well. The observation was further verified by Alizarin Red S staining, which showed that IMD1-53 reduced positive red nodules among calcified VSMCs. In conclusion, IMD1-53 attenuated VSMC calcification by inhibiting ER stress through cAMP/PKA signalling.

  15. Intermedin1–53 Protects Against Myocardial Fibrosis by Inhibiting Endoplasmic Reticulum Stress and Inflammation Induced by Homocysteine in Apolipoprotein E-Deficient Mice

    PubMed Central

    Zhang, Jin-Sheng; Hou, Yue-Long; Lu, Wei-Wei; Ni, Xian-Qiang; Lin, Fan; Yu, Yan-Rong; Tang, Chao-Shu

    2016-01-01

    Aim: Endoplasmic reticulum stress (ERS) and inflammation participate in cardiac fibrosis. Importantly, a novel paracrine/autocrine peptide intermedin1–53 (IMD1–53) in the heart inhibits myocardial fibrosis in rats. However, the mechanisms are yet to be fully elucidated. Methods: Myocardial fibrosis in apolipoprotein E-deficient (ApoE -/-) mice and neonatal rat cardiac fibroblasts (CFs) were induced using homocysteine (Hcy). Results: IMD1–53 inhibited myocardial fibrosis in vivo and in vitro. Picrosirius red staining showed that IMD1–53 reduced myocardial interstitial collagen deposition in ApoE-/- mice treated with Hcy and decreased the expression of myocardial collagen I and III, which was further verified in rat CFs. IMD1–53 attenuated myocardial hypertrophy, as shown by cardiomyocyte cross-sectional area, ratio of heart weight to body weight, and mRNA levels of atrial natriuretic peptide and brain natriuretic peptide. IMD1–53 inhibited the upregulation of ERS hallmarkers such as glucose-regulated protein 78 (GRP78), GRP94, activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1α, spliced-X-box-binding protein-1, protein kinase receptor-like ER kinase, and eukaryotic translation initiation factor 2α in mouse myocardium and rat CFs treated with Hcy. In addition, IMD1–53 decreased the production of inflammatory factors such as tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-1β in the mouse myocardium and rat CFs treated with Hcy. Concurrently, IMD1–53 ameliorated the expression of nuclear factor-κB, transforming growth factor-β1, and c-Jun N-terminal kinase in the mouse myocardium and rat CFs treated with Hcy. Conclusions: IMD potentially protects against myocardial fibrosis induced by Hcy in ApoE-/- mice, possibly via attenuating myocardial ERS and inflammation. PMID:27052784

  16. Endoplasmic Reticulum Stress Regulates Adipocyte Resistin Expression

    PubMed Central

    Lefterova, Martina I.; Mullican, Shannon E.; Tomaru, Takuya; Qatanani, Mohammed; Schupp, Michael; Lazar, Mitchell A.

    2009-01-01

    OBJECTIVE Resistin is a secreted polypeptide that impairs glucose metabolism and, in rodents, is derived exclusively from adipocytes. In murine obesity, resistin circulates at elevated levels but its gene expression in adipose tissue is paradoxically reduced. The mechanism behind the downregulation of resistin mRNA is poorly understood. We investigated whether endoplasmic reticulum (ER) stress, which is characteristic of obese adipose tissue, regulates resistin expression in cultured mouse adipocytes. RESEARCH DESIGN AND METHODS The effects of endoplasmic stress inducers on resistin mRNA and secreted protein levels were examined in differentiated 3T3-L1 adipocytes, focusing on the expression and genomic binding of transcriptional regulators of resistin. The association between downregulated resistin mRNA and induction of ER stress was also investigated in the adipose tissue of mice fed a high-fat diet. RESULTS ER stress reduced resistin mRNA in 3T3-L1 adipocytes in a time- and dose-dependent manner. The effects of ER stress were transcriptional because of downregulation of CAAT/enhancer binding protein-α and peroxisome proliferator–activated receptor-γ transcriptional activators and upregulation of the transcriptional repressor CAAT/enhancer binding protein homologous protein-10 (CHOP10). Resistin protein was also substantially downregulated, showing a close correspondence with mRNA levels in 3T3-L1 adipocytes as well as in the fat pads of obese mice. CONCLUSIONS ER stress is a potent regulator of resistin, suggesting that ER stress may underlie the local downregulation of resistin mRNA and protein in fat in murine obesity. The paradoxical increase in plasma may be because of various systemic abnormalities associated with obesity and insulin resistance. PMID:19491212

  17. Diabetes: Targeting endoplasmic reticulum to combat juvenile diabetes.

    PubMed

    Urano, Fumihiko

    2014-03-01

    Limited options for clinical management of patients with juvenile-onset diabetes mellitus call for a novel therapeutic paradigm. Two innovative studies support endoplasmic reticulum as an emerging target for combating both autoimmune and heritable forms of this disease.

  18. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  19. Nox NADPH oxidases and the endoplasmic reticulum.

    PubMed

    Laurindo, Francisco R M; Araujo, Thaís L S; Abrahão, Thalita B

    2014-06-10

    Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology.

  20. Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

    PubMed

    Tufi, R; Panaretakis, T; Bianchi, K; Criollo, A; Fazi, B; Di Sano, F; Tesniere, A; Kepp, O; Paterlini-Brechot, P; Zitvogel, L; Piacentini, M; Szabadkai, G; Kroemer, G

    2008-02-01

    Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

  1. Equol Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice by Inhibiting Endoplasmic Reticulum Stress via Activation of Nrf2 in Endothelial Cells

    PubMed Central

    Shi, Linying; Qin, Li; Zhang, Qianyong; Mi, Mantian

    2016-01-01

    The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway. PMID:27907038

  2. The effect of inhibition of endoplasmic reticulum stress on lipolysis in white adipose tissue in a rat model of chronic kidney disease

    PubMed Central

    Zhu, Yan; Chen, Yu-ling; Li, Cong; Ding, Xiao-yan; Xu, Guo-yu; Hu, Li-li; Hou, Fan-fan; Zhou, Qiu-gen

    2014-01-01

    Aim: Lipolysis in fat tissue plays an important role in the development of metabolic disturbances, a characteristic feature of chronic kidney disease (CKD). In the present study, we tested the hypothesis that the inhibition of endoplasmic reticulum (ER) stress could alleviate lipolysis in white adipose tissue in a rat model of CKD. Methods: A rat model of CKD was established by a method of reduced renal mass (RRM). Lipolysis was measured as the release of glycerol in ex vivo fat pads and cultured primary adipocytes. The activity of lipases and markers of ER stress were measured by Western blotting and immunoprecipitation. Results: Our data showed that lipolysis in visceral white adipose tissue was increased in RRM rats compared with control rats. In addition, increased phosphorylation of hormone-sensitive lipase (HSL) and binding of adipose triglyceride lipase (ATGL) to comparative gene identification-58 (CGI-58) protein were observed in the RRM rats. The phosphorylation of ER stress markers, including IRE1α, PERK, and eukaryotic initiation factor (eIF) 2α, and the expression of ER stress marker 78 kDa glucose-regulated protein (GRP78) were significantly increased in RRM rats. Treatment with an inhibitor of ER stress partially but significantly alleviated lipolysis, and this alleviation was accompanied by reduced binding of ATGL to CGI-58. Conclusion: Our results showed that enhanced lipolysis and ER stress occurred in visceral white adipose tissue in a rat model of CKD. Moreover, inhibition of ER stress significantly alleviated lipolysis. These findings suggest that ER stress is a potential therapeutic target for the metabolic disturbances associated with CKD. PMID:24442147

  3. The effect of inhibition of endoplasmic reticulum stress on lipolysis in white adipose tissue in a rat model of chronic kidney disease.

    PubMed

    Zhu, Yan; Chen, Yu-ling; Li, Cong; Ding, Xiao-yan; Xu, Guo-yu; Hu, Li-li; Hou, Fan-fan; Zhou, Qiu-gen

    2014-03-01

    Lipolysis in fat tissue plays an important role in the development of metabolic disturbances, a characteristic feature of chronic kidney disease (CKD). In the present study, we tested the hypothesis that the inhibition of endoplasmic reticulum (ER) stress could alleviate lipolysis in white adipose tissue in a rat model of CKD. A rat model of CKD was established by a method of reduced renal mass (RRM). Lipolysis was measured as the release of glycerol in ex vivo fat pads and cultured primary adipocytes. The activity of lipases and markers of ER stress were measured by Western blotting and immunoprecipitation. Our data showed that lipolysis in visceral white adipose tissue was increased in RRM rats compared with control rats. In addition, increased phosphorylation of hormone-sensitive lipase (HSL) and binding of adipose triglyceride lipase (ATGL) to comparative gene identification-58 (CGI-58) protein were observed in the RRM rats. The phosphorylation of ER stress markers, including IRE1α, PERK, and eukaryotic initiation factor (eIF) 2α, and the expression of ER stress marker 78 kDa glucose-regulated protein (GRP78) were significantly increased in RRM rats. Treatment with an inhibitor of ER stress partially but significantly alleviated lipolysis, and this alleviation was accompanied by reduced binding of ATGL to CGI-58. Our results showed that enhanced lipolysis and ER stress occurred in visceral white adipose tissue in a rat model of CKD. Moreover, inhibition of ER stress significantly alleviated lipolysis. These findings suggest that ER stress is a potential therapeutic target for the metabolic disturbances associated with CKD.

  4. Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner.

    PubMed

    Li, Yi; Yang, Jie; Chen, Mei-Hong; Wang, Qiang; Qin, Min-Jian; Zhang, Tong; Chen, Xiao-Qing; Liu, Bao-Lin; Wen, Xiao-Dong

    2015-09-01

    Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endothelial homeostasis and its mechanisms. Palmitate (PA) stimulation induced endoplasmic reticulum stress (ER stress) and subsequent thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in endothelial cells, leading to endothelial dysfunction. Ilexgenin A enhanced LKB1-dependent AMPK activity and improved ER stress by suppression of ROS-associated TXNIP induction. However, these effects were blocked by knockdown of AMPKα, indicating AMPK is essential for its action in suppression of ER stress. Meanwhile, ilexgenin A inhibited NLRP3 inflammasome activation by down-regulation of NLRP3 and cleaved caspase-1 induction, and thereby reduced IL-1β secretion. It also inhibited inflammation and apoptosis exposed to PA insult. Consistent with these results in endothelial cells, ilexgenin A attenuated ER stress and restored the loss of eNOS activity in vascular endothelium, and thereby improved endothelium-dependent vasodilation in rat aorta. A further analysis in high-fat fed mice showed that oral administration of ilexgenin A blocked ER stress/NLRP3 activation with reduced ROS generation and increased NO production in vascular endothelium, well confirming the beneficial effect of ilexgenin A on endothelial homeostasis in vivo. Taken together, these results show ER stress-associated TXNIP/NLRP3 inflammasome activation was responsible for endothelial dysfunction and ilexgenin A ameliorated endothelial dysfunction by suppressing ER-stress and TXNIP/NLRP3 inflammasome activation with a regulation of AMPK. This finding suggests that the application of ilexgenin A is useful in the management of cardiovascular diseases in obesity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Dl-3-n-butylphthalide improves functional recovery in rats with spinal cord injury by inhibiting endoplasmic reticulum stress-induced apoptosis

    PubMed Central

    He, Zili; Zhou, Yulong; Huang, Yan; Wang, Qingqing; Zheng, Binbin; Zhang, Hongyu; Li, Jiawei; Liu, Yanlong; Wu, Fenzan; Zhang, Xie; Tong, Songlin; Wang, Maofeng; Wang, Zhouguang; He, Huacheng; Xu, Huazi; Xiao, Jian

    2017-01-01

    Endoplasmic reticulum (ER) stress-induced apoptosis occurs in the spinal cord following traumatic spinal cord injury (SCI). Dl-3-n-butylphthalide (NBP) exerts an neuroprotective effects against both ischemic brain injury and neurodegenerative diseases; however, the relationship between ER stress-induced apoptosis and the therapeutic effect of NBP in SCI remains unclear. In this study, moderate spinal cord injuries were induced in Sprague-Dawley (SD) rats with a vascular clip. NBP was administered by oral (80 mg/kg/d) gavage 2 h before injury and then once daily for 28 d thereafter. Neurological recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotion rating scale, the inclined plane test, and the footprint analysis. Neuronal cell death was examined by TUNEL staining at 7 days post-injury. ER stress and apoptosis-related proteins were quantified by immunofluorescence staining and western blotting both in vivo and in vitro. Our results showed that NBP significantly decreased spinal cord lesion cavity area and improved locomotor recovery in SD rats after SCI. NBP also decreased neuronal apoptosis and inhibited activation of the caspase 3 cascade. Upregulation of ER stress-related proteins, such as GRP78, ATF-6, ATF-4, PDI, XBP-1, and CHOP, was reversed by NBP treatment in SD rats with SCI. Similarly, NBP effectively ameliorated ER stress and apoptosis-related protein expression induced by incubation with thapsigargin (TG) in PC12 cells. Our findings demonstrate that NBP treatment alleviates secondary SCI by inhibiting ER stress-induced apoptosis, thereby promoting neurological and locomoter functional recovery. PMID:28386335

  6. Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes.

    PubMed

    Yang, Fan; Yu, Xiangjing; Li, Ting; Wu, Jianjun; Zhao, Yajun; Liu, Jiaqi; Sun, Aili; Dong, Shiyun; Wu, Jichao; Zhong, Xin; Xu, Changqing; Lu, Fanghao; Zhang, Weihua

    2017-03-01

    The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome c (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression. Copyright © 2017 the American Physiological Society.

  7. Transcriptional Regulation of the Ufm1 Conjugation System in Response to Disturbance of the Endoplasmic Reticulum Homeostasis and Inhibition of Vesicle Trafficking

    PubMed Central

    Zhang, Yinghua; Zhang, Mingsheng; Wu, Jianchun; Lei, Guohua; Li, Honglin

    2012-01-01

    Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network. PMID:23152784

  8. Transcriptional regulation of the Ufm1 conjugation system in response to disturbance of the endoplasmic reticulum homeostasis and inhibition of vesicle trafficking.

    PubMed

    Zhang, Yinghua; Zhang, Mingsheng; Wu, Jianchun; Lei, Guohua; Li, Honglin

    2012-01-01

    Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network.

  9. Inhibition of microsomal cortisol production by (-)-epigallocatechin-3-gallate through a redox shift in the endoplasmic reticulum--a potential new target for treating obesity-related diseases.

    PubMed

    Szelényi, Péter; Révész, Katalin; Konta, Laura; Tüttõ, Anna; Mandl, József; Kereszturi, Éva; Csala, Miklós

    2013-01-01

    Conversion of cortisone to cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in the endoplasmic reticulum (ER) of the target cells is a major determinant of glucocorticoid action, and plays an important role in the development of obesity-related diseases. Inhibition of 11βHSD1 activity is, therefore, considered as a promising novel strategy for the treatment of metabolic syndrome and diabetes. Tea flavanols and their major representative, epigallocatechin gallate are known as antiobesity and antidiabetic agents. Their impacts on blood glucose level, hepatic glucose production, and insulin responsiveness resemble those observed on inhibition or depletion of 11βHSD1. We aimed to study the effect of epigallocatechin gallate on 11βHSD1 activity in ER-derived rat liver microsomes by measuring cortisone and cortisol with HPLC. Cortisol production was efficiently suppressed in a concentration dependent manner in intact microsomal vesicles. However, this effect was abolished by membrane permeabilization; and the three proteins involved in the overall process (11βHSD1, hexose 6-phosphate dehydrogenase, and glucose 6-phosphate transporter) were not or only mildly affected. Further investigation revealed the oxidation of luminal NADPH to NADP⁺, which attenuates cortisone reduction and favors cortisol oxidation in this compartment. Such a redox shift in the ER lumen might contribute to the beneficial health effects of tea flavanols and should be regarded as a promising strategy for the development of novel selective 11βHSD1 inhibitors to treat obesity-related diseases.

  10. 4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.

    PubMed

    Liu, Liangming; Wu, Huiling; Zang, JiaTao; Yang, Guangming; Zhu, Yu; Wu, Yue; Chen, Xiangyun; Lan, Dan; Li, Tao

    2016-08-01

    Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. Laboratory investigation. State Key Laboratory of Trauma, Burns and Combined Injury. Sprague-Dawley rats. Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. Anti-endoplasmic

  11. Endoplasmic reticulum stress in periimplantation embryos.

    PubMed

    Michalak, Marek; Gye, Myung Chan

    2015-03-01

    Stress coping mechanisms are critical to minimize or overcome damage caused by ever changing environmental conditions. They are designed to promote cell survival. The unfolded protein response (UPR) pathway is mobilized in response to the accumulation of unfolded proteins, ultimately in order to regain endoplasmic reticulum (ER) homeostasis. Various elements of coping responses to ER stress including Perk, Ask1, Bip, Chop, Gadd34, Ire1, Atf4, Atf6, and Xbp1 have been identified and were found to be inducible in oocytes and preimplantation embryos, suggesting that, as a normal part of the cellular adaptive mechanism, these coping responses, including the UPR, play a pivotal role in the development of preimplantation embryos. As such, the UPR-associated molecules and pathways may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos. After implantation, ER stress-induced coping responses become physiologically important for a normal decidual response, placentation, and early organogenesis. Attenuation of ER stress coping responses by tauroursodeoxycholate and salubrinal was effective for prevention of cell death of cultured embryos. Further elucidation of new and relevant ER stress coping responses in periimplantation embryos might contribute to a comprehensive understanding of the regulation of normal development of embryonic development and potentiation of embryonic development in vitro.

  12. Endoplasmic reticulum stress causes EBV lytic replication

    PubMed Central

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K.; Rowe, David T.; Wadowsky, Robert M.

    2011-01-01

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress–dependent and –independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress. PMID:21849482

  13. Epidermal growth factor receptor inhibition slows progression of diabetic nephropathy in association with a decrease in endoplasmic reticulum stress and an increase in autophagy.

    PubMed

    Zhang, Ming-Zhi; Wang, Yinqui; Paueksakon, Paisit; Harris, Raymond C

    2014-06-01

    Previous studies by us and others have reported renal epidermal growth factor receptors (EGFRs) are activated in models of diabetic nephropathy. In the present study, we examined the effect of treatment with erlotinib, an inhibitor of EGFR tyrosine kinase activity, on the progression of diabetic nephropathy in a type 1 diabetic mouse model. Inhibition of renal EGFR activation by erlotinib was confirmed by decreased phosphorylation of EGFR and extracellular signal-related kinase 1/2. Increased albumin/creatinine ratio in diabetic mice was markedly attenuated by erlotinib treatment. Erlotinib-treated animals had less histological glomerular injury as well as decreased renal expression of connective tissue growth factor and collagens I and IV. Autophagy plays an important role in the pathophysiology of diabetes mellitus, and impaired autophagy may lead to increased endoplasmic reticulum (ER) stress and subsequent tissue injury. In diabetic mice, erlotinib-treated mice had evidence of increased renal autophagy, as indicated by altered expression and activity of ATG12, beclin, p62, and LC3A II, hallmarks of autophagy, and had decreased ER stress, as indicated by decreased expression of C/EBP homologous protein, binding immunoglobulin protein, and protein kinase RNA-like ER kinase. The mammalian target of rapamycin (mTOR) pathway, a key factor in the development of diabetic nephropathy and an inhibitor of autophagy, is inhibited by AMP-activated protein kinase (AMPK) activation. Erlotinib-treated mice had activated AMPK and inhibition of the mTOR pathway, as evidenced by decreased phosphorylation of raptor and mTOR and the downstream targets S6 kinase and eukaryotic initiation factor 4B. Erlotinib also led to AMPK-dependent phosphorylation of Ulk1, an initiator of mammalian autophagy. These studies demonstrate that inhibition of EGFR with erlotinib attenuates the development of diabetic nephropathy in type 1 diabetes, which is mediated at least in part by inhibition of m

  14. Caffeic acid attenuates the inflammatory stress induced by glycated LDL in human endothelial cells by mechanisms involving inhibition of AGE-receptor, oxidative, and endoplasmic reticulum stress.

    PubMed

    Toma, Laura; Sanda, Gabriela M; Niculescu, Loredan S; Deleanu, Mariana; Stancu, Camelia S; Sima, Anca V

    2017-07-28

    Type 2 diabetes mellitus is a worldwide epidemic and its atherosclerotic complications determine the high morbidity and mortality of diabetic patients. Caffeic acid (CAF), a phenolic acid present in normal diets, is known for its antioxidant properties. The aim of this study was to investigate CAF's anti-inflammatory properties and its mechanism of action, using cultured human endothelial cells (HEC) incubated with glycated low-density lipoproteins (gLDL). Levels of the receptor for advanced glycation end-products (RAGE), inflammatory stress markers (C reactive protein, CRP; vascular cell adhesion molecule-1, VCAM-1; monocyte chemoattractant protein-1, MCP-1), and oxidative stress and endoplasmic reticulum stress (ERS) markers were evaluated in gLDL-exposed HEC, in the presence/absence of CAF. RAGE silencing or blocking, specific inhibitors for oxidative stress (apocynin, N-acetyl-cysteine), and ERS (salubrinal) were used. The results showed that: (i) gLDL induced CRP synthesis and secretion through mechanisms involving NADPH oxidase-dependent oxidative stress and ERS in HEC; (ii) gLDL-RAGE interaction, oxidative stress, and ERS stimulated the secretion of VCAM-1 and MCP-1 in HEC; and (iii) CAF reduced the secretion of CRP, VCAM-1, and MCP-1 in gLDL-exposed HEC by inhibiting RAGE expression, oxidative stress, and ERS. In conclusion, CAF might be a promising alternative to ameliorate a wide spectrum of disorders due to its complex mechanisms of action resulting in anti-inflammatory and antioxidative properties. © 2017 BioFactors, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  15. Fumonisin B1 Inhibits Endoplasmic Reticulum Stress Associated-apoptosis After FoscanPDT Combined with C6-Pyridinium Ceramide or Fenretinide.

    PubMed

    Boppana, Nithin B; Kraveka, Jacqueline M; Rahmaniyan, Mehrdad; Li, L I; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Delor, Jeremy S; Zhang, Kezhong; Korbelik, Mladen; Separovic, Duska

    2017-02-01

    Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  16. Tauroursodeoxycholic Acid Attenuates Angiotensin II Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-deficient Mice by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Qin, Y; Wang, Y; Liu, O; Jia, L; Fang, W; Du, J; Wei, Y

    2017-03-01

    Abdominal aortic aneurysm (AAA) is characterised by the infiltration of smooth muscle cell (SMC) apoptosis, inflammatory cells, neovascularisation, and degradation of the extracellular matrix. Previous work has shown that endoplasmic reticulum (ER) stress and SMC apoptosis were increased both in a mouse model and human thoracic aortic aneurysm. However, whether the ER stress is activated in AAA formation and whether suppressing ER stress attenuates AAA is unknown. Human AAA and control aorta samples were collected. Expression of ER stress chaperones glucose-regulated protein (GRP)-78 and GRP-94 was detected by immunohistochemical staining. The effect of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) on AAA formation in angiotensin (Ang) II induced apolipoprotein E(-/-) mice was explored. Elastin staining was used to observe the rupture of elastic fragmentation. Immunohistochemistry and Western blot analysis were performed, to detect the protein expression of ER stress chaperones and apoptosis molecules. There was significant upregulation of GRP-78 and GRP-94 in aneurysmal areas of human AAA and Ang II induced ApoE(-/-) mice (p < .05). TUDCA significantly attenuated the maximum diameters of abdominal aortas in Ang II induced ApoE(-/-) mice (p < .05). TUDCA significantly reduced expression of ER stress chaperones and the apoptotic cell numbers (p < .05). Furthermore, TUDCA significantly reduced expression of apoptosis molecules, such as caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminal kinase activating transcription factor 4, X-box binding protein, and eukaryotic initiation factor 2α in Ang II induced ApoE(-/-) mice (p < .05). The results suggest that ER stress is involved in human and Ang II induced AAA formation in ApoE(-/-) mice. TUDCA attenuates Ang II induced AAA formation in ApoE(-/-) mice by inhibiting ER stress mediated apoptosis. Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights

  17. Inhibition of brain mitogen-activated protein kinase signaling reduces central endoplasmic reticulum stress and inflammation and sympathetic nerve activity in heart failure rats

    PubMed Central

    Wei, Shun-Guang; Yu, Yang; Weiss, Robert M.; Felder, Robert B.

    2015-01-01

    Mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress in the brain have been implicated in the pathophysiological mechanisms in hypertension. The present study determined whether ER stress occurs in subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN) in heart failure (HF), and how MAPK signaling interacts with ER stress and other inflammatory mediators. HF rats had significantly higher levels of the ER stress biomarkers (GRP78, ATF6, ATF4, XBP-1, P58IPK and CHOP) in SFO and PVN, which were attenuated by a 4-week intracerebroventricular (ICV) infusion of inhibitors selective for p44/42 MAPK (PD98059), p38 MAPK (SB203580) or JNK (SP600125). HF rats also had higher mRNA levels of tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2 and NF-κB p65 and lower mRNA level of IκB-α in SFO and PVN, compared with SHAM rats, and these indicators of increased inflammation were attenuated in the HF rats treated with the MAPK inhibitors. Plasma norepinephrine level was higher in HF than SHAM rats, but was reduced in the HF rats treated with PD98059 and SB203580. A 4-week ICV infusion of PD98059 also improved some hemodynamic and anatomic indicators of left ventricular function in HF rats. These data demonstrate that ER stress increases in the SFO and PVN of rats with ischemia-induced HF, and that inhibition of brain MAPK signaling reduces brain ER stress and inflammation and decreases sympathetic excitation in HF. An interaction between MAPK signaling and ER stress in cardiovascular regions of the brain may contribute to the development of HF. PMID:26573710

  18. Inhibition of Brain Mitogen-Activated Protein Kinase Signaling Reduces Central Endoplasmic Reticulum Stress and Inflammation and Sympathetic Nerve Activity in Heart Failure Rats.

