Expression Of CCAAT/enhancer-binding Protein Homologous Protein Research Articles

Dexmedetomidine-induced cardioprotection in a mouse model of lung ischemia-reperfusion: the relationship with endoplasmic reticulum stress

Objective To evaluate dexmedetomidine-induced cardioprotection in a mouse model of lung ischemia-reperfusion(I/R)and the relationship with endoplasmic reticulum stress. Methods Forty healthy SPF male C57BL/6J mice, weighing 20-24 g, aged 8-10 weeks, were divided into 4 groups(n=10 each)using a random number table: sham operation group(Sham group), lung I/R group(I/R group), dexmedetomidine group(Dex group)and dexmedetomidine plus atipamezole(specific α2-adrenergic receptor antagonist)group(DA group). The model of lung I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion.In group Sham, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min.In Dex and DA groups, dexmedetomidine 20 μg/kg and dexmedetomidine 20 μg/kg plus atipamezole 250 μg/kg were injected intraperitoneally, respectively, at 30 min before establishment of the model.At 180 min of reperfusion, blood samples were collected from the orbit for determination of creatine kinase-MB(CK-MB)and lactic dehydrogenase(LDH)activities in serum.The animals were then sacrificed, and hearts were removed for determination of apoptosis in cardiomyocytes(by TUNEL)and expression of phosphorylated c-Jun N-terminal kinase(p-JNK), caspase-12, CCAAT/enhancer-binding protein homologous protein(CHOP)and glucose-regulated protein 78(GRP78)in myocardial tissues(by Western blot), and expression of JNK, caspase-12, CHOP, GRP78 mRNA in myocardial tissues(by real-time polymerase chain reaction). Apoptosis index was calculated. Results Compared with Sham group, the serum CK-MB and LDH activities and apoptosis index were significantly increased, the expression of p-JNK, JNK mRNA, and caspase-12, CHOP and GRP78 protein and mRNA was up-regulated in I/R, Dex and DA groups(P 0.05). Compared with DEX group, the serum CK-MB and LDH activities and apoptosis index were significantly increased, the expression of p-JNK, JNK mRNA, and caspase-12 and CHOP protein and mRNA was up-regulated(P 0.05). Conclusion Dexmedetomidine can reduce myocardial injury induced by lung I/R, and the mechanism may be related to activation of α2-adrenergic receptors and inhibition of endoplasmic reticulum stress in myocardial cells of mice. Key words: Dexmedetomidine; Lung; Reperfusion injury; Myocardium; Endoplasmic reticulum; Stress

Modulation of the Unfolded Protein Response by Tauroursodeoxycholic Acid Counteracts Apoptotic Cell Death and Fibrosis in a Mouse Model for Secondary Biliary Liver Fibrosis.

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death.

Ginsenoside Rh2 Inhibited Proliferation by Inducing ROS Mediated ER Stress Dependent Apoptosis in Lung Cancer Cells.

Ginsenoside Rh2 (G-Rh2), a component extracted from roots of ginseng, exhibited anti-cancer pharmacological activities by inhibiting proliferation and inducing apoptosis in lung cancer cells. However, the mechanisms of G-Rh2 suppressing lung cancer development remained elusive. This study tried to investigate the possible mechanism involved in anti-proliferative effect of G-Rh2 in lung cancer cells. As results, G-Rh2 inhibited the proliferation of H1299 cells in a dose-dependent manner by inducing cell apoptosis. Activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP), and caspase-4 were involved in G-Rh2-induced apoptosis of H1299 cells. It was also found that G-Rh2 could up-regulate expressions of ATF4, CHOP and caspase-4 in H1299 cells in a dose-dependent manner. In addition, NAC (N-acetylcysteine, a reactive oxygen species (ROS) scavenger) treatment dramatically decreased ROS generation in H1299 cells; both of NAC and 4-PBA (4-phenylbutyrate, a specific endoplasmic reticulum (ER) stress inhibitor) administration impaired apoptosis and expression levels of ATF4, CHOP and caspase-4 in G-Rh2 incubated H1299 cells. In vivo assays extended the significance of these results, showing that G-Rh2 inhibited lung cancer growth and the inhibition effects of G-Rh2 in tumor growth were significantly reduced by inhibition of ER stress. In conclusion, G-Rh2 inhibited proliferation of H1299 cells by inducing ROS mediated ER stress dependent cell apoptosis.