    PubMed

    Wei, Shun-Guang; Yu, Yang; Weiss, Robert M; Felder, Robert B

    2016-01-01

    Mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress in the brain have been implicated in the pathophysiology of hypertension. This study determined whether ER stress occurs in subfornical organ and hypothalamic paraventricular nucleus in heart failure (HF) and how MAPK signaling interacts with ER stress and other inflammatory mediators. HF rats had significantly higher levels of the ER stress biomarkers (glucose-regulated protein 78, activating transcription factor 6, activating transcription factor 4, X-box binding protein 1, P58(IPK), and C/EBP homologous protein) in subfornical organ and paraventricular nucleus, which were attenuated by a 4-week intracerebroventricular infusion of inhibitors selective for p44/42 MAPK (PD98059), p38 MAPK (SB203580), or c-Jun N-terminal kinase (SP600125). HF rats also had higher mRNA levels of tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and nuclear factor-κB p65, and a lower mRNA level of IκB-α, in subfornical organ and paraventricular nucleus, compared with SHAM rats, and these indicators of increased inflammation were attenuated in the HF rats treated with the MAPK inhibitors. Plasma norepinephrine level was higher in HF rats than in SHAM rats but was reduced in the HF rats treated with PD98059 and SB203580. A 4-week intracerebroventricular infusion of PD98059 also improved some hemodynamic and anatomic indicators of left ventricular function in HF rats. These data demonstrate that ER stress increases in the subfornical organ and paraventricular nucleus of rats with ischemia-induced HF and that inhibition of brain MAPK signaling reduces brain ER stress and inflammation and decreases sympathetic excitation in HF. An interaction between MAPK signaling and ER stress in cardiovascular regions of the brain may contribute to the development of HF. © 2015 American Heart Association, Inc.

  19. Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

    PubMed

    Sbiera, Silviu; Leich, Ellen; Liebisch, Gerhard; Sbiera, Iuliu; Schirbel, Andreas; Wiemer, Laura; Matysik, Silke; Eckhardt, Carolin; Gardill, Felix; Gehl, Annemarie; Kendl, Sabine; Weigand, Isabel; Bala, Margarita; Ronchi, Cristina L; Deutschbein, Timo; Schmitz, Gerd; Rosenwald, Andreas; Allolio, Bruno; Fassnacht, Martin; Kroiss, Matthias

    2015-11-01

    Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

  20. Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

    PubMed

    Imai, Toru; Kosuge, Yasuhiro; Saito, Hiroaki; Uchiyama, Taketo; Wada, Taira; Shimba, Shigeki; Ishige, Kumiko; Miyairi, Shinichi; Makishima, Makoto; Ito, Yoshihisa

    2016-03-01

    S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

  1. Downregulation of miR-384-5p attenuates rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through inhibiting endoplasmic reticulum stress.

    PubMed

    Jiang, Mingfang; Yun, Qiang; Shi, Feng; Niu, Guangming; Gao, Yang; Xie, Shenghui; Yu, Shengyuan

    2016-05-01

    Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of Parkinson's disease (PD). However, the role of microRNAs (miRNAs) in this process involved in PD remains poorly understood. Recent studies indicate that miR-384-5p plays an important role for cell survival in response to different insults, but the role of miR-384-5p in PD-associated neurotoxicity remains unknown. In this study, we investigated the role of miR-384-5p in an in vitro model of PD using dopaminergic SH-SY5Y cells treated with rotenone. We found that miR-384-5p was persistently induced by rotenone in neurons. Also, the inhibition of miR-384-5p significantly suppressed rotenone-induced neurotoxicity, while overexpression of miR-384-5p aggravated rotenone-induced neurotoxicity. Through bioinformatics and dual-luciferase reporter assay, miR-384-5p was found to directly target the 3'-untranslated region of glucose-regulated protein 78 (GRP78), the master regulator of ER stress sensors. Quantitative polymerase chain reaction and Western blotting analysis showed that miR-384-5p negatively regulated the expression of GRP78. Inhibition of miR-384-5p remarkably suppressed rotenone-evoked ER stress, which was evident by a reduction in the phosphorylation of activating transcription factor 4 (ATF4) and inositol-requiring enzyme 1 (IRE1α). The downstream target genes of ER stress including CCAAT/enhancer-binding protein-homologous protein (CHOP) and X box-binding protein-1 (XBP-1) were also decreased by the miR-384-5p inhibitor. In contrast, overexpression of miR-384-5p enhanced ER stress signaling. In addition, knockdown of GRP78 significantly abrogated the inhibitory effect of miR-384-5p inhibitors on cell apoptosis and ER stress signaling. Moreover, we observed a significant increase of miR-384-5p expression in primary neurons induced by rotenone. Taken together, our results suggest that miR-384-5p mediated ER stress by negatively regulating GRP78 and that miR-384-5p inhibition might be a

  2. Sialic acid rescues repurified lipopolysaccharide-induced acute renal failure via inhibiting TLR4/PKC/gp91-mediated endoplasmic reticulum stress, apoptosis, autophagy, and pyroptosis signaling.

    PubMed

    Yang, Chih-Ching; Yao, Chien-An; Yang, Jyh-Chin; Chien, Chiang-Ting

    2014-09-01

    Lipopolysaccharides (LPS) through Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) activation induce systemic inflammation where oxidative damage plays a key role in multiple organ failure. Because of the neutralization of LPS toxicity by sialic acid (SA), we determined its effect and mechanisms on repurified LPS (rLPS)-evoked acute renal failure. We assessed the effect of intravenous SA (10 mg/kg body weight) on rLPS-induced renal injury in female Wistar rats by evaluating blood and kidney reactive oxygen species (ROS) responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. SA can interact with rLPS through a high binding affinity. rLPS dose- and time-dependently reduced arterial blood pressure, renal microcirculation and blood flow, and increased vascular resistance in the rats. rLPS enhanced monocyte/macrophage (ED-1) infiltration and ROS production and impaired kidneys by triggering p-IRE1α/p-JNK/CHOP/GRP78/ATF4-mediated endoplasmic reticulum (ER) stress, Bax/PARP-mediated apoptosis, Beclin-1/Atg5-Atg12/LC3-II-mediated autophagy, and caspase 1/IL-1β-mediated pyroptosis in the kidneys. SA treatment at 30 min, but not 60 min after rLPS stimulation, gp91 siRNA and protein kinase C-α (PKC) inhibitor efficiently rescued rLPS-induced acute renal failure via inhibition of TLR4/PKC/NADPH oxidase gp91-mediated ER stress, apoptosis, autophagy and pyroptosis in renal proximal tubular cells, and rat kidneys. In response to rLPS or IFNγ, the enhanced Atg5, FADD, LC3-II, and PARP expression can be inhibited by Atg5 siRNA. Albumin (10 mg/kg body weight) did not rescue rLPS-induced injury. In conclusion, early treatment (within 30 min) of SA attenuates rLPS-induced renal failure via the reduction in LPS toxicity and subsequently inhibiting rLPS-activated TLR4/PKC/gp91/ER stress/apoptosis/autophagy/pyroptosis signaling.

  3. Altered Endoplasmic Reticulum Calcium Pump Expression during Breast Tumorigenesis

    PubMed Central

    Papp, Béla; Brouland, Jean-Philippe

    2011-01-01

    Endoplasmic reticulum calcium homeostasis is involved in several essential cell functions including cell proliferation, protein synthesis, stress responses or secretion. Calcium uptake into the endoplasmic reticulum is performed by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). In order to study endoplasmic reticulum calcium homeostasis in situ in mammary tissue, in this work SERCA3 expression was investigated in normal breast and in its benign and malignant lesions in function of the cell type, degree of malignancy, and histological and molecular parameters of the tumors. Our data indicate, that although normal breast acinar epithelial cells express SERCA3 abundantly, its expression is strongly decreased already in very early non-malignant epithelial lesions such as adenosis, and remains low in lobular carcinomas. Whereas normal duct epithelium expresses significant amounts of SERCA3, its expression is decreased in several benign ductal lesions, as well as in ductal adenocarcinoma. The loss of SERCA3 expression is correlated with Elston-Ellis grade, negative hormone receptor expression or triple negative status in ductal carcinomas. The concordance between decreased SERCA3 expression and several histological, as well as molecular markers of ductal carcinogenesis indicates that endoplasmic reticulum calcium homeostasis is remodeled during tumorigenesis in the breast epithelium. PMID:21863130

  4. Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

    PubMed Central

    Allison, D S; Young, E T

    1989-01-01

    The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide. Images PMID:2513481

  5. Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition.

    PubMed

    Cerqua, Cristina; Anesti, Vassiliki; Pyakurel, Aswin; Liu, Dan; Naon, Deborah; Wiche, Gerhard; Baffa, Raffaele; Dimmer, Kai S; Scorrano, Luca

    2010-11-01

    Trichoplein/mitostatin (TpMs) is a keratin-binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)-dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca(2+)-dependent stimuli that require ER-mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria-ER juxtaposition.

  6. Lipid Transport between the Endoplasmic Reticulum and Mitochondria

    PubMed Central

    Flis, Vid V.

    2013-01-01

    Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis. PMID:23732475

  7. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway.

    PubMed

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-08-27

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced--turnover of LC3-II and--accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors--chloroquine and--bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that α-solanine induces

  8. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway

    PubMed Central

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-01-01

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced – turnover of LC3-II and – accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors – chloroquine and – bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that

  9. Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

    PubMed Central

    Mitsuda, Satoshi; Yokomichi, Tomonobu; Yokoigawa, Junpei; Kataoka, Takao

    2014-01-01

    Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum. PMID:24649404

  10. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  11. Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

    PubMed Central

    Terasaki, M; Slater, N T; Fein, A; Schmidek, A; Reese, T S

    1994-01-01

    Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions. Images PMID:7519781

  12. Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

    PubMed

    Chanat, Eric; Le Parc, Annabelle; Lahouassa, Hichem; Badaoui, Bouabid

    2016-06-01

    In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

  13. Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum.

    PubMed

    Zito, Ester; Buono, Mario; Pepe, Stefano; Settembre, Carmine; Annunziata, Ida; Surace, Enrico Maria; Dierks, Thomas; Monti, Maria; Cozzolino, Marianna; Pucci, Piero; Ballabio, Andrea; Cosma, Maria Pia

    2007-05-16

    Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.

  14. Hydrogen-rich water protects against inflammatory bowel disease in mice by inhibiting endoplasmic reticulum stress and promoting heme oxygenase-1 expression

    PubMed Central

    Shen, Nai-Ying; Bi, Jian-Bin; Zhang, Jing-Yao; Zhang, Si-Min; Gu, Jing-Xian; Qu, Kai; Liu, Chang

    2017-01-01

    -α, IL-6 and IL-1β compared with the DSS group (P < 0.05). In addition, the pivotal proteins involved in endoplasmic reticulum (ER) stress, including p-eIF2α, ATF4, XBP1s and CHOP, were dramatically reduced after HRW treatment in contrast to the control group (P < 0.05). Furthermore, HRW treatment markedly up-regulated HO-1 expression, and the use of ZnPP obviously reversed the protective role of HRW. In the DSS + HRW + ZnPP group, colon shortening and colonic wall thickening were significantly aggravated, and the macroscopic damage scores were similar to those of the DSS + HRW group (P < 0.05). The histological study also showed more serious colonic damage that was similar to the DSS group. CONCLUSION HRW has a significant therapeutic potential in IBD by inhibiting inflammatory factors, oxidative stress and ER stress and by up-regulating HO-1 expression. PMID:28293084

  15. Heparin-based coacervate of bFGF facilitates peripheral nerve regeneration by inhibiting endoplasmic reticulum stress following sciatic nerve injury

    PubMed Central

    Wu, Yanqing; Li, Yiyang; Khor, Sinan; Mao, Yuqin; He, Huacheng; Xu, Ke; Zhang, Hongyu; Li, Xiaokun; Wang, Jian; Jiang, Huai; Jin, Qike; Ye, Qingsong; Wang, Zhouguang; Xiao, Jian

    2017-01-01

    Creating a microenvironment at the injury site that favors axonal regrowth and remyelinationis pivotal to the success of therapeutic reinnervation. The mature myelin sheath of the peripheral nervous system depends on active participation of Schwann cells to form new cytoskeletal components and tremendous amounts of relevant neurotrophic factors. In this study, we utilized a new biomaterial for growth factor delivery consisting of a biocompatible polycation, poly(ethylene argininylaspartatediglyceride) and heparin. It is capable of binding a variety of growth factors to deliver basic fibroblast growth factor (bFGF) through polyvalent ionic interactions for nerve repair. In vitro assays demonstrated that the bFGF loading efficiency reached 10 μg and this delivery vehicle could control the release of bFGF. In vivo, the coacervate enhanced bFGF bioavailability, which improved both motor and sensory function. It could also acceleratemyelinated fiber regeneration and remyelination and promote Schwann cells proliferation. Furthermore, the neuroprotective effect of bFGF-coacervate in sciatic nerve injury was associated with the alleviation of endoplasmic reticulum stress signal. This heparin-based delivery platform leads to increased bFGF loading efficiency and better controls its release, which will provide an effective strategy for peripheral nerve injury regeneration therapy. PMID:28624802

  16. Retrograde transport of toxins across the endoplasmic reticulum membrane.

    PubMed

    Lord, J M; Deeks, E; Marsden, C J; Moore, K; Pateman, C; Smith, D C; Spooner, R A; Watson, P; Roberts, L M

    2003-12-01

    Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

  17. Regulation of endoplasmic reticulum turnover by selective autophagy.

    PubMed

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-06-18

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

  18. Low molecular weight Abeta induces collapse of endoplasmic reticulum.

    PubMed

    Lai, Cora Sau-Wan; Preisler, Julie; Baum, Larry; Lee, Daniel Hong-Seng; Ng, Ho-Keung; Hugon, Jacques; So, Kwok-Fai; Chang, Raymond Chuen-Chung

    2009-05-01

    The endoplasmic reticulum (ER) is a dynamic multifunction organelle that is responsible for Ca(2+) homeostasis, protein folding, post-translational modification, protein degradation, and transportation of nascent proteins. Disruption of ER architecture might affect the normal physiology of the cell. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. Here, we found that soluble low molecular weight of Abeta disrupted the anchoring between ER and microtubules (MT) and induced collapse of ER. In addition, it decreased the stability of MT. Subsequently, low molecular weight Abeta triggered autophagy and enhanced lysosomal degradation, as shown by electron microscopy and live-cell imaging. Dysfunction of ER can be further proved in postmortem AD brain and transgenic mice bearing APP Swedish mutation by immunohistochemical analysis of calreticulin. Treatment with Taxol, a MT-stabilizing agent, could partially inhibit collapse of the ER and induction of autophagy. The results show that Abeta-induced disruption of MT can affect the architecture of the ER. Collapse/aggregation of the ER may play an important role in Abeta peptide-triggered neurodegenerative processes.

  19. Thiamine Deficiency Induces Endoplasmic Reticulum Stress in Neurons

    PubMed Central

    Wang, Xin; Wang, Bingwei; Fan, Zhiqin; Shi, Xianglin; Ke, Zun-Ji; Luo, Jia

    2007-01-01

    Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as GRP78, GADD153/Chop, phosphorylation of eIF2α and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TD-induced brain damage. PMID:17137721

  20. Thiamine deficiency induces endoplasmic reticulum stress in neurons.

    PubMed

    Wang, X; Wang, B; Fan, Z; Shi, X; Ke, Z-J; Luo, J

    2007-02-09

    Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as glucose-regulated protein (GRP) 78, growth arrest and DNA-damage inducible protein or C/EBP-homologus protein (GADD153/Chop), phosphorylation of eIF2alpha and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TD-induced brain damage.

  1. The effect of calcitriol on endoplasmic reticulum stress response.

    PubMed

    Haddur, Ela; Ozkaya, Ali Burak; Ak, Handan; Aydin, Hikmet Hakan

    2015-06-01

    Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis, inhibition of angiogenesis, and metastasis. Calcitriol also increases intracellular calcium triggering apoptosis in a calpain-dependent manner. Since the main storage unit for cellular calcium is endoplasmic reticulum (ER) and a decrease in ER calcium levels might induce ER stress associated cell death, we hypothesized that the cellular actions of calcitriol occur via ER stress. We have evaluated induction of ER stress by assessing BIP expression and XBP-1 splicing in breast cancer cell lines (MCF-7 and MDA-MB-231) and mammary epithelial cell line MCF10A. Our results suggest that cytotoxic concentrations of calcitriol induce an ER stress related response indicated as increased BIP levels and XBP-1 splicing not only in breast cancer cells but also in mammary epithelial cell line. However, vehicle treatment also induced a similar response de-emphasizing the importance of such effect. Calcitriol also failed to activate calpains, further weakening the idea of ER stress as the main mechanism for apoptotic effects of calcitriol. Taken together our results suggest an association between ER stress and vitamin D signaling. However present data indicates that ER stress by itself is not sufficient to explain anticancer properties of calcitriol.

  2. MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.

    PubMed

    Sugiura, Ayumu; Nagashima, Shun; Tokuyama, Takeshi; Amo, Taku; Matsuki, Yohei; Ishido, Satoshi; Kudo, Yoshihisa; McBride, Heidi M; Fukuda, Toshifumi; Matsushita, Nobuko; Inatome, Ryoko; Yanagi, Shigeru

    2013-07-11

    The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. The endoplasmic reticulum stress response: A link with tuberculosis?

    PubMed

    Cui, Yongyong; Zhao, Deming; Barrow, Paul Andrew; Zhou, Xiangmei

    2016-03-01

    Tuberculosis (TB) remains a major cause of mortality and morbidity in the worldwide. The endoplasmic-reticulum stress (ERS) response constitutes a cellular process that is triggered by mycobacterial infection that disturbs the folding of proteins in the endoplasmic reticulum (ER). The unfolded protein response (UPR) is induced to suspend the synthesis of early proteins and reduce the accumulation of unfolded- or misfolded proteins in the ER restoring normal physiological cell function. Prolonged or uncontrolled ERS leads to the activation of three signaling pathways (IRE1, PERK and ATF6) which directs the cell towards apoptosis. The absence of this process facilitates spread of the mycobacteria within the body. We summarize here recent advances in understanding the signaling pathway diversity governing ERS in relation to TB.

  4. Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.

    PubMed

    Zhang, Ying; Zhang, KaiQiang; Ma, Lin; Gu, HeFeng; Li, Jian; Lei, Shuang

    2016-09-01

    The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. [Collagen abnormalities and endoplasmic reticulum stress in bone and cartilage].

    PubMed

    Furuichi, Tatsuya; Nishimura, Gen; Ikegawa, Shiro

    2013-11-01

    There are many steps in the post-translational modification of collagen molecules. When abnormality occurs in some step, the unfolded collagen molecules are accumulated in the endoplasmic reticulum (ER) , leading to ER stress. ER stress also occurs downstream of the defective modification of collagen in bone and cartilage. ER stress-induced apoptosis or ER stress response without inducing apoptosis may be associated with the pathogenesis of genetic collagen disorders in bone and cartilage.

  6. Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis

    PubMed Central

    Kaser, Arthur; Martínez-Naves, Eduardo; Blumberg, Richard S.

    2015-01-01

    Purpose of review To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). Recent findings The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. Summary Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders. PMID:20495455

  7. Isolation and characterization of the Neurospora crassa endoplasmic reticulum.

    PubMed Central

    Borgeson, C E; Bowman, B J

    1983-01-01

    The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions. Images PMID:6311800

  8. Lipolysis Response to Endoplasmic Reticulum Stress in Adipose Cells*

    PubMed Central

    Deng, Jingna; Liu, Shangxin; Zou, Liangqiang; Xu, Chong; Geng, Bin; Xu, Guoheng

    2012-01-01

    In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues. PMID:22223650

  9. Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

    PubMed

    Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

    2016-10-04

    Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.

  10. Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy

    PubMed Central

    Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

    2016-01-01

    Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies. PMID:27588471

  11. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells.

    PubMed

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb.

  12. Resveratrol prevents doxorubicin-induced cardiotoxicity in H9c2 cells through the inhibition of endoplasmic reticulum stress and the activation of the Sirt1 pathway.

    PubMed

    Lou, Yu; Wang, Zhen; Xu, Yi; Zhou, Ping; Cao, Junxian; Li, Yuanshi; Chen, Yeping; Sun, Junfeng; Fu, Lu

    2015-09-01

    Treatment with doxorubicin (DOX) is one of the major causes of chemotherapy-induced cardiotoxicity and is therefore, the principal limiting factor in the effectiveness of chemotherapy for cancer patients. DOX‑induced heart failure is thought to result from endoplasmic reticulum (ER) stress and cardiomyocyte apoptosis. Resveratrol (RV), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. The aim of the present study was to examine the effects of RV on DOX‑induced cardiotoxicity in H9c2 cells. We hypothesized that RV would protect H9c2 cells against DOX‑induced ER stress and subsequent cell death through the activation of the Sirt1 pathway. Our results demonstrated that the decrease observed in the viability of the H9c2 cells following exposure to DOX was accompanied by a significant increase in the expression of the ER stress‑related proteins, glucose‑regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). However, we found that RV downregulated the expression of ER stress marker protein in the presence of DOX and restored the viability of the H9c2 cells. Exposure to RV or DOX alone only slightly increased the protein expression of Sirt1, whereas a significant increase in Sirt1 protein levels was observed in the cells treated with both RV and DOX. The Sirt1 inhibitor, nicotinamide (NIC), partially neutralized the effects of RV on the expression of Sirt1 in the DOX‑treated cells and completely abolished the effects of RV on the expression of GRP78 and CHOP. The findings of our study suggest that RV protects H9c2 cells against DOX‑induced ER stress through ER stabilization, and more specifically through the activation of the Sirt1 pathway, thereby leading to cardiac cell survival.

  13. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

    PubMed Central

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  14. C1q/TNF-Related Protein 9 (CTRP9) attenuates hepatic steatosis via the autophagy-mediated inhibition of endoplasmic reticulum stress.

    PubMed

    Jung, Tae Woo; Hong, Ho Cheol; Hwang, Hwan-Jin; Yoo, Hye Jin; Baik, Sei Hyun; Choi, Kyung Mook

    2015-12-05

    C1q/TNF-Related Protein (CTRP) 9, the closest paralog of adiponectin, has been reported to protect against diet-induced obesity and non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. We explored the protective effect of CTRP9 against hepatic steatosis and apoptosis, and identified the mechanisms through autophagy and endoplasmic reticulum (ER) stress using in vitro and in vivo experiments. Treating HepG2 cells with human recombinant CTRP9 significantly ameliorated palmitate- or tunicamycin-induced dysregulation of lipid metabolism, caspase 3 activity and chromatin condensation, which lead to reduction of hepatic triglyceride (TG) accumulation. CTRP9 treatment induced autophagy markers including LC3 conversion, P62 degradation, Beclin1 and ATG7 through AMPK phosphorylation in human primary hepatocytes. Furthermore, CTRP9 decreased palmitate- or tunicamycin-induced ER stress markers, such as eIF2α, CHOP and IRE-1, in HepG2 cells. Compound C, an AMPK inhibitor, and 3 methyladenine (3 MA), an autophagy inhibitor, canceled the effects of CTRP9 on ER stress, apoptosis and hepatic steatosis. In the livers of HFD-fed mice, adenovirus-mediated CTRP9 overexpression significantly induced AMPK phosphorylation and autophagy, whereas suppressed ER stress markers. In addition, both SREBP1-mediated lipogenic gene expression and apoptosis were significantly attenuated, which result in improvement in hepatic steatosis by overexpression of CTRP9. These results demonstrate that CTRP9 alleviates hepatic steatosis through relief of ER stress via the AMPK-mediated induction of autophagy.

  15. Crude Saponins of Panax notoginseng Have Neuroprotective Effects To Inhibit Palmitate-Triggered Endoplasmic Reticulum Stress-Associated Apoptosis and Loss of Postsynaptic Proteins in Staurosporine Differentiated RGC-5 Retinal Ganglion Cells.

    PubMed

    Wang, Dan-dan; Zhu, Hua-zhang; Li, Shi-wei; Yang, Jia-ming; Xiao, Yang; Kang, Qiang-rong; Li, Chen-yang; Zhao, Yun-shi; Zeng, Yong; Li, Yan; Zhang, Jian; He, Zhen-dan; Ying, Ying

    2016-02-24

    Increased apoptosis of retinal ganglion cells (RGCs) contributes to the gradual loss of retinal neurons at the early phase of diabetic retinopathy (DR). There is an urgent need to search for drugs with neuroprotective effects against apoptosis of RGCs for the early treatment of DR. This study aimed to investigate the neuroprotective effects of saponins extracted from Panax notoginseng, a traditional Chinese medicine, on apoptosis of RGCs stimulated by palmitate, a metabolic factor for the development of diabetes and its complications, and to explore the potential molecular mechanism. We showed that crude saponins of P. notoginseng (CSPN) inhibited the increased apoptosis and loss of postsynaptic protein PSD-95 by palmitate in staurosporine-differentiated RGC-5 cells. Moreover, CSPN suppressed palmitate-induced reactive oxygen species generation and endoplasmic reticulum stress-associated eIF2α/ATF4/CHOP and caspase 12 pathways. Thus, our findings address the potential therapeutic significance of CSPN for the early stage of DR.

  16. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

    PubMed Central

    Zhang, Jintao; Yi, Man; Zha, Longying; Chen, Siqiang; Li, Zhijia; Li, Cheng; Gong, Mingxing; Deng, Hong; Chu, Xinwei; Chen, Jiehua; Zhang, Zheqing; Mao, Limei; Sun, Suxia

    2016-01-01

    Purpose Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. Methods Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. Results Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin

  17. GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy

    PubMed Central

    Holczer, Marianna; Bánhegyi, Gábor; Kapuy, Orsolya

    2016-01-01

    The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress. PMID:27992581

  18. Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib.

    PubMed

    Coca, Ramón; Soler, Fernando; Cortés-Castell, Ernesto; Gil-Guillén, Vicente; Fernández-Belda, Francisco

    2014-01-01

    Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+-pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and

  19. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.

    PubMed

    Field, F J; Born, E; Murthy, S; Mathur, S N

    1998-02-01

    The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells. The appearance of newly synthesized cholesterol on the cell surface was rapid. By 30 min, 50% of the total labeled cholesterol was observed in the plasma membrane. The arrival of cholesterol at the plasma membrane was independent of new protein synthesis, a functional Golgi apparatus, or microtubular function. Progesterone, verapamil, and trifluoperazine, inhibitors of p-glycoprotein which are known to inhibit cholesterol transport from the plasma membrane to the endoplasmic reticulum, reduced the amount of newly synthesized cholesterol reaching the plasma membrane. The p-glycoprotein inhibitors, however, caused the accumulation of sterol intermediates in the plasma membrane, suggesting that sterol trafficking to the plasma membrane remained intact, but that trafficking from the plasma membrane to the endoplasmic reticulum was disrupted. In contrast, nigericin, another potent inhibitor of cholesterol movement from the plasma membrane to the endoplasmic reticulum, did not alter the transport of newly synthesized cholesterol to the plasma membrane. Moreover, promoting cholesterol transport from the plasma membrane to the endoplasmic reticulum by sphingomyelin hydrolysis or by micellar cholesterol influx did not alter the percent of newly synthesized cholesterol transported to the plasma membrane. Likewise, preventing plasma membrane cholesterol from reaching the endoplasmic reticulum by incubating cells with lysophosphatidylcholine, filipin, or digitonin did not alter the arrival of newly synthesized cholesterol to the plasma membrane. The results suggest that the amount of cholesterol moving to the plasma membrane from the endoplasmic reticulum is constitutive and regulated at the level of cholesterol synthesis and not at the level of the transport process. The pathways of cholesterol transport to and from the

  20. Protein misfolding in the endoplasmic reticulum as a conduit to human disease.

    PubMed

    Wang, Miao; Kaufman, Randal J

    2016-01-21

    In eukaryotic cells, the endoplasmic reticulum is essential for the folding and trafficking of proteins that enter the secretory pathway. Environmental insults or increased protein synthesis often lead to protein misfolding in the organelle, the accumulation of misfolded or unfolded proteins - known as endoplasmic reticulum stress - and the activation of the adaptive unfolded protein response to restore homeostasis. If protein misfolding is not resolved, cells die. Endoplasmic reticulum stress and activation of the unfolded protein response help to determine cell fate and function. Furthermore, endoplasmic reticulum stress contributes to the aetiology of many human diseases.

  1. Sertraline induces endoplasmic reticulum stress in hepatic cells.

    PubMed

    Chen, Si; Xuan, Jiekun; Couch, Letha; Iyer, Advait; Wu, Yuanfeng; Li, Quan-Zhen; Guo, Lei

    2014-08-01

    Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity. Published by Elsevier Ireland Ltd.

  2. Characterization of Ca2+ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

    PubMed Central

    Buckhout, Thomas J.

    1984-01-01

    The characteristics of Ca2+ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca2+ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0. The Ca2+-dependent modulation protein, calmodulin, was tested for its effect on Ca2+ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca2+ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca2+ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca2+ transport activity. The inhibition of Ca2+ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca2+ uptake into root endoplasmic reticulum. PMID:16663981

  3. Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum.

    PubMed

    Johannes, Ludger; Pezo, Valérie; Mallard, Frédéric; Tenza, Danièle; Wiltz, Aimée; Saint-Pol, Agnès; Helft, Julie; Antony, Claude; Benaroch, Philippe

    2003-05-01

    HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb3, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, beta COP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed.

  4. Inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus NL63 spike protein-mediated entry by altering the glycan processing of angiotensin I-converting enzyme 2.

    PubMed

    Zhao, Xuesen; Guo, Fang; Comunale, Mary Ann; Mehta, Anand; Sehgal, Mohit; Jain, Pooja; Cuconati, Andrea; Lin, Hanxin; Block, Timothy M; Chang, Jinhong; Guo, Ju-Tao

    2015-01-01

    Endoplasmic reticulum (ER)-resident glucosidases I and II sequentially trim the three terminal glucose moieties on the N-linked glycans attached to nascent glycoproteins. These reactions are the first steps of N-linked glycan processing and are essential for proper folding and function of many glycoproteins. Because most of the viral envelope glycoproteins contain N-linked glycans, inhibition of ER glucosidases with derivatives of 1-deoxynojirimycin, i.e., iminosugars, efficiently disrupts the morphogenesis of a broad spectrum of enveloped viruses. However, like viral envelope proteins, the cellular receptors of many viruses are also glycoproteins. It is therefore possible that inhibition of ER glucosidases not only compromises virion production but also disrupts expression and function of viral receptors and thus inhibits virus entry into host cells. Indeed, we demonstrate here that iminosugar treatment altered the N-linked glycan structure of angiotensin I-converting enzyme 2 (ACE2), which did not affect its expression on the cell surface or its binding of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein. However, alteration of N-linked glycans of ACE2 impaired its ability to support the transduction of SARS-CoV and human coronavirus NL63 (HCoV-NL63) spike glycoprotein-pseudotyped lentiviral particles by disruption of the viral envelope protein-triggered membrane fusion. Hence, in addition to reducing the production of infectious virions, inhibition of ER glucosidases also impairs the entry of selected viruses via a post-receptor-binding mechanism.

  5. Endoplasmic reticulum stress: an unrecognized actor in solid organ transplantation.

    PubMed

    Pallet, Nicolas; Fougeray, Sophie; Beaune, Philippe; Legendre, Christophe; Thervet, Eric; Anglicheau, Dany

    2009-09-15

    Endoplasmic reticulum (ER) stress is an adaptive response to the accumulation of misfolded proteins within the ER, which can trigger cell dedifferentiation and cell suicide. Increasing evidences suggest its implication in mediating allograft injury. Herein, we summarize the mechanisms of ER stress and discuss its implication in allograft injury. Increasing our understanding of the cellular and molecular mechanisms of acute and chronic allograft damages could lead to the development of new biomarkers and to the discovery of new therapeutic strategies to prevent the initiation of graft dysfunction or to promote the tissue regeneration after injury.

  6. Lipid trafficking at endoplasmic reticulum-chloroplast membrane contact sites.

    PubMed

    Block, Maryse A; Jouhet, Juliette

    2015-08-01

    Glycerolipid synthesis in plant cells is characterized by an intense trafficking of lipids between the endoplasmic reticulum (ER) and chloroplasts. Initially, fatty acids are synthesized within chloroplasts and are exported to the ER where they are used to build up phospholipids and triacylglycerol. Ultimately, derivatives of these phospholipids return to chloroplasts to form galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, the main and essential lipids of photosynthetic membranes. Lipid trafficking was proposed to transit through membrane contact sites (MCSs) connecting both organelles. Here, we review recent insights into ER-chloroplast MCSs and lipid trafficking between chloroplasts and the ER.

  7. Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics.

    PubMed

    Pizzo, Paola; Pozzan, Tullio

    2007-10-01

    Mitochondria and endoplasmic reticulum (ER) have different roles in living cells but they interact both physically and functionally. A key aspect of the mitochondria-ER relationship is the modulation of Ca(2+) signaling during cell activation, which thus affects a variety of physiological processes. We focus here on the molecular aspects that control the dynamics of the organelle-organelle interaction and their relationship with Ca(2+) signals, also discussing the consequences that these phenomena have, not only for cell physiology but also in the control of cell death.

  8. Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

    PubMed Central

    Cortés-Castell, Ernesto; Gil-Guillén, Vicente; Fernández-Belda, Francisco

    2014-01-01

    Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+–pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and

  9. Melatonin Induces Anti-Inflammatory Effects to Play a Protective Role via Endoplasmic Reticulum Stress in Acute Pancreatitis.

    PubMed

    Chen, Yina; Zhang, Jie; Zhao, Qian; Chen, Qinfen; Sun, Yangjie; Jin, Yin; Wu, Jiansheng

    2016-01-01

    Melatonin, which is mainly secreted by the pineal gland and released into blood, has anti-inflammatory properties in acute pancreatitis. Many studies show that melatonin can relieve inflammation in taurocholate-induced acute pancreatitis. However, the mechanisms of its anti-inflammatory effects are still undefined, especially the relationship between melatonin and endoplasmic reticulum stress. We explored the anti-inflammatory activity of melatonin in AR42J and rat models. The CCK-8 assay was used to assess effects of melatonin on AR42J cell viability. Inflammatory degree and the expressions of endoplasmic reticulum stress related molecules were examined by quantitative RT-PCR and western blotting. The degree of inflammation in the tissue was also accessed by pathological grading. Finally, we used the western blotting method to verify apoptosis and autophagy. Endoplasmic reticulum stress was obviously activated in early stage inflammation in AR42J and rat models. Melatonin could induce anti-inflammatory effects via endoplasmic reticulum stress. Melatonin significantly inhibited inflammatory cytokines and the expression of ERS-related molecules. Finally, it played a protective role by promoting apoptosis and autophagy of the cells, which were damaged in the process of inflammatory reaction. Melatonin induces anti-inflammatory effects via endoplasmic reticulum stress in acute pancreatitis to play a protective role. © 2016 The Author(s) Published by S. Karger AG, Basel.

  10. Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum.

    PubMed

    Marcolongo, Paola; Senesi, Silvia; Gava, Barbara; Fulceri, Rosella; Sorrentino, Vincenzo; Margittai, Eva; Lizák, Beáta; Csala, Miklós; Bánhegyi, Gábor; Benedetti, Angelo

    2008-08-01

    Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11beta-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11beta-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

  11. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    PubMed

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  12. Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood-Brain Barrier Damage.

    PubMed

    Qie, Xiaojuan; Wen, Di; Guo, Hongyan; Xu, Guanjie; Liu, Shuai; Shen, Qianchao; Liu, Yi; Zhang, Wenfang; Cong, Bin; Ma, Chunling

    2017-01-01

    Methamphetamine (METH) abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood-brain barrier (BBB). Herein, we explored the potential mechanism of endoplasmic reticulum (ER) stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3) cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS) and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo) and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption.

  13. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.

    PubMed

    Underwood, K W; Jacobs, N L; Howley, A; Liscum, L

    1998-02-13

    We have studied the movement of low density lipoprotein (LDL)-derived cholesterol in cultured Chinese hamster ovary cells. Our hypothesis is that when LDL cholesterol is effluxed from lysosomes, the bulk of LDL cholesterol is mobilized to the plasma membrane, while another pathway delivers LDL cholesterol from lysosomes to acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum. Three lines of evidence support this model. First, LDL cholesterol transport to ACAT can be blocked without inhibiting the movement of cholesterol from lysosomes to plasma membrane or from plasma membrane to endoplasmic reticulum. Second, LDL cholesterol transport to ACAT is normal in a Chinese hamster ovary mutant with defective plasma membrane-to-ACAT movement. Third, LDL cholesterol is not diluted by the plasma membrane cholesterol pool before reaching ACAT. Our evidence supports a vesicular model of cholesterol transport from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane.

  14. [Involvement of endoplasmic reticulum stress in solid organ transplantation].

    PubMed

    Pallet, Nicolas; Bouvier, Nicolas; Beaune, Philippe; Legendre, Christophe; Anglicheau, Dany; Thervet, Eric

    2010-04-01

    Endoplasmic reticulum (ER) stress is a situation caused by the accumulation of unfolded proteins in the endoplasmic reticulum, triggering an evolutionary conserved adaptive response termed the unfolded protein response. When adaptation fails, excessive and prolonged ER stress triggers cell suicide. Important roles for ER-initiated cell death pathways have been recognized for several diseases, including diabetes, hypoxia, ischemia/reperfusion injury, neurodegenerative and heart diseases. The implication of the ER stress is not well recognized in solid organ transplantation, but increasing evidence suggests its implication in mediating allograft injury. The purpose of this review is to summarize the mechanisms of ER stress and to discuss its implication during tissue injury in solid organ transplantation. The possible implications of the ER stress in the modifications of cell functional properties and phenotypic changes are also discussed beyond the scope of adaptation and cell death. Increasing the understanding of the cellular and molecular mechanisms of acute and chronic allograft damages could lead to the development of new biomarkers and to the discovery of new therapeutic strategies to prevent the initiation of graft dysfunction or to promote the tissue regeneration after injury.

  15. Endoplasmic Reticulum Calcium Pumps and Cancer Cell Differentiation

    PubMed Central

    Papp, Béla; Brouland, Jean-Philippe; Arbabian, Atousa; Gélébart, Pascal; Kovács, Tünde; Bobe, Régis; Enouf, Jocelyne; Varin-Blank, Nadine; Apáti, Ágota

    2012-01-01

    The endoplasmic reticulum (ER) is a major intracellular calcium storage pool and a multifunctional organelle that accomplishes several calcium-dependent functions involved in many homeostatic and signaling mechanisms. Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA)-type calcium pumps. SERCA activity can determine ER calcium content available for intra-ER functions and for calcium release into the cytosol, and can shape the spatiotemporal characteristics of calcium signals. SERCA function therefore constitutes an important nodal point in the regulation of cellular calcium homeostasis and signaling, and can exert important effects on cell growth, differentiation and survival. In several cell types such as cells of hematopoietic origin, mammary, gastric and colonic epithelium, SERCA2 and SERCA3-type calcium pumps are simultaneously expressed, and SERCA3 expression levels undergo significant changes during cell differentiation, activation or immortalization. In addition, SERCA3 expression is decreased or lost in several tumor types when compared to the corresponding normal tissue. These observations indicate that ER calcium homeostasis is remodeled during cell differentiation, and may present defects due to decreased SERCA3 expression in tumors. Modulation of the state of differentiation of the ER reflected by SERCA3 expression constitutes an interesting new aspect of cell differentiation and tumor biology. PMID:24970132

  16. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori.

    PubMed

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-11-05

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H₂O₂, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

  17. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori

    PubMed Central

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-01-01

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis. PMID:26556347

  18. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

    PubMed

    Manfredi, Giovanni; Kawamata, Hibiki

    2016-06-01

    Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS.

  19. Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders

    PubMed Central

    Volpi, Vera G.; Touvier, Thierry; D'Antonio, Maurizio

    2017-01-01

    Reaching the correct three-dimensional structure is crucial for the proper function of a protein. The endoplasmic reticulum (ER) is the organelle where secreted and transmembrane proteins are synthesized and folded. To guarantee high fidelity of protein synthesis and maturation in the ER, cells have evolved ER-protein quality control (ERQC) systems, which assist protein folding and promptly degrade aberrant gene products. Only correctly folded proteins that pass ERQC checkpoints are allowed to exit the ER and reach their final destination. Misfolded glycoproteins are detected and targeted for degradation by the proteasome in a process known as endoplasmic reticulum-associated degradation (ERAD). The excess of unstructured proteins in the ER triggers an adaptive signal transduction pathway, called unfolded protein response (UPR), which in turn potentiates ERQC activities in order to reduce the levels of aberrant molecules. When the situation cannot be restored, the UPR drives cells to apoptosis. Myelin-forming cells of the central and peripheral nervous system (oligodendrocytes and Schwann cells) synthesize a large amount of myelin proteins and lipids and therefore are particularly susceptible to ERQC failure. Indeed, deficits in ERQC and activation of ER stress/UPR have been implicated in several myelin disorders, such as Pelizaeus-Merzbacher and Krabbe leucodystrophies, vanishing white matter disease and Charcot-Marie-Tooth neuropathies. Here we discuss recent evidence underlying the importance of proper ERQC functions in genetic disorders of myelinating glia. PMID:28101003

  20. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    SciTech Connect

    Liu Qiong; Zhan Jinbiao . E-mail: jzhan2k@zju.edu.cn; Chen Xinhong; Zheng Shu

    2006-05-12

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.

  1. Gating Behavior of Endoplasmic Reticulum Potassium Channels of Rat Hepatocytes in Diabetes

    PubMed Central

    Ghasemi, Maedeh; Khodaei, Naser; Salari, Sajjad; Eliassi, Afsaneh; Saghiri, Reza

    2014-01-01

    Background: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Method: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Results: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. Conclusion: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans. PMID:24842143

  2. Sigma-1 receptors (sigma(1) binding sites) form raft-like microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export.

    PubMed

    Hayashi, Teruo; Su, Tsung-Ping

    2003-08-01

    The brain sigma-1 receptors can bind neurosteroids and psychotropic drugs, including neuroleptics and cocaine and are implicated in schizophrenia, depression, and drug dependence. In this study, we found that sigma-1 receptors specifically target lipid storage sites (lipid droplets) on the endoplasmic reticulum by forming a distinct class of lipid microdomains. Both endogenously expressing sigma-1 receptors and transfected C-terminally enhanced yellow fluorescent protein (EYFP)-tagged sigma-1 receptors (Sig-1R-EYFP) target unique "ring-like" structures associated with endoplasmic reticulum reticular networks in NG108-15 cells. The ring-like structures contain neutral lipids and are enlarged by the oleate treatment, indicating that they are endoplasmic reticulum-associated lipid droplets (ER-LDs). sigma-1 receptors colocalize with caveolin-2, a cholesterol-binding protein in lipid rafts on the ER-LDs, but not with adipocyte differentiation-related protein (ADRP), a cytosolic lipid droplet (c-LD)-specific protein. When the double-arginine ER retention signal on the N terminus of sigma-1 receptors is truncated, sigma-1 receptors no longer exist on ER-LDs, but predominantly target c-LDs, which contain ADRP. sigma-1 receptors on ER-LDs form detergent-resistant raft-like lipid microdomains, the buoyancy of which is different from that of plasma membrane lipid rafts. (+)-Pentazocine causes sigma-1 receptors to disappear from the microdomains. N-Terminally EYFP-tagged sigma-1 receptors (EYFP-Sig-1R) failed to target ER-LDs. EYFP-Sig-1R-transfected cells showed an unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, decreases in c-LDs and cholesterol in plasma membranes, and the bulbous aggregation of endoplasmic reticulum. Thus, sigma-1 receptors are unique endoplasmic reticulum proteins that regulate the compartmentalization of lipids on the endoplasmic reticulum and their export from the endoplasmic reticulum to plasma membrane and c-LDs.

  3. Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

    PubMed

    Bravo-Sagua, R; Rodriguez, A E; Kuzmicic, J; Gutierrez, T; Lopez-Crisosto, C; Quiroga, C; Díaz-Elizondo, J; Chiong, M; Gillette, T G; Rothermel, B A; Lavandero, S

    2013-02-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

  4. Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

    PubMed Central

    Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.

    2014-01-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  5. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

    PubMed

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M; Neuhaus, H Ekkehard

    2013-07-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

  6. Endoplasmic reticulum protein ERp46 in prostate adenocarcinoma

    PubMed Central

    Duivenvoorden, Wilhelmina C.M.; Hopmans, Sarah N.; Austin, Richard C.; Pinthus, Jehonathan H.

    2017-01-01

    Endoplasmic reticulum (ER) protein ERp46 is a member of the protein disulfide isomerase family of oxidoreductases, which facilitates the reduction of disulfides in proteins and their folding. Accumulation of misfolded proteins has been implicated in cancer. The objectives of the present study were to investigate the role of ERp46 in prostate cancer, its expression and its effects on prostate cancer growth. A tissue microarray with human prostate cancer and normal prostate tissue samples was stained for ERp46 followed by image analysis. Human prostate adenocarcinoma 22Rv1 cells were stably transfected with short hairpin RNA (shRNA) specific for ERp46, a non-effective scrambled control or a plasmid containing full-length human ERp46 cDNA, and cell growth was determined. Subcloned cells were treated with thapsigargin or tunicamycin to induce ER stress and lysates were subjected to western blot analysis for ER stress proteins. Subcutaneous xenografts of parental 22Rv1, ERp46-overexpressing (ERp46+), shERp46 or scrambled control cells were established in male inbred BALB/c nude mice (n=10/group). Tumor growth curves of the xenografts were constructed over a period of 30 days and subsequently the mice were sacrificed and the amount of serum prostate-specific antigen was determined. The results demonstrated increased ERp46 expression levels in prostate cancer tissue samples of Gleason ≥7 compared with normal prostate tissue samples. When ERp46 was stably knocked down using shRNA or overexpressed in prostate carcinoma 22Rv1 cells, tumor growth in vitro and in BALB/c nude mice was inhibited and accelerated, respectively. ERp46 overexpression led to reduced sensitivity to ER stress as indicated by higher half maximal inhibitory concentrations for tunicamycin and thapsigargin in ERp46+ cells. The shERp46 cells lost the ability to upregulate protein disulfide isomerase following tunicamycin-induced ER stress. The present study suggests a role for ERp46 as a therapeutic

  7. Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response

    PubMed Central

    Gardner, Brooke M.; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M.; Walter, Peter

    2013-01-01

    Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1’s sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review. PMID:23388626

  8. Endoplasmic reticulum stress: The cause and solution to Huntington's disease?

    PubMed

    Jiang, Yuwei; Chadwick, Sarah R; Lajoie, Patrick

    2016-10-01

    Accumulation of misfolded proteins is a hallmark of many human diseases, including several incurable neurological disorders, such as Huntington's disease (HD). In HD, expansion of a polyglutamine stretch within the first exon of the Huntingtin protein (Htt) leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Several studies in various organisms (from yeast to humans) have identified the accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER stress) as a crucial determinant of cellular toxicity in HD. In this review, we highlight the recent research linking HD to ER stress. We also discuss how the modulation of signaling pathways responsible for coping with misfolded protein accumulation in the ER may constitute attractive methods to reduce toxicity and identify new therapeutic targets for treatment of HD. This article is part of a Special Issue entitled SI:ER stress.

  9. Co-chaperones of the mammalian endoplasmic reticulum.

    PubMed

    Melnyk, Armin; Rieger, Heiko; Zimmermann, Richard

    2015-01-01

    In mammalian cells, the rough endoplasmic reticulum or ER plays a central role in the biogenesis of most extracellular plus many organellar proteins and in cellular calcium homeostasis. Therefore, this organelle comprises molecular chaperones that are involved in import, folding/assembly, export, and degradation of polypeptides in millimolar concentrations. In addition, there are calcium channels/pumps and signal transduction components present in the ER membrane that affect and are affected by these processes. The ER lumenal Hsp70, termed immunoglobulin-heavy chain binding protein or BiP, is the central player in all these activities and involves up to seven different co-chaperones, i.e. ER-membrane integrated as well as ER-lumenal Hsp40s, which are termed ERj or ERdj, and two nucleotide exchange factors.

  10. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs

    PubMed Central

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W.; Schalek, Richard; Hayworth, Kenneth J.; Hand, Arthur R.; Yankova, Maya; Huber, Greg; Lichtman, Jeff W.; Rapoport, Tom A.; Kozlov, Michael M.

    2013-01-01

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. PMID:23870120

  11. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

    PubMed

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W; Schalek, Richard; Hayworth, Kenneth J; Hand, Arthur R; Yankova, Maya; Huber, Greg; Lichtman, Jeff W; Rapoport, Tom A; Kozlov, Michael M

    2013-07-18

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.

  12. Endoplasmic Reticulum Calcium, Stress and Cell-to-Cell Adhesion

    PubMed Central

    Mauro, Theodora

    2014-01-01

    Darier's Disease (DD) is caused by mutations in the endoplasmic reticulum (ER) Ca2+ ATPase ATP2A2 (protein SERCA2). Current treatment modalities are ineffective for many patients. This report shows that impaired SERCA2 function, both in DD keratinocytes and in normal keratinocytes treated with the SERCA2-inhibitor thapsigargin, depletes ER Ca2+ stores, leading to constitutive ER stress and increased sensitivity to ER stressors. ER stress, in turn, leads to abnormal cell-to-cell adhesion via impaired redistribution of desmoplakin, desmoglein 3, desmocollin 3 and E-cadherin to the plasma membrane. This report illustrates how ER Ca2+ depletion and the resulting ER stress are central to the pathogenesis of the disease. Additionally, the authors introduce a possible new therapeutic agent, Miglustat. PMID:24924761

  13. Crystalloid smooth endoplasmic reticulum in the quail uropygial gland.

    PubMed

    Fringes, B; Gorgas, K

    1993-06-01

    The occurrence, localization and organization of crystalloid smooth endoplasmic reticulum (SER) membrane aggregates in the male quail uropygial gland was investigated by electron microscopy. The lattice-like structures exhibiting a hexagonal honeycomb pattern are regularly found in the perinuclear region of the fully developed intermediate cell (type II) which is most effective in lipid biosynthesis and constitutes the middle layers of the stratified glandular epithelium undergoing sebaceous transformation. The crystalloids frequently exhibit a rectangular shape and tend to cluster, the latter exceeding 5 microns in length. They are composed of sets of highly ordered and densely packed tubular SER profiles. Diaminobenzidine (DAB) stained peroxisomes exhibit a close spatial relationship to the borders of crystalloids, but the organelles do not participate in the formation of these grid-like structures. The functional significance of the conformational change of the SER organization is not known. Local accumulation of specific lipogenic enzymes within this functional SER domain is discussed.

  14. WLS retrograde transport to the endoplasmic reticulum during Wnt secretion.

    PubMed

    Yu, Jia; Chia, Joanne; Canning, Claire Ann; Jones, C Michael; Bard, Frédéric A; Virshup, David M

    2014-05-12

    Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

  15. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain.

  16. Terasaki spiral ramps in the rough endoplasmic reticulum.

    PubMed

    Guven, Jemal; Huber, Greg; Valencia, Dulce María

    2014-10-31

    We present a model describing the morphology as well as the assembly of "Terasaki ramps," the recently discovered helicoidal connections linking adjacent sheets of the rough endoplasmic reticulum (ER). The fundamental unit is a localized symmetric double-ramped "parking garage" formed by two separated gently pitched, approximately helicoidal, ramps of opposite chiralities. This geometry is stabilized by a short-range repulsive interaction between ramps associated with bending energy which opposes the long-range attraction associated with tension. The ramp inner boundaries are themselves stabilized by the condensation of membrane-shaping proteins along their length. A mechanism for parking garage self-assembly is proposed involving the nucleation of dipoles at the center of tubular three-way junctions within the smooth ER. Our predictions are compared with the experimental data.

  17. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

    PubMed

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-07-25

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

  18. Interaction of the smooth endoplasmic reticulum and mitochondria.

    PubMed

    Goetz, J G; Nabi, I R

    2006-06-01

    The ER (endoplasmic reticulum) is composed of multiple domains including the nuclear envelope, ribosome-studded rough ER and the SER (smooth ER). The SER can also be functionally segregated into domains that regulate ER-Golgi traffic (transitional ER), ERAD (ER-associated degradation), sterol and lipid biosynthesis and calcium sequestration. The last two, as well as apoptosis, are critically regulated by the close association of the SER with mitochondria. Studies with AMFR (autocrine motility factor receptor) have defined an SER domain whose integrity and mitochondrial association can be modulated by ilimaquinone as well as by free cytosolic calcium levels in the normal physiological range. AMFR is an E3 ubiquitin ligase that targets its ligand directly to the SER via a caveolae/raft-dependent pathway. In the present review, we will address the relationship between the calcium-dependent morphology and mitochondrial association of the SER and its various functional roles in the cell.

  19. Terasaki Spiral Ramps in the Rough Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Guven, Jemal; Huber, Greg; Valencia, Dulce María

    2014-10-01

    We present a model describing the morphology as well as the assembly of "Terasaki ramps," the recently discovered helicoidal connections linking adjacent sheets of the rough endoplasmic reticulum (ER). The fundamental unit is a localized symmetric double-ramped "parking garage" formed by two separated gently pitched, approximately helicoidal, ramps of opposite chiralities. This geometry is stabilized by a short-range repulsive interaction between ramps associated with bending energy which opposes the long-range attraction associated with tension. The ramp inner boundaries are themselves stabilized by the condensation of membrane-shaping proteins along their length. A mechanism for parking garage self-assembly is proposed involving the nucleation of dipoles at the center of tubular three-way junctions within the smooth ER. Our predictions are compared with the experimental data.