Effects of FTY720 pretreatment on lung injury induced by limb ischemia-reperfusion in rats

Objective To evaluate the effects of FTY720 pretreatment on lung injury induced by limb ischemia-reperfusion (I/R) in rats. Methods Thirty healthy male Sprague-Dawley rats, weighing 230-260 g, were divided into 3 groups (n=10 each) using a random number table: sham operation group (group S), I/R group and FTY720 group (group F). Bilateral hind limb I/R was induced by applying rubber band tourniquet high around each thigh for 3 h followed by 3 h of reperfusion.FTY720 was given by intragastric gavage at 3 mg·kg-1·d-1 for 7 consecutive days before I/R in group F, while the equal volume of normal saline was given instead in group I/R.At the end of reperfusion, blood samples were collected from the carotid artery for blood gas analysis, and oxygenation index (OI) was calculated.The rats were then sacrificed and lungs removed for examination of pathological changes (with light microscope) and for determination of apoptosis rate (using flow cytometry) and expression of CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, c-Jun N-terminal kinase (JNK), Bcl-2 and Bax protein in lung tissues (using Western blot). Bax/Bcl-2 ratio was calculated. Results Compared with group S, OI was significantly decreased in group I/R, and apoptosis rate was increased, the expression of CHOP, caspase-12, JNK and Bax protein was up-regulated, and the expression of Bcl-2 was down-regulated, and Bax/Bcl-2 ratio was increased in I/R and F groups (P<0.05). Compared with group I/R, OI was significantly increased, apoptosis rate was decreased, the expression of caspase-12, CHOP, Bax and JNK protein was down-regulated, and the expression of Bcl-2 was up-regulated, and Bax/Bcl-2 ratio was decreased in group F (P<0.05). The pathological changes of lungs were significantly attenuated in group F as compared with group I/R. Conclusion FTY720 pretreatment can attenuate lung injury induced by limb I/R in rats, and the mechanism may be related to inhibition of endoplasmic reticulum stress-related cell apoptosis. Key words: Extremities; Reperfusion injury; Respiratory distress syndrome, adult; FTY720

Evaluation and Treatment of Endoplasmic Reticulum (ER) Stress in Right Ventricular Dysfunction during Monocrotaline-Induced Rat Pulmonary Arterial Hypertension.

Endoplasmic reticulum (ER) stress contributes to pulmonary artery hypertension (PAH). However, the exact roles of ER stress in right ventricular (RV) dysfunction, which is strongly associated with PAH, are largely unknown. Here, we aimed to explore how ER stress affects RV function in a rat PAH model and evaluated the effects of an ER stress inhibitor on RV dysfunction. We examined expression changes of an ER marker: chaperone glucose-regulated protein 78 (GRP78), three ER stress sensor proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), and a key ER stress-induced apoptosis indicator: CCAAT/enhancer-binding protein homologous protein (CHOP), with inflammation indicators: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) in RV at 3, 7, 14 and 28days following a single dose of monocrotaline (MCT) injection, with or without a preventive treatment [4-phenylbutyric acid (PBA)]. RV function was evaluated by histological, molecular and echocardiographic analysis. 1) GRP78 protein expression started to increase (1.5±0.06 fold change) at 3d post MCT injection, even before the formation of PAH. 2) ATF6, IRE1, and PERK showed distinctive expression patterns post MCT injection. 3) CHOP expression remained low at day 3 & 7, but significantly increased at day 14 (p<0.05), along with the peak of RV cardiomyocytes apoptosis. 4) PBA inhibited ER stress and alleviated remodeling and dysfunction in the RV. The early phase of ER stress might benefit RV function, whereas the extended phase led to RV cardiomyocyte apoptosis and dysfunction. Inhibition of ER stress by PBA during PAH directly improved RV function.

Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways.

Retinal ganglion cells (RGCs) consume large quantities of energy to convert light information into a neuronal signal, which makes them highly susceptible to hypoxic injury. This study aimed to investigate the potential protection by baclofen, a GABAB receptor agonist of RGCs against hypoxia-induced apoptosis. Cobalt chloride (CoCl2) was applied to mimic hypoxia. Primary rat RGCs were subjected to CoCl2 with or without baclofen treatment, and RNA interference techniques were used to knock down the GABAB2 gene in the primary RGCs. The viability and apoptosis of RGCs were assessed using cell viability and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, Hoechst staining, and flow cytometry. The expression of cleaved caspase-3, bcl-2, bax, Akt, phospho-Akt, protein kinase RNA (PKR)-like ER kinase (PERK), phospho-PERK, eIF2α, phospho-eIF2α, ATF-4 and CCAAT/enhancer-binding protein homologous protein (CHOP) were measured using western blotting. GABAB2 mRNA expression was determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Our study revealed that CoCl2 significantly induced RGC apoptosis and that baclofen reversed these effects. CoCl2-induced reduction of Akt activity was also reversed by baclofen. Baclofen prevented the activation of the PERK pathway and the increase in CHOP expression induced by CoCl2. Knockdown of GABAB2 and the inactivation of the Akt pathway by inhibitors reduced the protective effect of baclofen on CoCl2-treated RGCs. Taken together, these results demonstrate that baclofen protects RGCs from CoCl2-induced apoptosis by increasing Akt activity and by suppressing the PERK pathway and CHOP activation.