  20. Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders

    PubMed Central

    Cai, Yu; Arikkath, Jyothi; Yang, Lu; Guo, Ming-Lei; Periyasamy, Palsamy; Buch, Shilpa

    2016-01-01

    ABSTRACT The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies. PMID:26902584

  1. Endoplasmic reticulum stress, pancreatic β-cell degeneration, and diabetes.

    PubMed

    Papa, Feroz R

    2012-09-01

    Overwhelming of protein folding in the endoplasmic reticulum (ER)--referred to as "ER stress"--activates a set of intracellular signaling pathways termed the unfolded protein response (UPR). Beneficial outputs of the UPR promote adaptation in cells experiencing manageably low levels of ER stress. However, if ER stress reaches critically high levels, the UPR uses destructive outputs to trigger programmed cell death. Genetic mutations in various UPR components cause inherited syndromes of diabetes mellitus in both rodents and humans, implicating the UPR in the proper functioning and survival of pancreatic islet β cells. Markers of chronically elevated ER stress, terminal UPR signaling, and apoptosis are evident in β cells in these rare disorders; these markers are similarly present in islets of human patients with common forms of diabetes. These findings promise to enhance our molecular understanding of human diabetes significantly and may lead to new and effective therapies.

  2. Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

    Wang, Pengwei; Hawes, Chris; Hussey, Patrick J

    2017-04-01

    The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants.

  3. Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum*

    PubMed Central

    Rose, April E.; Zhao, Chenguang; Turner, Elizabeth M.; Steyer, Anna M.; Schlieker, Christian

    2014-01-01

    Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures. PMID:24275647

  4. Novel ubiquitin-dependent quality control in the endoplasmic reticulum.

    PubMed

    Feldman, M; van der Goot, F Gisou

    2009-08-01

    Proteins of the endomembrane system undergo assisted folding in the endoplasmic reticulum (ER), then quality-control and, if misfolded, ER-associated degradation (ERAD). Recent findings on the biogenesis of a type-I membrane protein (an LRP6 mutant) lead us to hypothesize the existence of a novel mechanism promoting folding of membrane proteins from the cytosolic side of the ER. The proposed folding mechanism involves cycles of chaperone binding through mono-ubiquitylation and de-ubiquitylation, followed eventually by poly-ubiquitylation and ERAD. This suggests a novel dual role for ubiquitylation in the ER - dependent on the type of ubiquitin chains involved - in folding and in degradation, and highlights the potential importance of de-ubiquitylating enzymes.

  5. Endoplasmic reticulum stress in diabetes: New insights of clinical relevance.

    PubMed

    Balasubramanyam, Muthuswamy; Lenin, Raji; Monickaraj, Finny

    2010-04-01

    The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. Accumulating evidence suggests that ER stress plays a role in the pathogenesis of diabetes, contributing to pancreatic β-cell loss and insulin resistance. ER stress may also link obesity, inflammation and insulin resistance in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response in diabetes and its complications. Special attention was given to elucidate how ER stress could explain some of the 'clinical paradoxes' such as secondary sulfonylurea failure, initial worsening of retinopathy during tight glycemic control, insulin resistance induced by protease inhibitors and other clinically relevant observations.

  6. Assembly of MHC class I molecules within the endoplasmic reticulum.

    PubMed

    Zhang, Yinan; Williams, David B

    2006-01-01

    MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.

  7. Stress Responses from the Endoplasmic Reticulum in Cancer

    PubMed Central

    Kato, Hironori; Nishitoh, Hideki

    2015-01-01

    The endoplasmic reticulum (ER) is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR). The UPR also contributes to the regulation of various intracellular signaling pathways such as calcium signaling and lipid signaling. More recently, the mitochondria-associated ER membrane (MAM), which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signaling, inflammatory signaling, the autophagic response, and the UPR. Interestingly, in cancer, these signaling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signaling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM. PMID:25941664

  8. Pharmacological Modulators of Endoplasmic Reticulum Stress in Metabolic Diseases

    PubMed Central

    Jung, Tae Woo; Choi, Kyung Mook

    2016-01-01

    The endoplasmic reticulum (ER) is the principal organelle responsible for correct protein folding, a step in protein synthesis that is critical for the functional conformation of proteins. ER stress is a primary feature of secretory cells and is involved in the pathogenesis of numerous human diseases, such as certain neurodegenerative and cardiometabolic disorders. The unfolded protein response (UPR) is a defense mechanism to attenuate ER stress and maintain the homeostasis of the organism. Two major degradation systems, including the proteasome and autophagy, are involved in this defense system. If ER stress overwhelms the capacity of the cell’s defense mechanisms, apoptotic death may result. This review is focused on the various pharmacological modulators that can protect cells from damage induced by ER stress. The possible mechanisms for cytoprotection are also discussed. PMID:26840310

  9. Maternal obesity alters endoplasmic reticulum homeostasis in offspring pancreas.

    PubMed

    Soeda, Jumpei; Mouralidarane, Angelina; Cordero, Paul; Li, Jiawei; Nguyen, Vi; Carter, Rebeca; Kapur, Sabrina R; Pombo, Joaquim; Poston, Lucilla; Taylor, Paul D; Vinciguerra, Manlio; Oben, Jude A

    2016-06-01

    The prevalence of non-alcoholic fatty pancreas disease (NAFPD) is increasing in parallel with obesity rates. Stress-related alterations in endoplasmic reticulum (ER), such as the unfolded protein response (UPR), are associated with obesity. The aim of this study was to investigate ER imbalance in the pancreas of a mice model of adult and perinatal diet-induced obesity. Twenty female C57BL/6J mice were assigned to control (Con) or obesogenic (Ob) diets prior to and during pregnancy and lactation. Their offspring were weaned onto Con or Ob diets up to 6 months post-partum. Then, after sacrifice, plasma biochemical analyses, gene expression, and protein concentrations were measured in pancreata. Offspring of Ob-fed mice had significantly increased body weight (p < 0.001) and plasma leptin (p < 0.001) and decreased insulin (p < 0.01) levels. Maternal obesogenic diet decreased the total and phosphorylated Eif2α and increased spliced X-box binding protein 1 (XBP1). Pancreatic gene expression of downstream regulators of UPR (EDEM, homocysteine-responsive endoplasmic reticulum-resident (HERP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP)) and autophagy-related proteins (LC3BI/LC3BII) were differently disrupted by obesogenic feeding in both mothers and offspring (from p < 0.1 to p < 0.001). Maternal obesity and Ob feeding in their offspring alter UPR in NAFPD, with involvement of proapoptotic and autophagy-related markers. Upstream and downstream regulators of PERK, IRE1α, and ATF6 pathways were affected differently following the obesogenic insults.

  10. Carbon monoxide-releasing molecules reverse leptin resistance induced by endoplasmic reticulum stress.

    PubMed

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Kim, Seul-Ki; Uddin, Md Jamal; Rhew, Hyunyul; Kim, Taeksang; Ryter, Stefan W; Chung, Hun Taeg

    2013-04-01

    Leptin, a circulating hormone, regulates food intake and body weight. While leptin resistance represents a major cause of obesity, the underlying mechanisms remain unclear. Endoplasmic reticulum (ER) stress can contribute to leptin resistance. Carbon monoxide (CO), a gaseous molecule, exerts antiapoptotic and anti-inflammatory effects in animal models of tissue injury. We hypothesized that CO could inhibit leptin resistance during ER stress. Thapsigargin or tunicamycin was used to induce ER stress in human cells expressing the leptin receptor. These agents markedly inhibited leptin-induced STAT3 phosphorylation, confirming that ER stress induces leptin resistance. The CO-releasing molecule CORM-2 blocked the ER stress-dependent inhibition of leptin-induced STAT3 phosphorylation. CORM-2 treatment induced the phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), and eukaryotic translation initiation factor-2α and enhanced PERK phosphorylation during ER stress. Furthermore, CORM-2 inhibited X-box binding protein-1 expression, activating transcription factor-6 cleavage, and inositol-requiring enzyme (IRE)1α phosphorylation induced by ER stress. IRE1α knockdown rescued leptin resistance, whereas PERK knockdown blocked CO-dependent regulation of IRE1α. In vivo, CO inhalation normalized body weight in animals fed high-fat diets. Furthermore, CO modulated ER stress pathways and rescued leptin resistance in vivo. In conclusion, the pathological mechanism of leptin resistance may be ameliorated by the pharmacological application of CO.

  11. The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum.

    PubMed Central

    Thiyagarajah, P; Lim, S C

    1986-01-01

    A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [( Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane. PMID:2943271

  12. Selenium suppresses oxidative-stress-enhanced vascular smooth muscle cell calcification by inhibiting the activation of the PI3K/AKT and ERK signaling pathways and endoplasmic reticulum stress.

    PubMed

    Liu, Hongmei; Li, Xiaoming; Qin, Fei; Huang, Kaixun

    2014-03-01

    Vascular calcification is a prominent feature of many diseases, including atherosclerosis, and it has emerged as a powerful predictor of cardiovascular morbidity and mortality. A number of studies have examined the association between selenium and risk of cardiovascular diseases, but little is known about the role of selenium in vascular calcification. To determine the role of selenium in regulating vascular calcification, we assessed the effect of sodium selenite on oxidative-stress-enhanced vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Oxidative stress induced by xanthine/xanthine oxidase increased apoptosis, as determined by Hoechst 33342 staining and annexin V/propidium iodide staining, and it enhanced osteoblastic differentiation and calcification of VSMCs, on the basis of alkaline phosphatase activity, the expression of Runx2 and type I collagen, and calcium deposition. These effects of oxidative stress were significantly inhibited by selenite. The following processes may explain the inhibitory effects of selenite: (1) selenite significantly suppressed oxidative stress, as evidenced by the decrease of the oxidative status of the cell and lipid peroxidation levels, as well as by the increase of the total protein thiol content and the activity of the antioxidant selenoenzyme glutathione peroxidase; (2) selenite significantly attenuated oxidative-stress-induced activation of the phosphatidylinositol 3-kinase/AKT and extracellular-signal-regulated kinase signaling pathways, resulting in decreased osteoblastic differentiation of VSMCs; (3) selenite significantly inhibited oxidative-stress-activated endoplasmic reticulum stress, thereby leading to decreased apoptosis. Our results suggest a potential role of selenium in the prevention of vascular calcification, which may provide more mechanistic insights into the relationship between selenium and cardiovascular diseases.

  13. Toll-like receptor 4-induced endoplasmic reticulum stress contributes to endothelial dysfunction

    USDA-ARS?s Scientific Manuscript database

    Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Endoplasmic reticulum (ER) stress is implicated as one of the mechanisms for pathophysiology of various cardiometabolic syndromes, including insulin resistance and endothelial dysfunction. ...

  14. Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

    PubMed

    Maltseva, D V; Krainova, N A; Khaustova, N A; Nikulin, S V; Tonevitskaya, S A; Poloznikov, A A

    2017-08-01

    Viscumin (mistletoe lectin I, MLI) in concentrations of 10(-11)-10(-7) M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

  15. Endoplasmic Reticulum as a Site of Phenylpropanoid and Flavonoid Metabolism in Hippeastrum1

    PubMed Central

    Wagner, George J.; Hrazdina, Geza

    1984-01-01

    The nature of bound forms of enzymes of phenylpropanoid and flavonoid metabolism have been investigated in Hippeastrum CV Dutch Red Hybrid. Particulate components of petal homogenates were fractionated on sucrose gradients and the EDTA shift method was employed to characterize membranes of the endoplasmic reticulum. In magnesiumcontaining gradients, a portion of phenylalanine ammonia lyase, chalcone synthase, glucosyl transferase, and all of the trans-cinnamate 4-monooxygenase and NADH Cytochrome c reductase (the last an endoplasmic reticulum marker) were associated with membranes equilibrating at 1.18 specific gravity. In gradients lacking magnesium and containing EDTA, the above activities—except chalcone synthase, which was lost—and protein were diminished at 1.18 specific gravity and enhanced at lower densities characteristic of membranes of the smooth endoplasmic reticulum. These results are consistent with the contention that endoplasmic reticulum is a site of phenylpropanoid and flavonoid metabolism in Hippeastrum. PMID:16663530

  16. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    DTIC Science & Technology

    2015-07-01

    1 AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...COVERED 07/01/2014-06/30/2015 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a...NUMBER(S) 13. SUPPLEMENTARY NOTES 14. ABSTRACT We hypothesize that ER stress induced by glucose in diabetes promotes diabetic CKD through CRT stimulation

  17. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    DTIC Science & Technology

    2016-07-01

    AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...1 July 2015- 30 June 2016 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a...We hypothesize that ER stress induced by glucose in diabetes promotes diabetic CKD through CRT stimulation of TGF-beta-dependent calcium/NFAT

  18. GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na(+)-Ca(2+) exchanger and endoplasmic reticulum Ca(2+) refilling.

    PubMed

    Bondarenko, Alexander I; Montecucco, Fabrizio; Panasiuk, Olga; Sagach, Vadim; Sidoryak, Nataliya; Brandt, Karim J; Mach, François

    2017-02-01

    Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na(+)/Ca(2+) exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPβS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca(2+)-dependent K(+) channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca(2+)-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca(2+)-re-addition following Ca(2+) stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca(2+) refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Inhibition of endoplasmic reticulum chaperone protein glucose-regulated protein 78 potentiates anti-angiogenic therapy in renal cell carcinoma through inactivation of the PERK/eIF2α pathway

    PubMed Central

    Han, Kyung Seok; Li, Na; Raven, Pater A.; Fazli, Ladan; Frees, Sebastian; Ettinger, Susan; Park, Ki Chung; Hong, Sung Joon; Gleave, Martin E.; So, Alan I.

    2015-01-01

    Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC. PMID:26472187

  20. Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway

    PubMed Central

    Yu, Xin-Shuang; Du, Juan; Fan, Yu-Jun; Liu, Feng-Jun; Cao, Li-Li; Liang, Ning; Xu, De-Guo; Zhang, Jian-Dong

    2016-01-01

    Objective This study aims to investigate the effects of endoplasmic reticulum stress (ERS) on autophagy, apoptosis and chemoresistance of human small cell lung cancer (SCLC) cells via the PI3K/AKT/mTOR signaling pathway. Results The expressions of ERS-related proteins (PEAK, eIF2α and CHOP) up-regulated, autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis-related proteins (Bax and procaspase-3) down-regulated in NCI-H446 and H69 cells after tunicamycin treatment for 24 h. Compared with the blank group, the tunicamycin, BEZ235 and tunicamycin + BEZ235 groups exhibited decreased expressions of p-PI3K, p-AKT and p-mTOR, and increased expressions of autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis proteins (Bax and procaspase-3), and the most obvious changes were observed in the tunicamycin + BEZ235 group. Materials and Methods CCK-8 assay was applied to select the best cell line from five SCLC cell lines (NCI-H446, H69, H526, H146 and H209). Finally, NCI-H446 and H69 cells were selected for further experiments. NCI-H446/CDDP and H69/CDDP were selected and divided into the blank group, tunicamycin (an ESR inducer) group, BEZ235 (inhibitors of PI3K/AKT/mTOR pathway) group and tunicamycin + BEZ235 group. Cell apoptosis was detected by flow cytometry. Autophagy was observed by fluorescence microscopy and flow cytometry. Western blotting was used to detect the expressions of ERS-related proteins, autophagy-related proteins, apoptosis-related proteins and PI3K/AKT/mTOR pathway-related proteins. Conclusions Our findings provide evidence that the activation of ERS could promote autophagy and apoptosis and reverse chemoresistance of human SCLC cells by inhibiting the PI3K/AKT/mTOR pathway. PMID:27765907

  1. Inhibition of endoplasmic reticulum stress-activated IRE1α-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rat model of Parkinson's disease.

    PubMed

    Tong, Qiang; Wu, Liang; Jiang, Teng; Ou, Zhou; Zhang, Yingdong; Zhu, Dongya

    2016-04-05

    Telmisartan, one unique angiotensin II type 1 receptor blocker, has been attracting attention due to its putative peroxisome proliferator-activated receptor (PPAR)-γ or β/δ actions. Recently, telmisartan has been reported to exert neuroprotective effects in animal models of Parkinson's disease (PD). However, the underlying mechanisms have not been fully clarified. Recently, accumulating evidence has shown that endoplasmic reticulum (ER) stress plays a crucial role in rotenone-induced neuronal apoptosis. Additionally, studies have revealed that inositol-requiring enzyme/endonuclease 1α (IRE1α) is necessary and sufficient to trigger ER stress. In the present study, we aimed to determine whether ER stress-activated IRE1α-mediated apoptotic pathway is involved in the neuroprotection of telmisartan in the rotenone rats of PD and explore the possible involvement of PPAR-β/δ activation. The catalepsy tests were performed to test the catalepsy symptom. The dopamine content and α-synuclein expression were ascertained through high-performance liquid chromatography and immunohistochemistry, respectively. The expression of IRE1α, TNF receptor associated factor 2 (TRAF2), caspase-12 and PPAR-β/δ was detected by western blot. Neuronal apoptosis was assessed by TUNEL and immunohistochemistry. Our results show that telmisartan ameliorated the catalepsy symptom and attenuated dopamine depletion as well as α-synuclein accumulation. Moreover, telmisartan decreased ER stress-mediated neuronal apoptosis. Furthermore, telmisartan inhibited IRE1α-TRAF2-caspase-12 apoptotic signaling pathway. Additionally, telmisartan activated PPAR β/δ, implying that PPAR-β/δ activation properties of telmisartan are possibly or partially involved in the neuroprotective effects. In conclusion, our findings suggest that suppressing ER stress-activated IRE1α-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rats of PD. Copyright

  2. The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

    PubMed

    Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

    2016-09-15

    Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q(2)cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as

  3. Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology

    PubMed Central

    Chong, Wai Chin; Shastri, Madhur D.; Eri, Rajaraman

    2017-01-01

    The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases. PMID:28379196

  4. Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

    PubMed

    Korkhov, Vladimir M; Zuber, Benoît

    2009-08-24

    Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

  5. Endoplasmic Reticulum Stress Plays a Key Role in the Pathogenesis of Diabetic Peripheral Neuropathy

    PubMed Central

    Lupachyk, Sergey; Watcho, Pierre; Stavniichuk, Roman; Shevalye, Hanna; Obrosova, Irina G.

    2013-01-01

    Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins impairs metabolism, transcriptional regulation, and gene expression, and it is a key mechanism of cell injury. Endoplasmic reticulum stress plays an important role in cardiovascular and neurodegenerative diseases, cancer, and diabetes. We evaluated the role for this phenomenon in diabetic peripheral neuropathy. Endoplasmic reticulum stress manifest in upregulation of multiple components of unfolded protein response was identified in neural tissues (sciatic nerve, spinal cord) of streptozotocin diabetic rats and mice. A chemical chaperone, trimethylamine oxide, administered for 12 weeks after induction of diabetes (110 mg⋅kg−1⋅d−1, a prevention paradigm) attenuated endoplasmic reticulum stress, peripheral nerve dysfunction, intraepidermal nerve fiber loss, and sciatic nerve and spinal cord oxidative-nitrative stress in streptozotocin diabetic rats. Similar effects on diabetes-induced endoplasmic reticulum stress and peripheral nerve dysfunction were observed with a structurally unrelated chemical chaperone, 4-phenylbutyric acid (100 mg⋅kg−1⋅d−1, intraperitoneal). CCAAT/enhancer-binding protein homologous protein (CHOP)−/− mice made diabetic with streptozotocin displayed less severe sciatic nerve oxidative-nitrative stress and peripheral neuropathy than the wild-type (C57Bl6/J) mice. Neither chemical chaperones nor CHOP gene deficiency reduced diabetic hyperglycemia. Our findings reveal an important role of endoplasmic reticulum stress in the development of diabetic peripheral neuropathy and identify a potential new therapeutic target. PMID:23364451

  6. HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

    SciTech Connect

    Luo, Ying; Li, Shu-Jun; Yang, Jian; Qiu, Yuan-Zhen; Chen, Fang-Ping

    2013-09-06

    Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

  7. Vesicular trafficking of incoming human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-secretase activity.

    PubMed

    Zhang, Wei; Kazakov, Teymur; Popa, Andreea; DiMaio, Daniel

    2014-09-16

    The route taken by papillomaviruses from the cell surface to the nucleus during infection is incompletely understood. Here, we developed a novel human papillomavirus 16 (HPV16) pseudovirus in which the carboxy terminus of the minor capsid protein L2 is exposed on the exterior of the intact capsid prior to cell binding. With this pseudovirus, we used the proximity ligation assay immune detection technique to demonstrate that during entry HPV16 L2 traffics into and out of the early endosome prior to Golgi localization, and we demonstrated that L2 enters the endoplasmic reticulum during entry. The cellular membrane-associated protease, γ-secretase, is required for infection by HPV16 pseudovirus and authentic HPV16. We also showed that inhibition of γ-secretase does not interfere substantively with virus internalization, initiation of capsid disassembly, entry into the early endosome, or exit from this compartment, but γ-secretase is required for localization of L2 and viral DNA to the Golgi apparatus and the endoplasmic reticulum. These results show that incoming HPV16 traffics sequentially from the cell surface to the endosome and then to the Golgi apparatus and the endoplasmic reticulum prior to nuclear entry. The human papillomaviruses are small nonenveloped DNA viruses responsible for approximately 5% of all human cancer deaths, but little is known about the process by which these viruses transit from the cell surface to the nucleus. Here we show that incoming HPV16, the most common high-risk HPV, traffics though a series of vesicular compartments during infectious entry, including the endosome, Golgi apparatus, and endoplasmic reticulum. Furthermore, we show that γ-secretase, a cellular membrane-associated protease, is required for entry of the L2 minor capsid protein and viral DNA into the Golgi apparatus and endoplasmic reticulum. These studies reveal a new pathway of cell entry by DNA viruses and suggest that components of this pathway are candidate

  8. Apoptosis, autophagy & endoplasmic reticulum stress in diabetes mellitus

    PubMed Central

    Demirtas, Levent; Guclu, Aydin; Erdur, Fatih Mehmet; Akbas, Emin Murat; Ozcicek, Adalet; Onk, Didem; Turkmen, Kultigin

    2016-01-01

    The prevalence of diabetes mellitus (DM) is increasing secondary to increased consumption of food and decreased physical activity worldwide. Hyperglycaemia, insulin resistance and hypertrophy of pancreatic beta cells occur in the early phase of diabetes. However, with the progression of diabetes, dysfunction and loss of beta cells occur in both types 1 and 2 DM. Programmed cell death also named apoptosis is found to be associated with diabetes, and apoptosis of beta cells might be the main mechanism of relative insulin deficiency in DM. Autophagic cell death and apoptosis are not entirely distinct programmed cell death mechanisms and share many of the regulator proteins. These processes can occur in both physiologic and pathologic conditions including DM. Besides these two important pathways, endoplasmic reticulum (ER) also acts as a cell sensor to monitor and maintain cellular homeostasis. ER stress has been found to be associated with autophagy and apoptosis. This review was aimed to describe the interactions between apoptosis, autophagy and ER stress pathways in DM. PMID:28256459

  9. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  10. Unique defense strategy by the endoplasmic reticulum body in plants.

    PubMed

    Yamada, Kenji; Hara-Nishimura, Ikuko; Nishimura, Mikio

    2011-12-01

    The endoplasmic reticulum (ER) is a site for the production of secretory proteins. Plants have developed ER subdomains for protein storage. The ER body is one such structure, which is observed in Brassicaceae plants. ER bodies accumulate in seedlings and roots or in wounded leaves in Arabidopsis. ER bodies contain high amounts of the β-glucosidases PYK10/BGLU23 in seedlings and roots or BGLU18 in wounded tissues. These results suggest that ER bodies are involved in the metabolism of glycoside molecules, presumably to produce repellents against pests and fungi. When Arabidopsis roots are homogenized, PYK10 formed large protein aggregates that include other β-glucosidases (BGLU21 and BGLU22), GDSL lipase-like proteins (GLL22) and cytosolic jacalin-related lectins (PBP1/JAL30, JAL31, JAL33, JAL34 and JAL35). Glucosidase activity increases by the aggregate formation. NAI1, a basic helix-loop-helix transcription factor, regulates the expression of the ER body proteins PYK10 and NAI2. Reduced expression of NAI2, PYK10 and BGLU21 resulted in abnormal ER body formation, indicating that these components regulate ER body formation. PYK10, BGLU21 and BGLU22 possess hydrolytic activity for scopolin, a coumaroyl glucoside that accumulates in the roots of Arabidopsis, and nai1 and pyk10 mutants are more susceptible to the symbiotic fungus Piriformospora indica. Therefore, it appears that the ER body is a unique organelle of Brassicaceae plants that is important for defense against pests and fungi.

  11. Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

    PubMed Central

    Swanson, M S; Isberg, R R

    1995-01-01

    Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication. PMID:7642298

  12. Coordination of Endoplasmic Reticulum (ER) Signaling During Maize Seed Development

    SciTech Connect

    Boston, Rebecca S.

    2010-11-20

    Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesize storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies. Our underlying hypothesis is that the endoplasmic reticulum (ER) stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presence of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly. The capacity of the ER to deposit these aberrant proteins in protein bodies during a period that extends several weeks provides an excellent model for deconvoluting the ER stress response of plants. We have focused in this project on the means by which the ER senses and responds to functional perturbations and the underlying intracellular communication that occurs among biosynthetic, trafficking and degradative pathways for proteins during seed development.

  13. Hydrogen sulfide, endoplasmic reticulum stress and alcohol mediated neurotoxicity.

    PubMed

    George, Akash K; Behera, Jyotirmaya; Kelly, Kimberly E; Zhai, Yuankun; Tyagi, Neetu

    2017-02-14

    Alcohol is one of the most socially accepted addictive drugs in modern society. Its abuse affects virtually all organ systems with the central nervous system (CNS) being particularly vulnerable to excessive alcohol exposure. Alcohol exposure also causes profound damage to both the adult and developing brain. Excessive alcohol consumption induces numerous pathophysiological stress responses, one of which is the endoplasmic reticulum (ER) stress response. Potential mechanisms that trigger the alcohol induced ER stress response are either directly or indirectly related to alcohol metabolism, which include toxic levels of acetaldehyde and homocysteine, oxidative stress and abnormal epigenetic modifications. Growing evidence suggests that H2S is the most recently recognized gasotransmitter with tremendous physiological protective functions against oxidative stress induced neurotoxicity. In this review we address the alcohol induced oxidative stress mediated ER stress and the role of H2S in its mitigation in the context of alcohol neurotoxicity. Interruption of ER stress triggers is anticipated to have therapeutic benefits for alcohol mediated diseases and disorders.

  14. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    DOE PAGES

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; ...

    2016-05-23

    Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). Here in this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered intomore » all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.« less

  15. Heme oxygenase-1 comes back to endoplasmic reticulum

    SciTech Connect

    Kim, Hong Pyo; Pae, Hyun-Ock; Back, Sung Hun; Chung, Su Wol; Woo, Je Moon; Son, Yong; Chung, Hun-Taeg

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  16. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

  17. The Role of the Endoplasmic Reticulum in Peroxisome Biogenesis

    PubMed Central

    Dimitrov, Lazar; Lam, Sheung Kwan; Schekman, Randy

    2013-01-01

    Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis. PMID:23637287

  18. The role of the endoplasmic reticulum in peroxisome biogenesis.

    PubMed

    Dimitrov, Lazar; Lam, Sheung Kwan; Schekman, Randy

    2013-05-01

    Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis.

  19. Apoptosis, autophagy & endoplasmic reticulum stress in diabetes mellitus.

    PubMed

    Demirtas, Levent; Guclu, Aydin; Erdur, Fatih Mehmet; Akbas, Emin Murat; Ozcicek, Adalet; Onk, Didem; Turkmen, Kultigin

    2016-10-01

    The prevalence of diabetes mellitus (DM) is increasing secondary to increased consumption of food and decreased physical activity worldwide. Hyperglycaemia, insulin resistance and hypertrophy of pancreatic beta cells occur in the early phase of diabetes. However, with the progression of diabetes, dysfunction and loss of beta cells occur in both types 1 and 2 DM. Programmed cell death also named apoptosis is found to be associated with diabetes, and apoptosis of beta cells might be the main mechanism of relative insulin deficiency in DM. Autophagic cell death and apoptosis are not entirely distinct programmed cell death mechanisms and share many of the regulator proteins. These processes can occur in both physiologic and pathologic conditions including DM. Besides these two important pathways, endoplasmic reticulum (ER) also acts as a cell sensor to monitor and maintain cellular homeostasis. ER stress has been found to be associated with autophagy and apoptosis. This review was aimed to describe the interactions between apoptosis, autophagy and ER stress pathways in DM.

  20. Endoplasmic Reticulum Stress Interacts With Inflammation in Human Diseases.

    PubMed

    Cao, Stewart Siyan; Luo, Katherine L; Shi, Lynn

    2016-02-01

    The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro-apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways.

  1. Endoplasmic reticulum stress response in yeast and humans

    PubMed Central

    Wu, Haoxi; Ng, Benjamin S. H.; Thibault, Guillaume

    2014-01-01

    Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases. PMID:24909749

  2. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    PubMed

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  3. Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.

    PubMed

    Missiroli, Sonia; Morganti, Claudia; Giorgi, Carlotta; Pinton, Paolo

    2016-01-01

    Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.

  4. Live cell imaging of protein dislocation from the endoplasmic reticulum.

    PubMed

    Zhong, Yongwang; Fang, Shengyun

    2012-08-10

    Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.

  5. Small GTPases and Brucella entry into the endoplasmic reticulum.

    PubMed

    de Bolle, Xavier; Letesson, Jean-Jacques; Gorvel, Jean-Pierre

    2012-12-01

    A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

  6. Chlorpyrifos induces endoplasmic reticulum stress in JEG-3 cells.

    PubMed

    Reyna, Luciana; Flores-Martín, Jésica; Ridano, Magali E; Panzetta-Dutari, Graciela M; Genti-Raimondi, Susana

    2017-04-01

    Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50μM or 100μM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53.

  7. Arachidonoyl-Specific Diacylglycerol Kinase ε and the Endoplasmic Reticulum

    PubMed Central

    Nakano, Tomoyuki; Matsui, Hirooki; Tanaka, Toshiaki; Hozumi, Yasukazu; Iseki, Ken; Kawamae, Kaneyuki; Goto, Kaoru

    2016-01-01

    The endoplasmic reticulum (ER) comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA) serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK) that phosphorylates diacylglycerol (DG) to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology, and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER. PMID:27917381

  8. Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.

    PubMed

    Rieusset, Jennifer

    2017-06-01

    The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca(2+)) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca(2+) signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.

    PubMed

    Xue, Yong; Schmollinger, Stefan; Attar, Narsis; Campos, Oscar A; Vogelauer, Maria; Carey, Michael F; Merchant, Sabeeha S; Kurdistani, Siavash K

    2017-08-11

    The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Altered localization of amyloid precursor protein under endoplasmic reticulum stress.

    PubMed

    Kudo, Takashi; Okumura, Masayo; Imaizumi, Kazunori; Araki, Wataru; Morihara, Takashi; Tanimukai, Hitoshi; Kamagata, Eiichiro; Tabuchi, Nobuhiko; Kimura, Ryo; Kanayama, Daisuke; Fukumori, Akio; Tagami, Shinji; Okochi, Masayasu; Kubo, Mikiko; Tanii, Hisashi; Tohyama, Masaya; Tabira, Takeshi; Takeda, Masatoshi

    2006-06-02

    Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.

  11. Naltrexone attenuates endoplasmic reticulum stress induced hepatic injury in mice.

    PubMed

    Moslehi, A; Nabavizadeh, F; Nabavizadeh, Fatemeh; Dehpour, A R; Dehpou, A R; Tavanga, S M; Hassanzadeh, G; Zekri, A; Nahrevanian, H; Sohanaki, H

    2014-09-01

    Endoplasmic reticulum (ER) stress provides abnormalities in insulin action, inflammatory responses, lipoprotein B100 degradation and hepatic lipogenesis. Excess accumulation of triglyceride in hepatocytes may also lead to disorders such as non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Opioid peptides are involved in triglyceride and cholesterol dysregulation, inflammation and cell death. In this study, we evaluated Naltrexone effects on ER stress induced liver injury. To do so, C57/BL6 mice received saline, DMSO and Naltrexone, as control groups. ER stress was induced by tunicamycin (TM) injection. Naltrexone was given before TM administration. Liver blood flow and biochemical serum analysis were measured. Histopathological evaluations, TNF-α measurement and Real-time RT-PCR were also performed. TM challenge provokes steatosis, cellular ballooning and lobular inflammation which significantly reduced in Naltrexone treated animals. ALT, AST and TNF-α increased in the TM group and improved in the Naltrexone plus TM group. Triglyceride and cholesterol levels decreased in TM treated mice with no increase in Naltrexone treated animals. In the Naltrexone plus TM group, gene expression of Bax/Bcl-2 ratio and caspase3 significantly lowered compared with the TM group. In this study, we found that Naltrexone had a notable alleviating role in ER stress induced steatosis and liver injury.

  12. Protein misfolding and endoplasmic reticulum stress in chronic lung disease.

    PubMed

    Wei, James; Rahman, Sadaf; Ayaub, Ehab A; Dickhout, Jeffrey G; Ask, Kjetil

    2013-04-01

    The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or "proteostasis," prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases.

  13. Protein quality control, retention, and degradation at the endoplasmic reticulum.

    PubMed

    Benyair, Ron; Ron, Efrat; Lederkremer, Gerardo Z

    2011-01-01

    In order to maintain proper cellular functions, all living cells, from bacteria to mammalian cells, must carry out a rigorous quality control process in which nascent and newly synthesized proteins are examined. An important role of this process is to protect cells against pathological accumulation of unfolded and misfolded proteins. The endoplasmic reticulum (ER) has evolved as a staging ground for secretory protein synthesis with distinct sites for entry, quality control, and exit. In the ER, most proteins are N-glycosylated, a posttranslational modification that defines the quality control pathway that the protein will undergo. The folding state of glycoproteins is revealed by specific modifications of their N-glycans. Regardless of size and posttranslational modifications, the folding states of all proteins must be identified as unfolded, properly folded, or terminally misfolded and accordingly subjected to ER retention and continued folding attempts, export and maturation, or retrotranslocation to the cytosol for degradation. These processes involve specialized machineries that utilize molecular chaperones, protein- and N-glycan-modifying enzymes, and lectins for protein folding and quality control and ubiquitination and degradation machineries for disposal. All these machineries are regulated by a signaling pathway, the unfolded protein response, which upregulates ER functions when under the stress of high protein load. Here, we describe the molecular mechanisms that are implicated and discuss recent data that underline the importance of compartmentalization in the segregation of the various functions of the ER for their correct function.

  14. Selective export of autotaxin from the endoplasmic reticulum.

    PubMed

    Lyu, Lin; Wang, Baolu; Xiong, Chaoyang; Zhang, Xiaotian; Zhang, Xiaoyan; Zhang, Junjie

    2017-04-28

    Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Prognosis of oocytes showing aggregation of smooth endoplasmic reticulum.

    PubMed

    Ebner, Thomas; Moser, Marianne; Shebl, Omar; Sommerguber, Michael; Tews, Gernot

    2008-01-01

    Few cytoplasmic dysmorphisms of oocytes have been reported to negatively influence the further fate of the ova. One such anomaly, namely the central aggregation of the smooth endoplasmic reticulum (SER), has recently been associated with suboptimal outcome in a limited number of patients. In order to increase prognostic value, it was decided to prospectively screen all intracytoplasmic sperm injection patients within 1 year for eggs showing aggregations of SER. In addition, all deliveries (obstetric and neonatal data) were analysed. Occurrence of SER cluster was related to duration (P < 0.001) and dosage (P < 0.01) of the stimulation. Fertilization (58.9%) and blastulation rate (44.0%) were lower (P < 0.01) in affected ova compared with unaffected counterparts (77.4 and 87.8%, respectively). Pregnancies in women with affected gametes were accompanied by a higher incidence of obstetric problems (P < 0.01) leading to a non-significant trend towards earlier delivery and significantly reduced birthweight (P < 0.05). It is strongly recommended to avoid transfer of embryos/blastocysts derived from SER cluster-positive gametes. Patients have to be informed that even transfer of sibling oocytes without this anomaly involves a higher risk of detrimental outcome.

  16. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

    PubMed Central

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-01-01

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function. DOI: http://dx.doi.org/10.7554/eLife.23882.001 PMID:28742022

  17. Causes and consequences of endoplasmic reticulum stress in rheumatic disease.

    PubMed

    Navid, Fatemeh; Colbert, Robert A

    2017-01-01

    Rheumatic diseases represent a heterogeneous group of inflammatory conditions, many of which involve chronic activation of both innate and adaptive immune responses by multiple genetic and environmental factors. These immune responses involve the secretion of excessive amounts of cytokines and other signalling mediators by activated immune cells. The endoplasmic reticulum (ER) is the cellular organelle that directs the folding, processing and trafficking of membrane-bound and secreted proteins, including many key components of the immune response. Maintaining homeostasis in the ER is critical to cell function and survival. Consequently, elaborate mechanisms have evolved to sense and respond to ER stress through three main signalling pathways that together comprise the unfolded protein response (UPR). Activation of the UPR can rapidly resolve the accumulation of misfolded proteins, direct permanent changes in the size and function of cells during differentiation, and critically influence the immune response and inflammation. Recognition of the importance of ER stress and UPR signalling pathways in normal and dysregulated immune responses has greatly increased in the past few years. This Review discusses several settings in which ER stress contributes to the pathogenesis of rheumatic diseases and considers some of the therapeutic opportunities that these discoveries provide.

  18. Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

    PubMed

    Song, Yanping; Jiang, Ying; Ying, Wantao; Gong, Yan; Yan, Yujuan; Yang, Dong; Ma, Jie; Xue, Xiaofang; Zhong, Fan; Wu, Songfeng; Hao, Yunwei; Sun, Aihua; Li, Tao; Sun, Wei; Wei, Handong; Zhu, Yunping; Qian, Xiaohong; He, Fuchu

    2010-03-05

    To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle.

  19. Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate

    PubMed Central

    Corazzari, Marco; Gagliardi, Mara; Fimia, Gian Maria; Piacentini, Mauro

    2017-01-01

    Perturbation of endoplasmic reticulum (ER) homeostasis results in a stress condition termed “ER stress” determining the activation of a finely regulated program defined as unfolded protein response (UPR) and whose primary aim is to restore this organelle’s physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s) regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch. PMID:28491820

  20. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    SciTech Connect

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; Kohalmi, Susanne E.; Menassa, Rima

    2016-05-23

    Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). Here in this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

  1. Proteostasis: bad news and good news from the endoplasmic reticulum.

    PubMed

    Noack, Julia; Brambilla Pisoni, Giorgia; Molinari, Maurizio

    2014-01-01

    The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms' homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

  2. Coping with endoplasmic reticulum stress in the cardiovascular system.

    PubMed

    Groenendyk, Jody; Agellon, Luis B; Michalak, Marek

    2013-01-01

    The endoplasmic reticulum (ER) is a multifunctional intracellular organelle, a component of the cellular reticular network that allows cells to adjust to a wide variety of conditions. The cardiomyocyte reticular network is the ideal location of sensors for both intrinsic and extrinsic factors that disrupt energy and/or nutrient homeostasis and lead to ER stress, a disturbance in ER function. ER stress has been linked to both physiological and pathological states in the cardiovascular system; such states include myocardial infarction, oxygen starvation (hypoxia) and fuel starvation, ischemia, pressure overload, dilated cardiomyopathy, hypertrophy, and heart failure. The ER stress coping response (e.g., the unfolded protein response) is composed of discrete pathways that are controlled by a collection of common regulatory components that may function as a single entity involved in reacting to ER stress. These corrective strategies allow the cardiomyocyte reticular network to restore energy and/or nutrient homeostasis and to avoid cell death. Therefore, the identities of the ER stress corrective strategies are important targets for the development of therapeutic approaches for cardiovascular and other acquired disorders.

  3. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    PubMed Central

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; Kohalmi, Susanne E.; Menassa, Rima

    2016-01-01

    Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles. PMID:27242885

  4. A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress

    PubMed Central

    Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d’Hellencourt, Christian; Ravanan, Palaniyandi

    2014-01-01

    Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress. PMID:25120434

  5. PROTEOMICS ANALYSIS OF ROUGH ENDOPLASMIC RETICULUM IN PANCREATIC BETA CELLS

    PubMed Central

    Lee, Jin-sook; Wu, Yanning; Skallos, Patracia; Fang, Jingye; Zhang, Xuebao; Karnovsky, Alla; Woods, James; Stemmer, Paul M.; Liu, Ming; Zhang, Kezhong; Chen, Xuequn

    2015-01-01

    Pancreatic beta cells have well-developed endoplasmic reticulum (ER) to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by one dimensional SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. Gene ontology analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. PMID:25546123

  6. The Mammalian Endoplasmic Reticulum-Associated Degradation System

    PubMed Central

    Olzmann, James A.; Kopito, Ron R.; Christianson, John C.

    2013-01-01

    The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells. PMID:23232094

  7. Terasaki Ramps in the Endoplasmic Reticulum: Structure, Function and Formation

    NASA Astrophysics Data System (ADS)

    Huber, Greg; Guven, Jemal; Valencia, Dulce-Maria

    2015-03-01

    The endoplasmic reticulum (ER) has long been considered an exceedingly important and complex cellular organelle in eukaryotes (like you). It is a membrane structure, part folded lamellae, part tubular network, that both envelopes the nucleus and threads its way outward, all the way to the cell's periphery. Despite the elegant mechanics of bilayer membranes offered by the work of Helfrich and Canham, as far as the ER is concerned, theory has mostly sat on the sidelines. However, refined imaging of the ER has recently revealed beautiful and subtle geometrical forms - simple geometries, from the mathematical point of view - which some have called a ``parking garage for ribosomes.'' I'll review the discovery and physics of Terasaki ramps and discuss their relation to cell-biological questions, such as ER and nuclear-membrane re-organization during mitosis. Rather than being a footnote in a textbook on differential geometry, these structures suggest answers to a number of the ER's structure-function problems.

  8. PERK-opathies: An Endoplasmic Reticulum Stress Mechanism Underlying Neurodegeneration.

    PubMed

    Bell, Michelle C; Meier, Shelby E; Ingram, Alexandria L; Abisambra, Jose F

    2016-01-01

    The unfolded protein response (UPR) plays a vital role in maintaining cell homeostasis as a consequence of endoplasmic reticulum (ER) stress. However, prolonged UPR activity leads to cell death. This time-dependent dual functionality of the UPR represents the adaptive and cytotoxic pathways that result from ER stress. Chronic UPR activation in systemic and neurodegenerative diseases has been identified as an early sign of cellular dyshomeostasis. The Protein Kinase R-like ER Kinase (PERK) pathway is one of three major branches in the UPR, and it is the only one to modulate protein synthesis as an adaptive response. The specific identification of prolonged PERK activity has been correlated with the progression of disorders such as diabetes, Alzheimer's disease, and cancer, suggesting that PERK plays a role in the pathology of these disorders. For the first time, the term "PERK-opathies" is used to group these diseases in which PERK mediates detriment to the cell culminating in chronic disorders. This article reviews the literature documenting links between systemic disorders with the UPR, but with a specific emphasis on the PERK pathway. Then, articles reporting links between the UPR, and more specifically PERK, and neurodegenerative disorders are presented. Finally, a therapeutic perspective is discussed, where PERK interventions could be potential remedies for cellular dysfunction in chronic neurodegenerative disorders.

  9. Thermal Shock Induces Host Proteostasis Disruption and Endoplasmic Reticulum Stress in the Model Symbiotic Cnidarian Aiptasia.

    PubMed

    Oakley, Clinton A; Durand, Elysanne; Wilkinson, Shaun P; Peng, Lifeng; Weis, Virginia M; Grossman, Arthur R; Davy, Simon K

    2017-06-02

    Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 and 33.5 °C, 12 days) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation and degrading damaged proteins, which is indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress.

  10. The protective effect of the earthworm active ingredients on hepatocellular injury induced by endoplasmic reticulum stress.

    PubMed

    Wang, Qi; Duan, Leng-Xin; Xu, Zheng-Shun; Wang, Jian-Gang; Xi, Shou-Min

    2016-08-01

    The earthworm is a widely used Chinese herbal medicine. There are more than 40 prescriptions including earthworms in the "Compendium of Materia Medica". TCM theory holds that earthworms exert antispasmodic and antipyretic effects through the liver meridian to calm the liver. However, the clinical effect of earthworms on liver injury has not been clearly demonstrated. We have previously established a method to extract the active ingredients from earthworms (hereinafter referred to as EWAs) [1]. In the present study, we observed protective effect of the EWAs on tunicamycin-induced ERS (endoplasmic reticulum stress) model in human hepatic L02 cells. The results showed that the EWAs promote proliferation and reduced apoptosis of ERS model in L02 cells (P<0.01). The up-regulation of ERS-related proteins, including PERK (protein kinase RNA-like endoplasmic reticulum kinase), eIF2a (eukaryotic translation initiation factor 2a), ATF4 (activating transcription factor 4) and CHOP (CCAAT/enhancer binding protein homologous protein), in L02 cell under ERS was inhibited by treatment of the EWAs (P<0.01). In summary, our data suggest the EWAs can significant attenuate ERS-induced hepatocyte injury via PERK-eIF2a-ATF4 pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.

    PubMed

    Mund, Thomas; Gewies, Andreas; Schoenfeld, Nicole; Bauer, Manuel K A; Grimm, Stefan

    2003-04-01

    We have isolated Spike, a novel and evolutionary conserved BH3-only protein. BH3-only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3-only proteins, Spike could not interact with all tested Bcl-2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro-caspase-8 and Bcl-XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl-XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down-regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3-only and antiapoptotic Bcl-2 proteins regulate cell death.

  12. Gelatin Nanostructured Lipid Carriers Incorporating Nerve Growth Factor Inhibit Endoplasmic Reticulum Stress-Induced Apoptosis and Improve Recovery in Spinal Cord Injury.

    PubMed

    Zhu, Si-Pin; Wang, Zhou-Guang; Zhao, Ying-Zheng; Wu, Jiang; Shi, Hong-Xue; Ye, Li-Bing; Wu, Fen-Zan; Cheng, Yi; Zhang, Hong-Yu; He, Songbin; Wei, Xiaojie; Fu, Xiao-Bing; Li, Xiao-Kun; Xu, Hua-Zi; Xiao, Jian

    2016-09-01

    Clinical translation of growth factor therapies faces multiple challenges; the most significant one is the short half-life of the naked protein. Gelatin nanostructured lipid carriers (GNLs) had previously been used to encapsulate the basic fibroblast growth factor to enhance the functional recovery in hemiparkinsonian rats. In this research, we comparatively study the enhanced therapy between nerve growth factor (NGF) loaded GNLs (NGF-GNLs) and NGF only in spinal cord injury (SCI). The effects of NGF-GNLs and NGF only were tested by the Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test, and footprint analysis. Western blot analysis and immunofluorescent staining were further performed to identify the expression of ER stress-related proteins, neuron-specific marker neuronal nuclei (NeuN), and growth-associated protein 43 (GAP43). Correlated downstream signals Akt/GSK-3β and ERK1/2 were also analyzed with or without inhibitors. Results showed that NGF-GNLs, compared to NGF only, enhanced the neuroprotection effect in SCI rats. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 inhibited by NGF-GNL treatment were more obvious. Meanwhile, NGF-GNLs in the recovery of SCI are related to the inhibition of ER stress-induced cell death via the activation of downstream signals PI3K/Akt/GSK-3β and ERK1/2.

  13. Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

    SciTech Connect

    Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

    2008-01-15

    The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.

  14. CDIP1-BAP31 complex transduces apoptotic signals from endoplasmic reticulum to mitochondria under endoplasmic reticulum stress.

    PubMed

    Namba, Takushi; Tian, Fang; Chu, Kiki; Hwang, So-Young; Yoon, Kyoung Wan; Byun, Sanguine; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W

    2013-10-31

    Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

  15. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib.

    PubMed

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy.

  16. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    PubMed Central

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  17. Tumor Hypoxia Blocks Wnt Processing and Secretion through the Induction of Endoplasmic Reticulum Stress▿

    PubMed Central

    Verras, Meletios; Papandreou, Ioanna; Lim, Ai Lin; Denko, Nicholas C.

    2008-01-01

    Poorly formed tumor blood vessels lead to regions of microenvironmental stress due to depletion of oxygen and glucose and accumulation of waste products (acidosis). These conditions contribute to tumor progression and correlate with poor patient prognosis. Here we show that the microenvironmental stresses found in the solid tumor are able to inhibit the canonical Wnt/β-catenin signaling pathway. However, tumor cells harboring common β-catenin pathway mutations, such as loss of adenomatous polyposis coli, are insensitive to this novel hypoxic effect. The underlying mechanism responsible is hypoxia-induced endoplasmic reticulum (ER) stress that inhibits normal Wnt protein processing and secretion. ER stress causes dissociation between GRP78/BiP and Wnt, an interaction essential for its correct posttranslational processing. Microenvironmental stress can therefore block autocrine and paracrine signaling of the Wnt/β-catenin pathway and negatively affect tumor growth. This study provides a general paradigm relating oxygen status to ER function and growth factor signaling. PMID:18824543

  18. Ethanol extract of propolis protects macrophages from oxidized low density lipoprotein-induced apoptosis by inhibiting CD36 expression and endoplasmic reticulum stress-C/EBP homologous protein pathway.

    PubMed

    Tian, Hua; Sun, Hong-Wei; Zhang, Jia-Jun; Zhang, Xiao-Wei; Zhao, Li; Guo, Shou-Dong; Li, Yan-Yan; Jiao, Peng; Wang, Hao; Qin, Shu-Cun; Yao, Shu-Tong

    2015-07-14

    Ethanol extract of propolis (EEP), rich in flavones, has been known for various biological activities including antioxidant, antiinflammatory and antibiotic activities. Our previous studies have shown that EEP protects endothelial cells from oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and inhibits atherosclerotic lesion development. In this present study, we explored the protective effect of EEP on ox-LDL-induced cytotoxicity in macrophages and specifically the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis. EEP was prepared and the total flavonoids content of EEP was determined by the colorimetric method of Chinese Standard (GB/T 20574-2006). The effects of EEP on lipid accumulation, cytotoxicity and apoptosis in RAW264.7 cells induced by ox-LDL or tunicamycin (TM, an ER stress inducer) were assayed using oil red O staining, MTT assay, flow cytometric analysis and so on. Immunofluorescence, Western blot and real time-PCR analysis were then used to further investigate the molecular mechanisms by which EEP protects macrophages from ox-LDL-induced apoptosis. 4-phenylbutyric acid (PBA), an ER stress inhibitor, was used as a positive control. EEP (7.5, 15 and 30 mg/L) not only attenuated ox-LDL-induced lipid accumulation in RAW264.7 macrophages in a dose-dependent manner but also inhibited the decreased cell viability and the increased lactate dehydrogenase (LDH) leakage, caspase-3 activation and apoptosis induced by ox-LDL or tunicamycin (TM, a classical ER stress inducer), which were similar to 4-phenylbutyric acid (PBA, an inhibitor of ER stress) treatment. In addition, like PBA, EEP significantly suppressed the ox-LDL- or TM-induced activation of ER stress signaling pathway including the phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) as well as upregulation of glucose regulated protein 78 (GRP78) and the pro

  19. Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

    PubMed

    Fu, Hai Ying; Sanada, Shoji; Matsuzaki, Takashi; Liao, Yulin; Okuda, Keiji; Yamato, Masaki; Tsuchida, Shota; Araki, Ryo; Asano, Yoshihiro; Asanuma, Hiroshi; Asakura, Masanori; French, Brent A; Sakata, Yasushi; Kitakaze, Masafumi; Minamino, Tetsuo

    2016-03-04

    Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment. © 2016 American Heart Association, Inc.

  20. Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

    PubMed Central

    Chalat, Madhavan; Menon, Indu; Turan, Zeynep; Menon, Anant K.

    2012-01-01

    Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity. PMID:22427661

  1. Endoplasmic reticulum stress activation during total knee arthroplasty

    PubMed Central

    Hocker, Austin D; Boileau, Ryan M; Lantz, Brick A; Jewett, Brian A; Gilbert, Jeffrey S; Dreyer, Hans C

    2013-01-01

    Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (−28%, P < 0.05; −20%, P < 0.05, respectively) along with an increase in eIF2α phosphorylation (64%, P < 0.05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, P = 0.056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, P < 0.05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, P < 0.05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated. PMID:24159375

  2. Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.

    PubMed

    Melnik, Bodo C

    2014-12-01

    Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population.

  3. Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

    PubMed

    Okeke, Emmanuel; Dingsdale, Hayley; Parker, Tony; Voronina, Svetlana; Tepikin, Alexei V

    2016-06-01

    Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  4. Klotho ameliorates chemically induced endoplasmic reticulum (ER) stress signaling.

    PubMed

    Banerjee, Srijita; Zhao, Yanhua; Sarkar, Partha S; Rosenblatt, Kevin P; Tilton, Ronald G; Choudhary, Sanjeev

    2013-01-01

    Both endoplasmic reticulum (ER) stress, a fundamental cell response associated with stress-initiated unfolded protein response (UPR), and loss of Klotho, an anti-aging hormone linked to NF-κB-induced inflammation, occur in chronic metabolic diseases such as obesity and type 2 diabetes. We investigated if the loss of Klotho is causally linked to increased ER stress. We treated human renal epithelial HK-2, alveolar epithelial A549, HEK293, and SH-SH-SY5Y neuroblastoma cells with ER stress-inducing agents, thapsigargin and/or tunicamycin. Effects of overexpression or siRNA-mediated knockdown of Klotho on UPR signaling was investigated by immunoblotting and Real-time PCR. Elevated Klotho levels in HK-2 cells decreased expression of ER stress markers phospho--IRE1, XBP-1s, BiP, CHOP, pJNK, and phospho-p38, all of which were elevated in response to tunicamycin and/or thapsigargin. Similar results were observed using A549 cells for XBP-1s, BiP, and CHOP in response to thapsigargin. Conversely, knockdown of Klotho in HEK 293 cells using siRNA caused further thapsigargin-induced increases in pIRE-1, XBP-1s, and BiP. Klotho overexpression in A549 cells blocked thapsigargin-induced caspase and PARP cleavage and improved cell viability. Our data indicate that Klotho has an important role in regulating ER stress and that loss of Klotho is causally linked to ER stress-induced apoptosis. Copyright © 2013 S. Karger AG, Basel.

  5. Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling.

    PubMed

    Filadi, Riccardo; Greotti, Elisa; Turacchio, Gabriele; Luini, Alberto; Pozzan, Tullio; Pizzo, Paola

    2015-04-28

    The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.

  6. Effect of pulsed electromagnetic fields on endoplasmic reticulum stress.

    PubMed

    Keczan, E; Keri, G; Banhegyi, G; Stiller, I

    2016-10-01

    The maintenance of protein homeostasis in the endoplasmic reticulum (ER) is crucial in cell life. Disruption of proteostasis results in ER stress that activates the unfolded protein response (UPR); a signalling network assigned to manage the accumulated misfolded or unfolded proteins. Prolonged or unresolved ER stress leads to apoptotic cell death that can be the basis of many serious diseases. Our aim was to study the effect of pulsed electromagnetic fields (PEMF), an alternative, non-invasive therapeutic method on ER stressed cell lines. First, the effect of PEMF treatment on the expression of ER stress markers was tested in three different cell lines. PEMF had no remarkable effect on ER stress protein levels in human embryonic kidney (HEK293T) and human liver carcinoma (HepG2) cell lines. However, the expression of BiP, Grp94 and CHOP were increased in HeLa cells upon PEMF exposure. Therefore, HepG2 cell line was selected for further experiments. Cells were stressed by tunicamycin and exposed to PEMF. Grp94, PDI, CHOP and PARP expression as markers of stress were monitored by Western blot and cell viability was also investigated. Tunicamycin treatment, as expected, increased the expression of Grp94, PDI, CHOP and inactivated PARP. Analysis of protein expression showed that PEMF was able to decrease the elevated level of ER chaperons Grp94, PDI and the apoptosis marker CHOP. The truncated, inactive form of PARP was also decreased. Accordingly, cell viability was also improved by PEMF exposure. These results indicate that PEMF is able to moderate ER stress induced by tunicamycin in HepG2 cells. However, our results clearly draw attention to that different cell lines may vary in the response to PEMF treatment.

  7. Endoplasmic Reticulum Stress Is Chronically Activated in Chronic Pancreatitis*

    PubMed Central

    Sah, Raghuwansh P.; Garg, Sushil K.; Dixit, Ajay K.; Dudeja, Vikas; Dawra, Rajinder K.; Saluja, Ashok K.

    2014-01-01

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T−/−), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T−/− mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies. PMID:25077966

  8. Endoplasmic reticulum stress is chronically activated in chronic pancreatitis.

    PubMed

    Sah, Raghuwansh P; Garg, Sushil K; Dixit, Ajay K; Dudeja, Vikas; Dawra, Rajinder K; Saluja, Ashok K

    2014-10-03

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

  9. Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

    PubMed Central

    Kaufman, Randal J.; Malhotra, Jyoti D.

    2014-01-01

    Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associate changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis [4] [5] [6] [7]. Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered [8] [9] [10]. The next questions are how they are regulated for exquisite tight control of ER – mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. PMID:24690484

  10. Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration.

    PubMed

    Bernard-Marissal, Nathalie; Médard, Jean-Jacques; Azzedine, Hamid; Chrast, Roman

    2015-04-01

    Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1(-/-) mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum-mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases.

  11. Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

    NASA Technical Reports Server (NTRS)

    Zheng, H. Q.; Staehelin, L. A.

    2001-01-01

    The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

  12. Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

    NASA Technical Reports Server (NTRS)

    Zheng, H. Q.; Staehelin, L. A.

    2001-01-01

    The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

  13. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis.

    PubMed

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-12-20

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis.

  14. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis

    PubMed Central

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-01-01

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. PMID:27999379

  15. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

    PubMed

    Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

    2014-10-01

    Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure.

  16. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  17. Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Leitman, Julia; Ulrich Hartl, F.; Lederkremer, Gerardo Z.

    2013-11-01

    In Huntington’s disease, as in other neurodegenerative diseases, it was initially thought that insoluble protein aggregates are the toxic species. However, growing evidence implicates soluble oligomeric polyglutamine-expanded huntingtin in cytotoxicity. Here we show that pathogenic huntingtin inhibits endoplasmic reticulum (ER)-associated degradation and induces ER stress before its aggregation into visible inclusions. All three branches of the unfolded protein response are activated. ER stress can be compensated by overexpression of p97/VCP, suggesting its sequestration by pathogenic huntingtin as a main cause. Stress correlates with the presence of huntingtin oligomers and is independent of continual huntingtin synthesis. Stress levels, measured in striatal neurons, are stabilized but only slowly subside on huntingtin aggregation into inclusions. Our results can be explained by the constant conversion of huntingtin monomers to toxic oligomers; large aggregates sequester the former, precluding further conversion, whereas pre-existing toxic oligomers are only gradually depleted.

  18. Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells**

    PubMed Central

    Matthew Meinig, J.; Fu, Liqiang; Peterson, Blake R.

    2015-01-01

    The endoplasmic reticulum (ER) plays critical roles in the processing of secreted and transmembrane proteins. To deliver small molecules to this organelle, we synthesized fluorinated hydrophobic analogues of the fluorophore rhodol. These cell-permeable fluorophores are exceptionally bright, with quantum yields of ~ 0.8, and specifically accumulate in the ER of living HeLa cells, as imaged by confocal laser scanning microscopy. To target a biological pathway controlled by the ER, we linked a fluorinated hydrophobic rhodol to 5-nitrofuran-2-acrylaldehyde. In contrast to an untargeted nitrofuran warhead, delivery of this electrophilic nitrofuran to the ER by the rhodol resulted in cytotoxicity comparable to the ER-targeted cytotoxin eeyarestatin I, and specifically inhibited protein processing by the ubiquitin-proteasome system. Fluorinated hydrophobic rhodols represent outstanding fluorophores that enable delivery of small molecules for targeting of ER-associated proteins and pathways. PMID:26118368

  19. Endoplasmic Reticulum Stress Signaling in Mammalian Oocytes and Embryos: Life in the Balance

    PubMed Central

    Latham, Keith E.

    2015-01-01

    Mammalian oocytes and embryos are exquisitely sensitive to a wide range of insults related to physical stress, chemical exposure, and exposures to adverse maternal nutrition or health status. Although cells manifest specific responses to various stressors, many of these stressors intersect at the endoplasmic reticulum, where disruptions in protein folding and production of reactive oxygen species initiate downstream signaling events. These signals modulate mRNA translation and gene transcription, leading to recovery, activation of autophagy, or with severe and prolonged stress, apoptosis. ER stress signaling has recently come to the fore as a major contributor to embryo demise. Accordingly, agents that modulate or inhibit ER stress signaling have yielded beneficial effects on embryo survival and long-term developmental potential. We review here the mechanisms of ER stress signaling, their connections to mammalian oocytes and embryos, and the promising indications that interventions in this pathway may provide new opportunities for improving mammalian reproduction and health. PMID:25805126

  20. Elevated mitochondria-coupled NAD(P)H in endoplasmic reticulum of dopamine neurons

    PubMed Central

    Tucker, Kristal R.; Cavolo, Samantha L.; Levitan, Edwin S.

    2016-01-01

    Pyridine nucleotides are redox coenzymes that are critical in bioenergetics, metabolism, and neurodegeneration. Here we use brain slice multiphoton microscopy to show that substantia nigra dopamine neurons, which are sensitive to stress in mitochondria and the endoplasmic reticulum (ER), display elevated combined NADH and NADPH (i.e., NAD(P)H) autofluorescence. Despite limited mitochondrial mass, organellar NAD(P)H is extensive because much of the signal is derived from the ER. Remarkably, even though pyridine nucleotides cannot cross mitochondrial and ER membranes, inhibiting mitochondrial function with an uncoupler or interrupting the electron transport chain with cyanide (CN−) alters ER NAD(P)H. The ER CN− response can occur without a change in nuclear NAD(P)H, raising the possibility of redox shuttling via the cytoplasm locally between neuronal mitochondria and the ER. We propose that coregulation of NAD(P)H in dopamine neuron mitochondria and ER coordinates cell redox stress signaling by the two organelles. PMID:27582392

  1. Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

    PubMed Central

    Orsi, Andrea

    2007-01-01

    Prion protein (PrP), the causative agent of transmissible spongiform encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation. PMID:19164910

  2. Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology.

    PubMed

    Guzel, Elif; Arlier, Sefa; Guzeloglu-Kayisli, Ozlem; Tabak, Mehmet Selcuk; Ekiz, Tugba; Semerci, Nihan; Larsen, Kellie; Schatz, Frederick; Lockwood, Charles Joseph; Kayisli, Umit Ali

    2017-04-08

    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

  3. Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

    SciTech Connect

    Isomura, Midori; Kotake, Yaichiro Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

    2013-10-01

    Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.

  4. [Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy].

    PubMed

    Yu, Y; Zhou, C L; Yu, T T; Han, X J; Shi, H Y; Wang, H Z; Shen, J J; He, J

    2017-06-25

    Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P<0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0

  5. [Endoplasmic reticulum stress in kidney diseases: a question of life and death?].

    PubMed

    Pallet, Nicolas; Bouvier, Nicolas; Beaune, Philippe; Legendre, Christophe; Thervet, Eric; Anglicheau, Dany

    2009-06-01

    Increasing our understanding of the cellular and molecular mechanisms of acute and chronic kidney diseases will lead to the development of new biomarkers of early kidney injury and to the discovery of new therapeutic strategies to prevent the initiation of renal failure or to promote the renal regeneration after injury. The implication of the endoplasmic reticulum stress in kidney diseases is not well recognized, but increasing experimental evidences suggest its implication in a wide array of kidney insults. Beside its role in the regulation of cell death, the UPR response induced by the endoplasmic reticulum stress alters many cellular functions and constitutes an important mediator of inflammation and/or epithelial to mesenchymal transition. The purpose of this review is to summarize the existing data concerning the role of the endoplasmic reticulum stress during kidney injury and to clarify its precise role in chronic kidney disease.

  6. Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

    PubMed Central

    Iturriaga, G; Jefferson, R A; Bevan, M W

    1989-01-01

    The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide. PMID:2535509

  7. Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids.

    PubMed

    Uchio, Naohiro; Oma, Yoko; Toriumi, Kazuya; Sasagawa, Noboru; Tanida, Isei; Fujita, Eriko; Kouroku, Yoriko; Kuroda, Reiko; Momoi, Takashi; Ishiura, Shoichi

    2007-11-01

    Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

  8. The Role of Endoplasmic Reticulum Stress and Unfolded Protein Response in Atherosclerosis

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

    Pathogenesis of atherosclerosis is a complex process involving several metabolic and signalling pathways. Accumulating evidence demonstrates that endoplasmic reticulum stress and associated apoptosis can be induced in the pathological conditions of atherosclerotic lesions and contribute to the disease progression. Notably, they may play a role in the development of vulnerable plaques that induce thrombosis and are therefore especially dangerous. Endoplasmic reticulum stress response is regulated by several signaling mechanisms that involve protein kinases and transcription factors. Some of these molecules can be regarded as potential therapeutic targets to improve treatment of atherosclerosis. In this review we will discuss the role of endoplasmic reticulum stress and apoptosis in atherosclerosis development in different cell types and summarize the current knowledge on potential therapeutic agents targeting molecules regulating these pathways and their possible use for anti-atherosclerotic therapy. PMID:26840309

  9. Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.

    PubMed

    Sainath, Rajiv; Armijo-Weingart, Lorena; Ketscheck, Andrea; Xu, Zhuxuan; Li, Shuxin; Gallo, Gianluca

    2017-09-13

    Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150(Glu) dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol, 2017. © 2017 Wiley Periodicals, Inc.

  10. Vesicular Trafficking of Incoming Human Papillomavirus 16 to the Golgi Apparatus and Endoplasmic Reticulum Requires γ-Secretase Activity

    PubMed Central

    Zhang, Wei; Kazakov, Teymur; Popa, Andreea

    2014-01-01

    ABSTRACT The route taken by papillomaviruses from the cell surface to the nucleus during infection is incompletely understood. Here, we developed a novel human papillomavirus 16 (HPV16) pseudovirus in which the carboxy terminus of the minor capsid protein L2 is exposed on the exterior of the intact capsid prior to cell binding. With this pseudovirus, we used the proximity ligation assay immune detection technique to demonstrate that during entry HPV16 L2 traffics into and out of the early endosome prior to Golgi localization, and we demonstrated that L2 enters the endoplasmic reticulum during entry. The cellular membrane-associated protease, γ-secretase, is required for infection by HPV16 pseudovirus and authentic HPV16. We also showed that inhibition of γ-secretase does not interfere substantively with virus internalization, initiation of capsid disassembly, entry into the early endosome, or exit from this compartment, but γ-secretase is required for localization of L2 and viral DNA to the Golgi apparatus and the endoplasmic reticulum. These results show that incoming HPV16 traffics sequentially from the cell surface to the endosome and then to the Golgi apparatus and the endoplasmic reticulum prior to nuclear entry. PMID:25227470

  11. Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes.

    PubMed

    Guan, Cuiping; Xu, Wen; Hong, Weisong; Zhou, Miaoni; Lin, Fuquan; Fu, Lifang; Liu, Dongyin; Xu, Aie

    2015-06-01

    Swollen endoplasmic reticulum (ER) is commonly observed in the melanocytes of vitiligo patients; however, the cause and proteins involved in this remain to be elucidated. Oxidative stress has been reported to be involved in the pathogenesis of vitiligo and previous studies have demonstrated that hydrogen peroxide (H2O2) induced melanocyte apoptosis, whereas quercetin exhibited cytoprotective activities against the effects of H2O2. The aim of the present study was to further investigate the role of H2O2 in the ER of melanocytes as well as its role in the export of tyrosinase from ER; in addition, the present study aimed to determine the mechanism by which quercetin protects against the effects of H2O2. The results demonstrated that melanocyte cells treated with H2O2 presented with swollen ER; however, a normal ER configuration was observed in untreated cells as well as quercetin/H2O2‑treated cells. Furthermore, H2O2 inhibited tyrosinase export from the ER and decreased expression levels of tyrosinase; however, quercetin was found to attenuate the effects induced by H2O2. In conclusion, the results of the present study confirmed the hypothesis that H2O2 induced ER dilation and hindered functional tyrosinase export from the ER of melanocytes. It was also found that quercetin significantly weakened these effects mediated by H2O2, therefore it may have the potential for use in the treatment of vitiligo.

  12. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Unusual configurations of endoplasmic reticulum in cells of acute promyelocytic leukemia.

    PubMed

    Parkin, J L; Brunning, R D

    1978-08-01

    An ultrastructural study of leukemia cells from 8 patients with acute promyelocytic leukemia revealed several features that have not previously been emphasized: prominent dilated rough endoplasmic reticulum and two unusual configurations of endoplasmic reticulum (ER). The two membrane structures, multilaminar ER and complex stellate arrangements of ER, appeared to be morphogenetically related. The multilaminar ER was observed in every mitotic cell and less frequently in interphase cells. The stellate ER complex was observed only in interphase cells. Ultrastructural evidence is presented to support the possible evolution of the stellate ER complex from the multilaminar ER.

  14. Endoplasmic reticulum proteins quality control and the unfolded protein response: the regulative mechanism of organisms against stress injuries.

    PubMed

    Fu, Xi Ling; Gao, Dong Sheng

    2014-01-01

    The endoplasmic reticulum is the cellular compartment in which secretory proteins are synthesized and folded. Perturbations of endoplasmic reticulum homeostasis lead to the accumulation of unfolded proteins. The activation of the unfolded protein response during endoplasmic reticulum stress transmits information about the status of protein folding to the cytosol and nucleus. The unfolded protein response leads to the upregulation of genes encoding endoplasmic reticulum chaperones, attenuation of translation, and initiation of the endoplasmic reticulum quality control system to restore endoplasmic reticulum homeostasis. When the unfolded protein response is insufficient to rebuild the steady state in endoplasmic reticulum, the programmed cell death or apoptosis would be initiated, by triggering cell injuries, even to cell death through apoptosis signals. In this review, we briefly outline research on the chaperones and foldases conserved in eukaryotes and plants, and describe the general principles and mechanisms of the endoplasmic reticulum quality control and the unfolded protein response. We describe the current models for the molecular mechanism of the unfolded protein response in plants, and emphasize the role of inositol requiring enzyme-1-dependent network in the unfolded protein response. Finally, we give a general overview of the directions for future research on the unfolded protein response in plants and its role in the response to environmental stresses. © 2014 International Union of Biochemistry and Molecular Biology.

  15. Selenoprotein S is required for clearance of C99 through endoplasmic reticulum-associated degradation.

    PubMed

    Jang, Jun Ki; Park, Ki Jun; Lee, Jea Hwang; Ko, Kwan Young; Kang, Seongman; Kim, Ick Young

    2017-04-29

    Amyloid beta precursor protein (APP) is normally cleaved by α-secretase, but can also be cleaved by β-secretase (BACE1) to produce C99 fragments in the endoplasmic reticulum (ER) membrane. C99 is subsequently cleaved to amyloid β (Aβ), the aggregation of which is known to cause Alzheimer's disease. Therefore, C99 removing is for preventing the disease. Selenoprotein S (SelS) is an ER membrane protein participating in endoplasmic reticulum-associated degradation (ERAD), one of the stages in resolving ER stress of misfolded proteins accumulated in the ER. ERAD has been postulated as one of the processes to degrade C99; however, it remains unclear if the degradation depends on SelS. In this study, we investigated the effect of SelS on C99 degradation. We observed that both SelS and C99 were colocalized in the membrane fraction of mouse neuroblastoma Neuro2a (N2a) cells. While the level of SelS was increased by ER stress, the level of C99 was decreased. However, despite the induction of ER stress, there was no change in the amount of C99 in SelS knock-down cells. The interaction of C99 with p97(VCP), an essential component of the ERAD complex, did not occur in SelS knock-down cells. The ubiquitination of C99 was decreased in SelS knock-down cells. We also found that the extracellular amount of Aβ(1-42) was relatively higher in SelS knock-down cells than in control cells. These results suggest that SelS is required for C99 degradation through ERAD, resulting in inhibition of Aβ production. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Rescue of Glaucomatous Neurodegeneration by Differentially Modulating Neuronal Endoplasmic Reticulum Stress Molecules

    PubMed Central

    Yang, Liu; Li, Shaohua; Miao, Linqing; Huang, Haoliang; Liang, Feisi; Teng, Xiuyin; Xu, Lin; Wang, Qizhao; Xiao, Weidong; Ridder, William H.; Ferguson, Toby A.; Chen, Dong Feng; Kaufman, Randal J.

    2016-01-01

    Axon injury is an early event in neurodegenerative diseases that often leads to retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. The connection of these two obviously sequential degenerative events, however, is elusive. Deciphering the upstream signals that trigger the neurodegeneration cascades in both neuronal soma and axon would be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic. We showed previously that optic nerve injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death. Using two in vivo mouse models of optic neuropathies (traumatic optic nerve injury and glaucoma) and adeno-associated virus–mediated RGC-specific gene targeting, we now show that differential manipulation of unfolded protein response pathways in opposite directions—inhibition of eukaryotic translation initiation factor 2α-C/EBP homologous protein and activation of X-box binding protein 1—promotes both RGC axons and somata survival and preserves visual function. Our results indicate that axon injury-induced neuronal ER stress plays an important role in both axon degeneration and neuron soma death. Neuronal ER stress is therefore a promising therapeutic target for glaucoma and potentially other types of neurodegeneration. SIGNIFICANCE STATEMENT Neuron soma and axon degeneration have distinct molecular mechanisms although they are clearly connected after axon injury. We previously demonstrated that axon injury induces neuronal endoplasmic reticulum (ER) stress and that manipulation of ER stress molecules synergistically promotes neuron cell body survival. Here we investigated the possibility that ER stress also plays a role in axon degeneration and whether ER stress modulation preserves neuronal function in neurodegenerative diseases. Our results suggest that neuronal ER stress is a general mechanism of degeneration for both neuronal

  17. Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

    PubMed Central

    Mentlein, R; Heymann, E

    1987-01-01

    The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function. Images Fig. 1. PMID:3663197

  18. Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis.

    PubMed

    Lumley, Emily C; Osborn, Acadia R; Scott, Jessica E; Scholl, Amanda G; Mercado, Vicki; McMahan, Young T; Coffman, Zachary G; Brewster, Jay L

    2017-01-01

    The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500-1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20-40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20-40 nM) and high doses (200-1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca(2+) release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of

  19. Astragaloside IV Attenuates Podocyte Apoptosis Mediated by Endoplasmic Reticulum Stress through Upregulating Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 Expression in Diabetic Nephropathy

    PubMed Central

    Guo, Hengjiang; Cao, Aili; Chu, Shuang; Wang, Yi; Zang, Yingjun; Mao, Xiaodong; Wang, Hao; Wang, Yunman; Liu, Cheng; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a central role in the pathogenesis of diabetes. This protein has been recognized as a potential target for diabetic therapy. In this study, we identified astragaloside IV (AS-IV) as a potent modulator of SERCA inhibiting renal injury in diabetic status. Increasing doses of AS-IV (2, 6, and 18 mg kg-1 day-1) were administered intragastrically to db/db mice for 8 weeks. Biochemical and histopathological approaches were conducted to evaluate the therapeutic effects of AS-IV. Cultured mouse podocytes were used to further explore the underlying mechanism in vitro. AS-IV dose-dependently increased SERCA activity and SERCA2 expression, and suppressed ER stress-mediated and mitochondria-mediated apoptosis in db/db mouse kidney. AS-IV also normalized glucose tolerance and insulin sensitivity, improved renal function, and ameliorated glomerulosclerosis and renal inflammation in db/db mice. In palmitate stimulated podocytes, AS-IV markedly improved inhibitions of SERCA activity and SERCA2 expression, restored intracellular Ca2+ homeostasis, and attenuated podocyte apoptosis in a dose-dependent manner with a concomitant abrogation of ER stress as evidenced by the downregulation of GRP78, cleaved ATF6, phospho-IRE1α and phospho-PERK, and the inactivation of both ER stress-mediated and mitochondria-mediated apoptotic pathways. Furthermore, SERCA2b knockdown eliminated the effect of AS-IV on ER stress and ER stress-mediated apoptotic pathway, whereas its overexpression exhibited an anti-apoptotic effect. Our data obtained from in vivo and in vitro studies demonstrate that AS-IV attenuates renal injury in diabetes subsequent to inhibiting ER stress-induced podocyte apoptosis through restoring SERCA activity and SERCA2 expression. PMID:28066247

  20. Brain Endoplasmic Reticulum Stress Mechanistically Distinguishes the Saline-Intake and Hypertensive Response to DOCA-Salt

    PubMed Central

    Jo, Fusakazu; Jo, Hiromi; Hilzendeger, Aline M.; Thompson, Anthony P.; Cassell, Martin D.; Rutkowski, D. Thomas; Davisson, Robin L.; Grobe, Justin L.; Sigmund, Curt D.

    2015-01-01

    Endoplasmic reticulum stress has become an important mechanism in hypertension. We examined the role of endoplasmic reticulum stress in mediating the increased saline intake and hypertensive effects in response to DOCA-salt. Intracerebroventricular delivery of the endoplasmic reticulum stress-reducing chemical chaperone Tauroursodeoxycholic acid did not affect the magnitude of hypertension, but markedly decreased saline intake in response to DOCA-salt. Increased saline intake returned after Tauroursodeoxycholic acid was terminated. Decreased saline intake was also observed after intracerebroventricular infusion of 4-phenylbutyrate, another chemical chaperone. Immunoreactivity to CHOP, a marker of irremediable endoplasmic reticulum stress, was increased in the subfornical organ and supraoptic nucleus of DOCA-salt mice, but the signal was absent in control and CHOP-deficient mice. Electron microscopy revealed abnormalities in endoplasmic reticulum structure (decrease in membrane length, swollen membranes, and decreased ribosome numbers) in the subfornical organ consistent with endoplasmic reticulum stress. Subfornical organ-targeted adenoviral delivery of GRP78, a resident endoplasmic reticulum chaperone, decreased DOCA-salt-induced saline intake. The increase in saline intake in response to DOCA-salt was blunted in CHOP-deficient mice, but these mice exhibited a normal hypertensive response. We conclude: 1) brain endoplasmic reticulum stress mediates the saline intake, but not blood pressure response to DOCA-salt, 2) DOCA-salt causes endoplasmic reticulum stress in the SFO which when attenuated by GRP78 blunts saline intake, and 3) CHOP may play a functional role in DOCA-salt-induced saline intake. The results suggest a mechanistic distinction between the importance of endoplasmic reticulum stress in mediating effects of DOCA-salt on saline intake and blood pressure. PMID:25895586

  1. Cyclosporine triggers endoplasmic reticulum stress in endothelial cells: a role for endothelial phenotypic changes and death.

    PubMed

    Bouvier, Nicolas; Flinois, Jean Pierre; Gilleron, Jerome; Sauvage, François-Ludovic; Legendre, Christophe; Beaune, Philippe; Thervet, Eric; Anglicheau, Dany; Pallet, Nicolas

    2009-01-01

    Calcineurin inhibitors cyclosporine and tacrolimus are effective immunosuppressants, but both substances have the same intrinsic nephrotoxic potential that adversely affects allograft survival in renal transplant patients and causes end-stage renal disease in other solid organ or bone marrow transplant recipients. Endothelial cells are the first biological interface between drugs and the kidney, and calcineurin inhibitors may influence endothelial function and viability in a number of ways. Notably, endothelial cells have recently been shown to contribute to the accumulation of interstitial fibroblasts in nonrenal models, through endothelial-to-mesenchymal transition. Here we demonstrate that cyclosporine, but not tacrolimus or its metabolites, induces morphological and phenotypic endothelial changes suggestive of a partial endothelial-to-mesenchymal transition in human umbilical arterial endothelial cells. We identify for the first time a contingent of interstitial myofibroblasts that coexpress endothelial markers in rat kidneys treated with cyclosporine, suggesting that endothelial-to-mesenchymal transition could occur in vivo. Finally, our findings suggest that endoplasmic reticulum stress triggered by cyclosporine induces endothelial cells to undergo endothelial phenotypic changes suggestive of a partial endothelial-to-mesenchymal transition, whereas salubrinal partially preserves the endothelial phenotype. Inversely, tacrolimus does not induce endothelial-to-mesenchymal transition or endoplasmic reticulum stress. In conclusion, this study demonstrates for the first time that cyclosporine, and not tacrolimus, induces endoplasmic reticulum stress in endothelial cells. Our findings also suggest that endoplasmic reticulum stress contributes to endothelial cell death and phenotypic changes similar to a partial endothelial-to-mesenchymal transition.

  2. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis

    PubMed Central

    Hughes, Alexandria; Oxford, Alexandra E.; Tawara, Ken; Jorcyk, Cheryl L.; Oxford, Julia Thom

    2017-01-01

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis. PMID:28335520

  3. Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress

    SciTech Connect

    Faria, Gisele; Cardoso, Cristina R.B.; Larson, Roy E.; Silva, Joao S.; Rossi, Marcos A.

    2009-01-15

    Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker of activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug.

  4. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

    PubMed

    Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom

    2017-03-20

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

  5. Ethanol stress impairs protein folding in the endoplasmic reticulum and activates Ire1 in Saccharomyces cerevisiae.

    PubMed

    Miyagawa, Ken-Ichi; Ishiwata-Kimata, Yuki; Kohno, Kenji; Kimata, Yukio

    2014-01-01

    Impaired protein folding in the endoplasmic reticulum (ER) evokes the unfolded protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane protein Ire1. Here, we report that ethanol stress damages protein folding in the ER, causing activation of Ire1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.

  6. Luteolin shows an antidepressant-like effect via suppressing endoplasmic reticulum stress.

    PubMed

    Ishisaka, Mitsue; Kakefuda, Kenichi; Yamauchi, Mika; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Tsuruta, Akifumi; Hara, Hideaki

    2011-01-01

    Depression is a significant public health problem and some reports indicate an association between depression and endoplasmic reticulum stress. Luteolin is a flavonoid contained in many plants and with a variety of known pharmacological properties such as anti-inflammatory, anti-anxiety, and memory-improving effects, suggesting that luteolin penetrates into the brain. In the present study, we investigated the effects of luteolin on endoplasmic reticulum stress-induced neuronal cell death. Luteolin significantly suppressed tunicamycin-induced cell death at 1 to 10 µM in human neuroblastoma cells. Luteolin increased in the expression of the 78 kDa glucose-regulated protein and 94 kDa glucose-regulated protein and decreased in the cleavage activation of caspase-3. Additionally, to investigate whether chronic luteolin treatment has an antidepression effect, we performed some behavioral tests. Chronic luteolin treatment showed antidepressant-like effects in behavioral tests and, luteolin attenuated the expression of endoplasmic reticulum stress-related proteins in the hippocampus of corticosterone-treated depression model mice. These findings indicate that luteolin has antidepressant-like effects, partly due to the suppression of endoplasmic reticulum stress.

  7. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2014-12-01

    Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung

  8. Cloning of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) from Caribbean spiny lobster Panulirus argus

    PubMed Central

    Arunachalam, S. C.; Meleshkevitch, E. A.; Mandal, P. K.; Boudko, D. Y.; Ahearn, G. A.

    2012-01-01

    We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using 45Ca2+ coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca2+-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene. PMID:18825387

  9. The combined therapeutic effects of bortezomib and fenretinide on neuroblastoma cells involve endoplasmic reticulum stress response.

    PubMed

    Pagnan, Gabriella; Di Paolo, Daniela; Carosio, Roberta; Pastorino, Fabio; Marimpietri, Danilo; Brignole, Chiara; Pezzolo, Annalisa; Loi, Monica; Galietta, Luis J V; Piccardi, Federica; Cilli, Michele; Nico, Beatrice; Ribatti, Domenico; Pistoia, Vito; Ponzoni, Mirco

    2009-02-15

    The proteasome inhibitor bortezomib inhibited cell growth and angiogenesis in neuroblastoma. Bortezomib has been shown to induce synergistic activity when combined with other antineoplastic agents. Here we have investigated the antitumor activity of bortezomib in combination with fenretinide, a synthetic retinoid, against neuroblastoma cells. Different neuroblastoma cell lines were tested for sensitivity to bortezomib and fenretinide, given alone or in different dose-dependent and time-dependent combination schedules. Cell proliferation, cell viability, and apoptosis were evaluated by measuring 3H-thymidine incorporation, trypan blue staining, DNA fragmentation, and western blot analysis. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. An orthotopic neuroblastoma mouse model was used to examine in vivo sensitivity. Each compound alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination. This inhibition was characterized by marked G2-M and G1 cell cycle arrest with nearly complete depletion of S phase. Bortezomib and fenretinide in association triggered an increased apoptosis through activation of specific genes of the endoplasmic reticulum stress compared with either drug tested alone. Tumor-bearing mice treated with bortezomib plus fenretinide lived statistically significantly longer than mice treated with each drug alone. Histologic evaluation and chorioallantoic membrane analysis of primary tumors showed that the combined therapeutic activity of bortezomib and fenretinide rested upon antitumor and antiangiogenic mechanisms. These findings provide the rationale for the development of a new therapeutic strategy for neuroblastoma based on this pharmacologic combination.

  10. Tauroursodeoxycholic Acid Attenuates Lipid Accumulation in Endoplasmic Reticulum-Stressed Macrophages

    PubMed Central

    Hua, Yinan; Kandadi, Machender R.; Zhu, Meijun; Ren, Jun; Sreejayan, Nair

    2011-01-01

    Background/Aim Recent evidence suggests that endoplasmic reticulum (ER) stress provoked under diabetic conditions augments the expression of scavenger receptors on macrophages, promoting the uptake of oxidized low-density lipoprotein (ox-LDL) uptake and atherogenesis. The aim of the present study was to test the hypothesis that the chemical chaperone tauroursodeoxycholic acid (TUDCA) attenuates lipid accumulation in macrophages subjected to ER stress. Methods Cultured human macrophages were subjected to ER-stress by treating them with tunicamycin. Lipid-uptake by macrophages subjected to ER-stress in the presence or absence of TUDCA was assessed by oil red O staining and by assessing the cellular uptake of Dil-ox-LDL by fluorescence measurement. Protein levels and phosphorylation status of ER stress markers, insulin-signalling molecules and scavenger receptor were assessed by Western blotting. Results Treatment of cultured human macrophages with the ER-stressor tunicamycin caused an increase in the protein levels of CD-36, and augmentation of lipid-uptake both of which were inhibited by TUDCA. TUDCA-treatment inhibited tunicamycin-induced ER-stress as evidenced by the attenuation of phosphorylation of eukaryotic translation initiation factor-2α and glucose reactive protein-78. In addition, TUDCA improved insulin signaling in macrophages by augmenting Akt-phosphorylation and blunting c-Jun N-terminal kinase activity. Conclusion Inhibition of macrophage ER-stress may represent a potential strategy in preventing atherogenesis under diabetic conditions. PMID:19834331

  11. Tauroursodeoxycholic acid attenuates endoplasmic reticulum stress and protects the liver from chronic intermittent hypoxia induced injury.

    PubMed

    Hou, Yanpeng; Yang, Huai'an; Cui, Zeshi; Tai, Xuhui; Chu, Yanling; Guo, Xing

    2017-09-01

    Obstructive sleep apnea that characterized by chronic intermittent hypoxia (CIH) has been reported to associate with chronic liver injury. Tauroursodeoxycholic acid (TUDCA) exerts liver-protective effects in various liver diseases. The purpose of this study was to test the hypothesis that TUDCA could protect liver against CIH injury. C57BL/6 mice were subjected to intermittent hypoxia for eight weeks and applied with TUDCA by intraperitoneal injection. The effect of TUDCA on liver histological changes, liver function, oxidative stress, inflammatory response, hepatocyte apoptosis and endoplasmic reticulum (ER) stress were investigated. The results showed that administration of TUDCA attenuated liver pathological changes, reduced serum alanine aminotransferase and aspartate aminotransferase level, suppressed reactive oxygen species activity, decreased tumor necrosis factor-α and interleukin-1β level and inhibited hepatocyte apoptosis induced by CIH. TUDCA also inhibited CIH-induced ER stress in liver as evidenced by decreased expression of ER chaperone 78 kDa glucose-related protein, unfolded protein response transducers and ER proapoptotic proteins. Altogether, the present study described a liver-protective effect of TUDCA in CIH mice model, and this effect seems at least partly through the inhibition of ER stress.

  12. Zonisamide suppresses endoplasmic reticulum stress-induced neuronal cell damage in vitro and in vivo.

    PubMed

    Tsujii, Saori; Ishisaka, Mitsue; Shimazawa, Masamitsu; Hashizume, Takanori; Hara, Hideaki

    2015-01-05

    Zonisamide has been reported to have protective effects on epilepsy and Parkinson׳s disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson׳s disease. Zonisamide not only suppressed MPP⁺-induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson׳s disease.

  13. 4-Phenylbutyrate Benefits Traumatic Hemorrhagic Shock in Rats by Attenuating Oxidative Stress, Not by Attenuating Endoplasmic Reticulum Stress.

    PubMed

    Yang, Guangming; Peng, Xiaoyong; Hu, Yi; Lan, Dan; Wu, Yue; Li, Tao; Liu, Liangming

    2016-07-01

    Vascular dysfunction such as vascular hyporeactivity following severe trauma and shock is a major cause of death in injured patients. Oxidative stress and endoplasmic reticulum stress play an important role in vascular dysfunction. The objective of the present study was to determine whether or not 4-phenylbutyrate can improve vascular dysfunction and elicit antishock effects by inhibiting oxidative and endoplasmic reticulum stress. Prospective, randomized, controlled laboratory experiment. State key laboratory of trauma, burns, and combined injury. Five hundred and fifty-two Sprague-Dawley rats. Rats were anesthetized, and a model of traumatic hemorrhagic shock was established by left femur fracture and hemorrhage. The effects of 4-phenylbutyrate (5, 20, 50, 100, 200, and 300 mg/kg) on vascular reactivity, animal survival, hemodynamics, and vital organ function in traumatic hemorrhagic shock rats and cultured vascular smooth muscle cells, and the relationship to oxidative stress and endoplasmic reticulum stress was observed. Lower doses of 4-phenylbutyrate significantly improved the vascular function, stabilized the hemodynamics, and increased the tissue blood flow and vital organ function in traumatic hemorrhagic shock rats, and markedly improved the survival outcomes. Among all dosages observed in the present study, 20 mg/kg of 4-phenylbutyrate had the best effect. Further results indicated that 4-phenylbutyrate significantly inhibited the oxidative stress, decreased shock-induced oxidative stress index such as the production of reactive oxygen species, increased the antioxidant enzyme levels such as superoxide dismutase, catalase, and glutathione, and improved the mitochondrial function by inhibiting the opening of the mitochondrial permeability transition pore in rat artery and vascular smooth muscle cells. In contrast, 4-phenylbutyrate did not affect the changes of endoplasmic reticulum stress markers following traumatic hemorrhagic shock. Furthermore, 4

  14. Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain.

    PubMed

    Kreiling, Jill A; Balantac, Zaneta L; Crawford, Andrew R; Ren, Yuexin; Toure, Jamal; Zchut, Sigalit; Kochilas, Lazaros; Creton, Robbert

    2008-01-01

    Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.

  15. Palmitate causes endoplasmic reticulum stress and apoptosis in human mesenchymal stem cells: prevention by AMPK activator.

    PubMed

    Lu, Jun; Wang, Qinghua; Huang, Lianghu; Dong, Huiyue; Lin, Lingjing; Lin, Na; Zheng, Feng; Tan, Jianming

    2012-11-01

    Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

  16. Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells.

    PubMed

    Li, Tianliang; Su, Ling; Zhong, Ning; Hao, Xuexi; Zhong, Diansheng; Singhal, Sunil; Liu, Xiangguo

    2013-07-01

    Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

  17. In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

    PubMed Central

    Hendershot, L M; Wei, J Y; Gaut, J R; Lawson, B; Freiden, P J; Murti, K G

    1995-01-01

    BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo. Images PMID:7612964

  18. A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress

    PubMed Central

    Cai, Lei; Zhang, Xin; Zhai, Yu; Liu, Jianren

    2016-01-01

    Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency. PMID:27708219

  19. Endoplasmic reticulum stress-regulated CXCR3 pathway mediates inflammation and neuronal injury in acute glaucoma.

    PubMed

    Ha, Y; Liu, H; Xu, Z; Yokota, H; Narayanan, S P; Lemtalsi, T; Smith, S B; Caldwell, R W; Caldwell, R B; Zhang, W

    2015-10-08

    Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.

  20. Endoplasmic Reticulum Chaperon Tauroursodeoxycholic Acid Attenuates Aldosterone-Infused Renal Injury

    PubMed Central

    Guo, Honglei; Li, Hongmei; Ling, Lilu

    2016-01-01

    Aldosterone (Aldo) is critically involved in the development of renal injury via the production of reactive oxygen species and inflammation. Endoplasmic reticulum (ER) stress is also evoked in Aldo-induced renal injury. In the present study, we investigated the role of ER stress in inflammation-mediated renal injury in Aldo-infused mice. C57BL/6J mice were randomized to receive treatment for 4 weeks as follows: vehicle infusion, Aldo infusion, vehicle infusion plus tauroursodeoxycholic acid (TUDCA), and Aldo infusion plus TUDCA. The effect of TUDCA on the Aldo-infused inflammatory response and renal injury was investigated using periodic acid-Schiff staining, real-time PCR, Western blot, and ELISA. We demonstrate that Aldo leads to impaired renal function and inhibition of ER stress via TUDCA attenuates renal fibrosis. This was indicated by decreased collagen I, collagen IV, fibronectin, and TGF-β expression, as well as the downregulation of the expression of Nlrp3 inflammasome markers, Nlrp3, ASC, IL-1β, and IL-18. This paper presents an important role for ER stress on the renal inflammatory response to Aldo. Additionally, the inhibition of ER stress by TUDCA negatively regulates the levels of these inflammatory molecules in the context of Aldo. PMID:27721575

  1. SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis

    PubMed Central

    Wang, Yuanzhong; Zhou, Dujin; Phung, Sheryl; Warden, Charles; Rashid, Rumana; Chan, Nymph; Chen, Shiuan

    2017-01-01

    Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer. PMID:28174265

  2. Repeated restraint stress increases seizure susceptibility by activation of hippocampal endoplasmic reticulum stress.

    PubMed

    Zhu, Xinjian; Dong, Jingde; Xia, Zhengrong; Zhang, Aifeng; Chao, Jie; Yao, Honghong

    2017-09-05

    A growing body of evidence suggests that stress triggers a variety of pathophysiological responses. Recent studies show that stress produces enduring effects on structure and function of hippocampus, which is one of the most important structures involved in epilepsy. In the present study, we determined the effect of repeated restraint stress exposure on the susceptibility of pentylenetetrazole (PTZ)-induced seizures and the possible mechanisms involved using a rodent model. Our results show that mice subjected to repeated restraint stress exhibited shorter latency to PTZ-induced tonic-clonic seizures and higher seizure severity, suggesting chronic restraint stress increases seizure susceptibility. Following repeated restraint stress, we observed an increased level of endoplasmic reticulum (ER) stress as well as oxidative stress in the hippocampus. Moreover, our results show that chronic restraint stress exposure causes neuron loss in the hippocampus. Inhibition of ER stress with chemical chaperone, tauroursodeoxycholic acid (TUDCA), however, protects against chronic restraint stress-induced neuron loss, suggesting repeated restraint stress-induced neuronal degeneration is dependent on ER stress activation. On the other hand, inhibition of ER stress with TUDCA suppresses restraint stress-induced seizure susceptibility. Taken together, these results indicate that repeated restraint stress increases seizure susceptibility by activation of hippocampal ER stress and ER stress mediated oxidative stress and neurodegeneration. Thus, attenuating ER stress may serve as a potential therapeutic strategy targeted to block stress-induced seizure activities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells

    PubMed Central

    Woźniak, Marcin J.; Bola, Becky; Brownhill, Kim; Yang, Yen-Ching; Levakova, Vesselina; Allan, Victoria J.

    2009-01-01

    Summary Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that ∼50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought. PMID:19454478

  4. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    PubMed

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  5. Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

    PubMed Central

    Bennett, Shauna M.; Jiang, Mengxi

    2013-01-01

    BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells. PMID:23740996

  6. A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress.

    PubMed

    Su, Jingjing; Shu, Liang; Zhang, Zhou; Cai, Lei; Zhang, Xin; Zhai, Yu; Liu, Jianren

    2016-11-22

    Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.

  7. Efavirenz Causes Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Endothelial Cells.

    PubMed

    Weiß, Marlene; Kost, Bernd; Renner-Müller, Ingrid; Wolf, Eckhard; Mylonas, Ioannis; Brüning, Ansgar

    2016-01-01

    The non-nucleoside reverse transcriptase inhibitor efavirenz is a widely prescribed antiretroviral drug used in combined antiretroviral therapy. Despite being an essential and life-saving medication, the required lifelong use of HIV drugs has been associated with a variety of adverse effects, including disturbances in lipid metabolism and increased cardiovascular risk. Efavirenz belongs to those HIV drugs for which cardiovascular and endothelial dysfunctions have been reported. It is here shown that elevated concentrations of efavirenz can inhibit endothelial meshwork formation on extracellular matrix gels by normal and immortalized human umbilical vein cells. This inhibition was associated with an increase in oxidative stress markers, endoplasmic reticulum (ER) stress markers, and autophagy. Induction of ER stress occurred at pharmacologically relevant concentrations of efavirenz and resulted in reduced proliferation and cell viability of endothelial cells, which worsened in the presence of elevated efavirenz concentrations. In combination with the HIV protease inhibitor nelfinavir, both oxidative stress and ER stress became elevated in endothelial cells. These data indicate that pharmacologically relevant concentrations of efavirenz can impair cell viability of endothelial cells and that these effects may be aggravated by either elevated concentrations of efavirenz or by a combined use of efavirenz with other oxidative stress-inducing medications.

  8. Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells

    PubMed Central

    Sheveleva, Elena V.; Landowski, Terry H.; Samulitis, Betty K.; Bartholomeusz, Geoffrey; Powis, Garth; Dorr, Robert T.

    2012-01-01

    Oxidative protein folding in the endoplasmic reticulum (ER) requires strict regulation of redox homeostasis. Disruption of the lumenal redox balance induces an integrated ER stress response that is associated with reduced protein translation, increased chaperone activity, and ultimately cell death. Imexon is a small molecule chemotherapeutic agent that has been shown to bind glutathione (GSH) and induce oxidative stress in tumor cells, however the mechanism of cytotoxicity is not well understood. This In this report, we investigate the effects of imexon on the integrated ER stress response in pancreatic carcinoma cells. Acute exposure to imexon induces an ER stress response characterized by accumulation of the oxidized form of the oxidoreductase Ero1α, phosphorylation of eIF2alpha and inhibition of protein synthesis. An RNA interference chemo-sensitization screen identified the eukaryotic translation initiation factor eIF2B5 as a target that enhanced imexon-induced growth inhibition of MiaPaCa-2 pancreatic cancer cells, but did not significantly augment the effects of imexon on protein synthesis. Concurrent reduction of intracellular thiols with N-acetyl cysteine reversed imexon activity, however co-treatment with superoxide scavengers had no effect, suggesting thiol binding may be a primary component of the oxidative effects of imexon. Moreover, the data suggest that disruption of the redox balance in the ER is a potential therapeutic target. PMID:22275514

  9. Endoplasmic Reticulum Stress Plays a Key Role in Rotenone-Induced Apoptotic Death of Neurons.

    PubMed

    Goswami, Poonam; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Swarnkar, Supriya; Nath, Chandishwar; Singh, Sarika

    2016-01-01

    Rotenone, a pesticide, causes neurotoxicity via the mitochondrial complex-I inhibition. The present study was conducted to evaluate the role of endoplasmic reticulum (ER) stress in rotenone-induced neuronal death. Cell viability, cytotoxicity, reactive oxygen species (ROS) generation, nitrite level, mitochondrial membrane potential (MMP), and DNA damage were assessed in rotenone-treated neuro-2A cells. Protein levels of ER stress markers glucose regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), and phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2-α) were estimated to assess the ER stress. To confirm the apoptotic death of neurons, mRNA levels of caspase-9, caspase-12 and caspase-3 were estimated. Further, to confirm the involvement of ER stress, neuro-2A cells were pretreated with ER stress inhibitor salubrinal. Co-treatment of antioxidant melatonin was also given to assess the role of oxidative stress in rotenone-induced apoptosis. Rotenone (0.1, 0.5, and 1 μM) treatment to neurons caused significantly decreased cell viability, increased cytotoxicity, increased ROS generation, increased expression of GRP78 and GADD, DNA damage and activation of caspase-12 and caspase-3 which were significantly attenuated by pretreatment of salubrinal (25 μM). Rotenone-induced dephosphorylation of eIF2α was also inhibited with salubrinal treatment. However, pretreatment of salubrinal did not affect the rotenone-induced increased nitrite levels, decreased MMP and caspase-9 activation. Co-treatment of antioxidant melatonin (1 mM) did not offer attenuation against rotenone-induced increased expression of caspase-9, caspase-12 and caspase-3. In conclusion, results indicated that ER stress plays a key role in rotenone-induced neuronal death, rather than oxidative stress. Graphical Abstract Pictorial presentation showed the involvement of endoplasmic reticulum (ER) stress, increased reactive oxygen species (ROS

  10. Effect of lithium chloride on endoplasmic reticulum stress-related PERK/ROCK signaling in a rat model of glaucoma.

    PubMed

    Sun, Xiao-Bo; Lu, Hong-E; Chen, Yuan; Fan, Xiao-Hui; Tong, Bin

    2014-12-01

    Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Lithium chloride (LiCl) inhibits glycogen synthase kinase-3 beta (GSK-3β) and attends PERK-induced endoplasmic reticulum stress (ERs) transition. PERK is a type I transmembrane protein located in the endoplasmic reticulum. PERK pathway activation takes place in ERs early inhibiting protein synthnesis to protect cell and promote cell survival. Here, we firstly evaluate that LiCl reduced IOP when administered intraperitoneally. After 6 weeks, IOP dropped by around 21.9% in LiCl treated rats. Then we investigated the effects of LiCI on PERK-mediated signaling pathways. LiCl treatment activated PERK and inhibited the expression of ROCK-1 and ROCK-2 in a rat model of glaucoma. Collectively, these results suggest that LiCl reduced the IOP through the phosphorylation of PERK by the regulation of PERK/ROCK signaling in glaucoma rat model.

  11. A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

    PubMed

    Takahashi, Nozomi; Harada, Miyuki; Hirota, Yasushi; Zhao, Lin; Azhary, Jerilee M K; Yoshino, Osamu; Izumi, Gentaro; Hirata, Tetsuya; Koga, Kaori; Wada-Hiraike, Osamu; Fujii, Tomoyuki; Osuga, Yutaka

    2017-01-01

    Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells. Copyright © 2017 by the Endocrine Society.

  12. Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid tumor cells.

    PubMed

    Nieto-Miguel, Teresa; Fonteriz, Rosalba I; Vay, Laura; Gajate, Consuelo; López-Hernández, Silvia; Mollinedo, Faustino

    2007-11-01

    The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G(2)-M arrest, depletion of ER-stored Ca(2+), Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage-inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis. Edelfosine prompted ER stress apoptotic signaling, but not the survival unfolded protein response. Edelfosine also induced persistent c-Jun NH(2)-terminal kinase (JNK) activation. Gene transfer-mediated overexpression of apoptosis signal-regulating kinase 1, which plays a crucial role in ER stress, enhanced edelfosine-induced JNK activation and apoptosis. Inhibition of JNK, caspase-4, or caspase-8 activation diminished edelfosine-induced apoptosis. Edelfosine treatment led to the generation of the p20 caspase-8 cleavage fragment of BAP31, directing proapoptotic signals between the ER and the mitochondria. bax(-/-)bak(-/-) double-knockout cells fail to undergo edelfosine-induced ER-stored Ca(2+) release and apoptosis. Wild-type and bax(-/-)bak(-/-) cells showed similar patterns of phosphatidylcholine and protein synthesis inhibition, despite their differences in drug sensitivity. Thus, edelfosine-induced apoptosis is dependent on Bax/Bak-mediated ER-stored Ca(2+) release, but phosphatidylcholine and protein synthesis inhibition is not critical. Transfection-enforced expression of Bcl-X(L), which localizes specifically in mitochondria, prevented apoptosis without inhibiting ER-stored Ca(2+) release. These data reveal that edelfosine induces an ER stress response in solid tumor cells, providing novel insights into the edelfosine-mediated antitumor activity. Our data also indicate that mitochondria are indispensable for this edelfosine-induced cell

  13. Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

    PubMed Central

    Skehel, P. A.; Fabian-Fine, R.; Kandel, E. R.

    2000-01-01

    VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum–resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis. PMID:10655491

  14. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    SciTech Connect

    Naranmandura, Hua; Xu, Shi; Koike, Shota; Pan, Li Qiang; Chen, Bin; Wang, Yan Wei; Rehman, Kanwal; Wu, Bin; Chen, Zhe; Suzuki, Noriyuki

    2012-05-01

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

  15. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    PubMed Central

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  16. Nucleocapsid protein from fig mosaic virus forms cytoplasmic agglomerates that are hauled by endoplasmic reticulum streaming.

    PubMed

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki; Namba, Shigetou

    2015-01-01

    Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we

  17. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    SciTech Connect

    Pan, Mu-Yun; Shen, Yuh-Chiang; Lu, Chien-Hsing; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified as an

  18. Increased monocyte-derived reactive oxygen species in type 2 diabetes: role of endoplasmic reticulum stress.

    PubMed

    Restaino, Robert M; Deo, Shekhar H; Parrish, Alan R; Fadel, Paul J; Padilla, Jaume

    2017-02-01

    What is the central question of this study? Patients with type 2 diabetes exhibit increased oxidative stress in peripheral blood mononuclear cells, including monocytes; however, the mechanisms remain unknown. What is the main finding and its importance? The main finding of this study is that factors contained within the plasma of patients with type 2 diabetes can contribute to increased oxidative stress in monocytes, making them more adherent to endothelial cells. We show that these effects are largely mediated by the interaction between endoplasmic reticulum stress and NADPH oxidase activity. Recent evidence suggests that exposure of human monocytes to glucolipotoxic media to mimic the composition of plasma of patients with type 2 diabetes (T2D) results in the induction of endoplasmic reticulum (ER) stress markers and formation of reactive oxygen species (ROS). The extent to which these findings translate to patients with T2D remains unclear. Thus, we first measured ROS (dihydroethidium fluorescence) in peripheral blood mononuclear cells (PBMCs) from whole blood of T2D patients (n = 8) and compared the values with age-matched healthy control subjects (n = 8). The T2D patients exhibited greater basal intracellular ROS (mean ± SD, +3.4 ± 1.4-fold; P < 0.05) compared with control subjects. Next, the increase in ROS in PBMCs isolated from T2D patients was partly recapitulated in cultured human monocytes (THP-1 cells) exposed to plasma from T2D patients for 36 h (+1.3 ± 0.08-fold versus plasma from control subjects; P < 0.05). In addition, we found that increased ROS formation in THP-1 cells treated with T2D plasma was NADPH oxidase derived and led to increased endothelial cell adhesion (+1.8 ± 0.5-fold; P < 0.05) and lipid uptake (+1.3 ± 0.3-fold; P < 0.05). Notably, we found that T2D plasma-induced monocyte ROS and downstream functional effects were abolished by treating cells with tauroursodeoxycholic acid, a chemical chaperone known to

  19. The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum.

    PubMed

    Lipschutz, Joshua H; Lingappa, Vishwanath R; Mostov, Keith E

    2003-06-06

    We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259-4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of m

  20. Protein disulfide isomerase-endoplasmic reticulum resident protein 57 regulates allergen-induced airways inflammation, fibrosis, and hyperresponsiveness.

    PubMed

    Hoffman, Sidra M; Chapman, David G; Lahue, Karolyn G; Cahoon, Jonathon M; Rattu, Gurkiranjit K; Daphtary, Nirav; Aliyeva, Minara; Fortner, Karen A; Erzurum, Serpil C; Comhair, Suzy A A; Woodruff, Prescott G; Bhakta, Nirav; Dixon, Anne E; Irvin, Charles G; Janssen-Heininger, Yvonne M W; Poynter, Matthew E; Anathy, Vikas

    2016-03-01

    Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum-localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown. Here we sought to examine the contribution of airway epithelium-specific ERp57 in the pathogenesis of allergic asthma. We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57. Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite-challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite-induced endoplasmic reticulum stress and airways fibrosis. Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier

  1. Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury.

    PubMed

    Mami, Iadh; Bouvier, Nicolas; El Karoui, Khalil; Gallazzini, Morgan; Rabant, Marion; Laurent-Puig, Pierre; Li, Shuping; Tharaux, Pierre-Louis; Beaune, Philippe; Thervet, Eric; Chevet, Eric; Hu, Guo-Fu; Pallet, Nicolas

    2016-03-01

    Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang(-/-)) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism. Copyright © 2016 by the American Society of Nephrology.

  2. Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury

    PubMed Central

    Mami, Iadh; Bouvier, Nicolas; El Karoui, Khalil; Gallazzini, Morgan; Rabant, Marion; Laurent-Puig, Pierre; Li, Shuping; Tharaux, Pierre-Louis; Beaune, Philippe; Thervet, Eric; Chevet, Eric; Hu, Guo-Fu

    2016-01-01

    Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang−/−) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism. PMID:26195817

  3. A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

    SciTech Connect

    Ganan, S.; Cazzulo, J.J.; Parodi, A.J. )

    1991-03-26

    N-Linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc{sub 1}Man{sub 5-9}GlcNAc{sub 2} and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man{sub 9}GlcNAc{sub 2} in protein N-glycosylation) were grown in the presence of ({sup 14}C)glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1,000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. No evidence for the presence of an endomannosidase yielding GlcMan from the glucosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-33% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.

  4. Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection.

    PubMed

    Grangeon, Romain; Agbeci, Maxime; Chen, Jun; Grondin, Gilles; Zheng, Huanquan; Laliberté, Jean-François

    2012-09-01

    The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.

  5. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells.

    PubMed

    Caballano-Infantes, Estefania; Terron-Bautista, José; Beltrán-Povea, Amparo; Cahuana, Gladys M; Soria, Bernat; Nabil, Hajji; Bedoya, Francisco J; Tejedo, Juan R

    2017-02-26

    Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca(2+) flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.

  6. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

    PubMed Central

    Caballano-Infantes, Estefania; Terron-Bautista, José; Beltrán-Povea, Amparo; Cahuana, Gladys M; Soria, Bernat; Nabil, Hajji; Bedoya, Francisco J; Tejedo, Juan R

    2017-01-01

    Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies. PMID:28289506

  7. Role of endoplasmic reticulum stress in light-induced photoreceptor degeneration in mice.

    PubMed

    Nakanishi, Tomohiro; Shimazawa, Masamitsu; Sugitani, Sou; Kudo, Takashi; Imai, Shunsuke; Inokuchi, Yuta; Tsuruma, Kazuhiro; Hara, Hideaki

    2013-04-01

    Exposure to excessive levels of light induces photoreceptor apoptosis and can be a causative factor in age-related macular degeneration (AMD). However, the cellular events that mediate this apoptotic response are poorly understood. Here, we investigated the roles of endoplasmic reticulum (ER) stress in light-induced cell death in the murine retina and murine photoreceptor cells (661W). Excessive light exposure induced retinal dysfunction, photoreceptor degeneration, and apoptosis. Furthermore, the accumulation of polyubiquitinated proteins and the transcriptional expression of ER stress-related factors, including 78-kDa glucose-regulated protein (GRP78)/immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP), were increased in light-exposed retinas. Light exposure also induced both cell death and up-regulation of polyubiquitinated proteins, S-opsin aggregation, bip and chop mRNAs in 661W cells in vitro. Knock-down of chop mRNA inhibited photoreceptor cell death induced by light exposure. Furthermore, treatment with BiP inducer X (BIX), an ER stress inhibitor, induced bip mRNA and reduced both chop expression and light-induced photoreceptor cell death. These data indicate that excessive ER stress may induce photoreceptor cell death in light-exposed retinas via activation of the CHOP-dependent apoptotic pathway, suggesting that the ER stress may play a pivotal role in light exposure-induced retinal damage. © 2012 International Society for Neurochemistry.

  8. Rab6 is increased in Alzheimer's disease brain and correlates with endoplasmic reticulum stress.

    PubMed

    Scheper, W; Hoozemans, J J M; Hoogenraad, C C; Rozemuller, A J M; Eikelenboom, P; Baas, F

    2007-10-01

    Alzheimer's disease (AD) is characterized by deposits of aggregated proteins. Accumulation of aggregation-prone proteins activates protein quality control mechanisms, such as the unfolded protein response (UPR) in the endoplasmic reticulum (ER). We previously reported upregulation of the UPR marker BiP in AD brain. In this study, we investigated the small GTPase Rab6, which is involved in retrograde Golgi-ER trafficking and may function as a post-ER quality control system. Using immunohistochemistry and semiquantitative Western blotting, the expression of Rab6 was analysed in hippocampus, entorhinal and temporal cortex of 10 AD patients and six nondemented control subjects. Rab6 is upregulated in AD temporal cortex from Braak stage 3/4, the same stage that UPR activation is found. We observe increased neuronal Rab6 immunoreactivity in all brain areas examined. Although some neurones show colocalization of immunoreactivity for Rab6 and hyperphosphorylated tau, strong Rab6 staining does not colocalize with tangles. We find a highly significant correlation between the Rab6 and BiP levels. In vitro data show that Rab6 is not upregulated as a result of UPR activation or proteasome inhibition indicating an independent regulatory mechanism. Our data suggest that ER and post-ER protein quality control mechanisms are activated early in the pathology of AD.

  9. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress.

    PubMed

    Zhang, Chao; Wang, Chendan; Ren, Jianbo; Guo, Xiangjie; Yun, Keming

    2016-10-24

    Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca(2+) release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca(2+) release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca(2+) overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca(2+) release and ER stress.

  10. Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

    PubMed Central

    De Miguel, Carmen; Hamrick, William C.; Hobbs, Janet L.; Pollock, David M.; Carmines, Pamela K.; Pollock, Jennifer S.

    2017-01-01

    Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury. PMID:28230089

  11. Acid Sphingomyelinase Mediates Oxidized-LDL Induced Apoptosis in Macrophage via Endoplasmic Reticulum Stress

    PubMed Central

    Zhao, Min; Pan, Wei; Shi, Rui-zheng; Bai, Yong-ping; You, Bo-yang; Zhang, Kai; Fu, Qiong-mei; Schuchman, Edward H.

    2016-01-01

    Aim: Macrophage apoptosis is a vital event in advanced atherosclerosis, and oxidized low-density lipoprotein (ox-LDL) is a major contributor to this process. Acid sphingomyelinase (ASM) and ceramide are also involved in the induction of apoptosis, particularly in macrophages. Our current study focuses on ASM and investigates its role in ox-LDL-induced macrophage apoptosis. Methods: Human THP-1 and mouse peritoneal macrophages were cultured in vitro and treated with ox-LDL. ASM activity and ceramide levels were quantified using ultra performance liquid chromatography. Protein and mRNA levels were analyzed using Western blot analysis and quantitative realtime PCR, respectively. Cell apoptosis was determined using Hoechst staining and flow cytometry. Results: Ox-LDL-induced macrophage apoptosis was triggered by profound endoplasmic reticulum (ER) stress, leading to an upregulation of ASM activity and ceramide levels at an early stage. ASM was inhibited by siRNA or desipramine (DES), and/or ceramide was degraded by recombinant acid ceramidase (AC). These events attenuated the effect of ox-LDL on ER stress. In contrast, recombinant ASM upregulated ceramide and ER stress. ASM siRNA, DES, recombinant AC, and ER stress inhibitor 4-phenylbutyric acid were blocked by elevated levels of C/EBP homologous protein (CHOP); ox-LDL induced elevated levels of CHOP. These events attenuated macrophage apoptosis. Conclusion: These results indicate that ASM/ceramide signaling pathway is involved in ox-LDL-induced macrophage apoptosis via ER stress pathway. PMID:26923251

  12. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

    PubMed Central

    Kawazoe, Nozomi; Kimata, Yukio; Izawa, Shingo

    2017-01-01

    Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH. PMID:28702017

  13. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae.

    PubMed

    Kawazoe, Nozomi; Kimata, Yukio; Izawa, Shingo

    2017-01-01

    Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

  14. (-)-Epicatechin mitigates high fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress

    PubMed Central

    Bettaieb, Ahmed; Vazquez Prieto, Marcela A.; Rodriguez Lanzi, Cecilia; Miatello, Roberto M.; Haj, Fawaz G.; Fraga, César G.; Oteiza, Patricia I.

    2014-01-01

    We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome (MetS). Adolescent rats were fed a regular chow diet without or with high fructose (HFr) (10% (w/v)) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor (IR), IRS-1, Akt and ERK1/2) was impaired, while negative regulators (PKC, IKK, JNK and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events which contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase, activation of redox-sensitive signals, expression of NF-κB-regulated pro-inflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant to define interventions that can prevent/mitigate MetS-associated insulin resistance. PMID:24746618

  15. Cellular Pathology of Pelizaeus-Merzbacher Disease Involving Chaperones Associated with Endoplasmic Reticulum Stress

    PubMed Central

    Inoue, Ken

    2017-01-01

    Disease-causing mutations in genes encoding membrane proteins may lead to the production of aberrant polypeptides that accumulate in the endoplasmic reticulum (ER). These mutant proteins have detrimental conformational changes or misfolding events, which result in the triggering of the unfolded protein response (UPR). UPR is a cellular pathway that reduces ER stress by generally inhibiting translation, increasing ER chaperones levels, or inducing cell apoptosis in severe ER stress. This process has been implicated in the cellular pathology of many neurological disorders, including Pelizaeus-Merzbacher disease (PMD). PMD is a rare pediatric disorder characterized by the failure in the myelination process of the central nervous system (CNS). PMD is caused by mutations in the PLP1 gene, which encodes a major myelin membrane protein. Severe clinical PMD phenotypes appear to be the result of cell toxicity, due to the accumulation of PLP1 mutant proteins and not due to the lack of functional PLP1. Therefore, it is important to clarify the pathological mechanisms by which the PLP1 mutants negatively impact the myelin-generating cells, called oligodendrocytes, to overcome this devastating disease. This review discusses how PLP1 mutant proteins change protein homeostasis in the ER of oligodendrocytes, especially focusing on the reaction of ER chaperones against the accumulation of PLP1 mutant proteins that cause PMD. PMID:28286750

  16. KUS121, a VCP modulator, attenuates ischemic retinal cell death via suppressing endoplasmic reticulum stress

    PubMed Central

    Hata, Masayuki; Ikeda, Hanako O.; Kikkawa, Chinami; Iwai, Sachiko; Muraoka, Yuki; Hasegawa, Tomoko; Kakizuka, Akira; Yoshimura, Nagahisa

    2017-01-01

    Ischemic neural damages cause several devastating diseases, including brain stroke and ischemic retinopathies, and endoplasmic reticulum (ER) stress has been proposed to be the underlying mechanism of the neuronal cell death of these conditions. We previously synthesized Kyoto University substances (KUSs) as modulators of valosin-containing protein (VCP); KUSs inhibit VCP ATPase activity and protect cells from different cell death-inducing insults. Here, we examined the efficacy of KUS121 in a rat model of retinal ischemic injury. Systemic administration of KUS121 to rats with ischemic retinal injury significantly suppressed inner retinal thinning and death of retinal ganglion and amacrine cells, with a significant functional maintenance of visual functions, as judged by electroretinography. Furthermore, intravitreal injection of KUS121, which is the clinically preferred route of drug administration for retinal diseases, appeared to show an equal or better neuroprotective efficacy in the ischemic retina compared with systemic administration. Indeed, induction of the ER stress marker C/EBP homologous protein (CHOP) after the ischemic insult was significantly suppressed by KUS121 administration. Our study suggests VCP modulation by KUS as a promising novel therapeutic strategy for ischemic neuronal diseases. PMID:28317920

  17. Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.

    PubMed

    Boussabbeh, Manel; Ben Salem, Intidhar; Prola, Alexandre; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2015-04-01

    Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. PPARδ Is Required for Exercise to Attenuate Endoplasmic Reticulum Stress and Endothelial Dysfunction in Diabetic Mice.

    PubMed

    Cheang, Wai San; Wong, Wing Tak; Zhao, Lei; Xu, Jian; Wang, Li; Lau, Chi Wai; Chen, Zhen Yu; Ma, Ronald Ching Wan; Xu, Aimin; Wang, Nanping; Tian, Xiao Yu; Huang, Yu

    2017-02-01

    Physical activity has profound benefits on health, especially on cardiometabolic wellness. Experiments in rodents with trained exercise have shown that exercise improves vascular function and reduces vascular inflammation by modulating the balance between nitric oxide (NO) and oxidative stress. However, the upstream regulator of exercise-induced vascular benefits is unclear. We aimed to investigate the involvement of peroxisome proliferator-activated receptor δ (PPARδ) in exercise-induced vascular functional improvement. We show that PPARδ is a crucial mediator for exercise to exert a beneficial effect on the vascular endothelium in diabetic mice. In db/db mice and high-fat diet-induced obese mice, 4 weeks of treadmill exercise restored endothelium-dependent vasodilation of aortas and flow-mediated vasodilation in mesenteric resistance arteries, whereas genetic ablation of Ppard abolished such improvements. Exercise induces AMPK activation and subsequent PPARδ activation, which help to reduce endoplasmic reticulum (ER) and oxidative stress, thus increasing NO bioavailability in endothelial cells and vascular tissues. Chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid decrease ER stress and protect against endothelial dysfunction in diabetic mice. The results demonstrate that PPARδ-mediated inhibition of ER stress contributes to the vascular benefits of exercise and provides potentially effective targets for treating diabetic vasculopathy.

  19. Endoplasmic reticulum stress protects human thyroid carcinoma cell lines against ionizing radiation-induced apoptosis.

    PubMed

    Wu, Xin-Yu; Fan, Rui-Tai; Yan, Xin-Hui; Cui, Jing; Xu, Jun-Ling; Gu, Hao; Gao, Yong-Ju

    2015-03-01

    Radiotherapy is one of the most effective forms of cancer treatment, used in the treatment of a number of malignant tumors. However, the resistance of tumor cells to ionizing radiation remains a major therapeutic problem and the critical mechanisms determining radiation resistance are poorly defined. In the present study, a cellular endoplasmic reticulum (ER) stress microenvironment was established through the pretreatment of cultured thyroid cancer cells with tunicamycin (TM) and thapsigargin (TG), in order to mimic the ER stress response in a tumor microenvironment. This microenviroment was confirmed through the X‑box binding protein 1 splice process, glucose‑regulated protein 78 kD and ER degradation‑enhancing α‑mannosidase‑like mRNA expression. A clonogenic assay was used to measure cancer cell resistance to 60Co‑γ following TM pretreatment; in addition, human C/EBP homologous protein (CHOP) mRNA expression was determined and apoptosis assays were performed. The results showed that TM or TG pretreatment inhibited CHOP expression and reduced the apoptotic rate of cells. Furthermore, the results demonstrated that the induced ER stress response rendered cancer cells more resistant to ionizing radiation‑induced apoptosis. Therefore, the ER stress pathway may be a potential therapeutic target in order to improve the clinical efficiency of radiotherapy.

  20. Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells.

    PubMed

    Márquez, Saioa; Fernández, José Javier; Terán-Cabanillas, Eli; Herrero, Carmen; Alonso, Sara; Azogil, Alicia; Montero, Olimpio; Iwawaki, Takao; Cubillos-Ruiz, Juan R; Fernández, Nieves; Crespo, Mariano Sánchez

    2017-01-01

    Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.

  1. Berberine reduces endoplasmic reticulum stress and improves insulin signal transduction in Hep G2 cells

    PubMed Central

    Wang, Zeng-si; Lu, Fu-er; Xu, Li-jun; Dong, Hui

    2010-01-01

    Aim: Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of insulin resistance and pancreatic β-cell dysfunction. The aim of this study is to investigate whether the insulin-sensitizing action of berberine is related to reducing ER stress. Methods: ER stress in cultured Hep G2 cells was induced with tunicamycin. Cells were pretreated with berberine in combination with or without insulin. The concentration of glucose was measured by glucose oxidase method. The molecular markers of ER stress, including ORP150, PERK, and eIF2α were analyzed by Western blot or real time PCR. The activity of JNK was also evaluated. Moreover, the insulin signaling proteins such as IRS-1 and AKT were determined by Western blot. Results: The production of glucose stimulated with insulin was reduced. The expressions of ORP150 was decreased both in gene and protein levels when cells were pretreated with berberine, while the activation of JNK was blocked. The levels of phosphorylation both on PERK and eIF2α were inhibited in cells pretreated with berberine. The level of IRS-1 ser307 phosphorylation was decreased, whereas IRS-1 tyr phosphorylation was increased notablely. AKT ser473 phosphorylation was also enhanced significantly in the presence of berberine. Conclusion: The antidiabetic effect of berberine in Hep G2 cells maybe related to attenuation of ER stress and improvement of insulin signal transduction. PMID:20383171

  2. Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

    PubMed Central

    Choukhi, Amélie; Ung, Sophana; Wychowski, Czeslaw; Dubuisson, Jean

    1998-01-01

    The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding. PMID:9557669

  3. Endoplasmic reticulum stress as a novel cellular response to di (2-ethylhexyl) phthalate exposure.

    PubMed

    Peropadre, Ana; Fernández Freire, Paloma; Pérez Martín, José Manuel; Herrero, Óscar; Hazen, María José

    2015-12-25

    Di (2-ethylhexyl) phthalate is a high-production chemical widely used as a plasticizer for polyvinyl chloride products. Due to its ubiquitous presence in environmental compartments and the constant exposure of the general population through ingestion, inhalation, and dermal absorption, this compound has been subjected to extensive in vivo and in vitro toxicological studies. Despite the available information, research on the cytotoxicity of di (2-ethylhexyl) phthalate in mammalian cells is relatively limited.In this paper, an in vitro multi-parametric approach was used to provide further mechanistic data on the toxic activity of this chemical in Vero and HaCaT cells. Our results reveal that a 24 h exposure to di (2-ethylhexyl) phthalate causes, in both cell lines, an inhibition of cell proliferation that was linked to cell cycle delay at the G1 phase. Concomitantly, the tested compound induces mild endoplasmic reticulum stress which leads to an adaptive rather than a pro-apoptotic response in mammalian cells. These findings demonstrate that there are multiple potential cellular targets of di (2-ethylhexyl) phthalate-induced toxicity and the need to develop further experimental studies for the risk assessment of this ubiquitous plasticizer.

  4. Distinct mechanisms controlling rough and smooth endoplasmic reticulum contacts with mitochondria.

    PubMed

    Wang, Peter T C; Garcin, Pierre O; Fu, Min; Masoudi, Matthew; St-Pierre, Pascal; Panté, Nelly; Nabi, Ivan R

    2015-08-01

    Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

  5. Bcl-XL specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum

    PubMed Central

    Klee, Martina; Pimentel-Muiños, Felipe X.

    2005-01-01

    Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-XL provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues. PMID:15728194

  6. Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

    PubMed Central

    Jiang, Chunping; Luo, Dongjun; Cao, Yin; Liu, Baorui

    2014-01-01

    Background. Hepatocellular carcinoma (HCC) remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR) markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs) were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER) stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC. PMID:24995326

  7. Induction of endoplasmic reticulum stress and the modulation of thioredoxin-1 in formaldehyde-induced neurotoxicity.

    PubMed

    Luo, Fu-Cheng; Zhou, Jia; Lv, Tao; Qi, Lei; Wang, Sheng-Dong; Nakamura, Hajime; Yodoi, Junji; Bai, Jie

    2012-06-01

    Formaldehyde (FA), a common environmental pollutant, has toxic effects on central nervous system. The detailed mechanisms on FA-induced neurotoxicity have not been fully elucidated. In this study, we found that glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) expression, biomarkers of endoplasmic reticulum (ER) stress, were increased and pro-caspase-12 was decreased after PC12 cells exposure to FA. These results suggest that FA actually induces ER stress. Thioredoxin-1 (Trx-1) has various biological activities, including the control of redox balance, the modulation of ER stress and inhibition of apoptosis. In the present study, Trx-1 expression was increased at early stage, but decreased at late stage after FA treatment. Knockdown of Trx-1 expression increased the susceptibility of PC12 cells to FA-induced neurotoxicity. We also found that ginsenoside Rg1 had the potential to induce Trx-1 expression and attenuated neurotoxicity induced by FA. ER stress caused by FA was suppressed by ginsenoside Rg1. These data indicate that Trx-1 is a therapeutic candidate for protecting against FA-induced neurotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. The Ca(2+)-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

    PubMed

    Espinoza-Fonseca, L Michel

    2017-03-28

    Ca(2+) transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca(2+) homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca(2+) uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca(2+) and other ions across the SR. During Ca(2+) uptake by the SR Ca(2+)-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca(2+) transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca(2+) release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca(2+) transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

  9. Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells

    PubMed Central

    Márquez, Saioa; Fernández, José Javier; Terán-Cabanillas, Eli; Herrero, Carmen; Alonso, Sara; Azogil, Alicia; Montero, Olimpio; Iwawaki, Takao; Cubillos-Ruiz, Juan R.; Fernández, Nieves; Crespo, Mariano Sánchez

    2017-01-01

    Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs. PMID:28674530

  10. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  11. Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

    PubMed Central

    Bertrand, Luc

    2015-01-01

    Increasing evidence demonstrates that the antiretroviral drugs (ARVds) used for human immunodeficiency virus (HIV) treatment have toxic effects that result in various cellular and tissue pathologies; however, their impact on the cells composing the blood-brain barrier is poorly understood. The current study focused on ARVds, used either in combination or alone, on the induction of endoplasmic reticulum (ER) stress responses in human brain endothelial cells. Among studied drugs (efavirenz, tenofovir, emtricitabine, lamivudine, and indinavir), only efavirenz increased ER stress via upregulation and activation of protein kinase-like ER kinase PERK and inositol requiring kinase 1α (IRE1α). At the same time, efavirenz diminished autophagic activity, a surprising result because typically the induction of ER stress is linked to enhanced autophagy. These results were confirmed in microvessels of HIV transgenic mice chronically administered with efavirenz. In a series of further experiments, we identified that efavirenz dysregulated ER stress and autophagy by blocking the activity of the Beclin-1/Atg14/PI3KIII complex in regard to synthesis of phosphatidylinositol 3-phosphate, a process that is linked to the formation of autophagosomes. Because autophagy is a protective mechanism involved in the removal of dysfunctional proteins and organelles, its inhibition can contribute to the toxicity of efavirenz or the development of neurodegenerative disease in HIV patients treated with this drug. PMID:25987489

  12. A novel animal model of hearing loss caused by acute endoplasmic reticulum stress in the cochlea.

    PubMed

    Fujinami, Yoshiaki; Mutai, Hideki; Mizutari, Kunio; Nakagawa, Susumu; Matsunaga, Tatsuo

    2012-01-01

    Many stimuli such as ischemia, hypoxia, heat shock, amino acid starvation, and gene mutation, exhibit a cellular response called endoplasmic reticulum (ER) stress. ER stress induces expression of a series of genes, leading to cell survival or apoptosis. Previously, we found that in an animal model of hearing loss caused by acute mitochondrial dysfunction, several ER stress markers including C/EBP homologous protein were induced in the cochlear lateral wall. To elucidate the mechanism of hearing loss caused by ER stress, we established a novel animal model of hearing loss by perilymphatic perfusion of tunicamycin, an ER stress activator that inhibits N-acetylglucosamine transferases. Subacute and progressive hearing loss was observed at all sound frequencies studied, and stimulation of ER stress marker genes was noted in the cochlea. The outer hair cells were the most sensitive to ER stress in the cochlea. Electron microscopic analysis demonstrated degeneration of the subcellular organelles of the inner hair cells and nerve endings of the spiral ganglion cells. This newly established animal model of hearing loss from ER stress will provide additional insight into the mechanism of sensorineural hearing loss.

  13. Endoplasmic reticulum stress in the peripheral nervous system is a significant driver of neuropathic pain.

    PubMed

    Inceoglu, Bora; Bettaieb, Ahmed; Trindade da Silva, Carlos A; Lee, Kin Sing Stephen; Haj, Fawaz G; Hammock, Bruce D

    2015-07-21

    Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

  14. Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease.

    PubMed

    Arduíno, Daniela Moniz; Esteves, A Raquel; Cardoso, Sandra M; Oliveira, Catarina R

    2009-09-01

    Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

  15. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    PubMed Central

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal MD; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-01-01

    Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome. DOI: http://dx.doi.org/10.7554/eLife.15887.001 PMID:27213518

  16. High concentration calcitriol induces endoplasmic reticulum stress related gene profile in breast cancer cells.

    PubMed

    Ozkaya, Ali Burak; Ak, Handan; Aydin, Hikmet Hakan

    2017-04-01

    C