Neurotoxin β‑N‑methylamino‑L‑alanine induces endoplasmic reticulum stress‑mediated neuronal apoptosis.

β-N-methylamino-L-alanine (BMAA) is a neurotoxin that is closely associated with the incidence of amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease. In cultured neuronal cells, BMAA notably induces the upregulation of endoplasmic reticulum (ER) chaperons and activates the unfolded protein response (UPR) receptor pathways of protein kinase RNA‑like endoplasmic reticulum kinase, inositol‑requiring kinase1 and transcription factor6. The ER stress‑specific protein CCAAT/‑enhancer‑binding protein homologous protein (CHOP) affords pro‑apoptotic responses that cause mitochondrial damage and caspase activation. BMAA also induces the activation of mitogen‑activated protein kinase member c‑JUN N‑terminal kinase, p38and extracellular signal‑regulated kinase, which have been suggested to be involved in the signaling pathway of UPR‑mediated apoptosis. Inhibition of ER stress using ER stress antagonist, salubrinal, attenuated the expression of CHOP and alleviated neuronal death. Overexpression of heat shock protein70suppressed the activation of UPR receptors and UPR‑evoked apoptotic signaling. The present findings demonstrated that ER stress induced by BMAA is the important mediator of neuronal injury and apoptotic death, and suggests development in novel therapeutic strategies for treatment.

Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells

Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycin-induced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor.

Expressions of CCAAT/enhancer-binding Protein Homologous Protein and Calnexin in the Hippocampus of a Mouse Model of Mesial Temporal Lobe Epilepsy.

Objective To explore the temporal and spatial distribution of CCAAT/enhancer-binding protein homologous protein (CHOP) and calnexin (CNX) in the dentate gyrus of mesial temporal lobe epilepsy (mTLE) mouse model. Methods We used kainic acid (KA) to induce acute phase (12 h and 24 h) mTLE mouse models and performed Western blotting and immunofluorescence to detect the different expressions and distribution pattern of CHOP and CNX in CA3 of the hippocampus. Results Compared with the controls,the expressions of CHOP(F=1.136,P=0.4069) and CNX (F=2.378,P=0.2087) did not increase in CA3 of hippocampus 12 h following KA injection in the acute phase of mTLE mouse models,whereas the expressions in CA1 and CA3 of hippocampus 24 h after injection were significantly higher (F=8.510,P=0.0362;F=6.968,P=0.0497,respectively). As shown by immunofluorescence analysis,CHOP was expressed mainly in CA3 of hippocampus 12 h after KA injection,and increased in CA1 and CA3 24 h after KA administration. Compared with the controls,the expressions of CHOP(F=24.480,P=0.0057) and CNX (F=7.149,P=0.0478) were significantly higher 24 h after KA injection.Conclusions The expression of CHOP increases along with the progression of seizures,indicating the increased level of endoplasmic reticulum stress. An increasing number of CNX,which serves as molecular chaperone,may be needed to facilitate the unfolded protein to complete the folding process.

Effect of sevoflurane preconditioning on lung injury induced by limb ischemia-reperfusion in rats and the relationship with endoplasmic reticulum stress

Objective To investigate the effect of sevoflurane preconditioning on lung injury induced by limb ischemia-reperfusion in rats and the relationship with endoplasmic reticulum stress. Methods Forty-five healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 250-300 g, were randomly allocated into 3 groups(n=15 each)using a random number table: sham operation group(group S), limb ischemia-reperfusion group(group LIR), and sevoflurane preconditioning group(group SP). Limb ischemia-reperfusion was induced by clamping bilateral femoral arteries for 3 h followed by 3 h reperfusion in LIR and SP groups.In group SP, 2.5% sevoflurane was inhaled for 30 min, and 15 min later the model was established.Before ischemia and at 3 h of reperfusion(at the corresponding time point in group S), arterial blood samples were collected for blood gas analysis, and arterial oxygen partial pressure(PaO2)and arterial carbon dioxide partial pressure(PaCO2)were recorded.The rats were sacrificed at 3 h of reperfusion, and lung specimens were obtained for determination of wet to dry lung weight ratio(W/D ratio)and total lung water content(TLW)and for microscopic examination of the pathological changes, and index of quantitative evaluation for alveolar damage(IQA)was calculated.The expression of glucose-regulated protein 78(GRP78)and CCAAT/enhancer-binding protein homologous protein(CHOP)protein and mRNA in lung tissues was detected by Western blot and real-time polymerase chain reaction, respectively. Results Compared to group S, the PaO2 was significantly decreased at 3 h of reperfusion, the W/D ratio, TLW and IQA were significantly increased, the expression of GRP78 and CHOP protein and mRNA was significantly up-regulated(P 0.05). Compared to group LIR, the PaO2 was significantly increased at 3 h of reperfusion, the W/D ratio, TLW and IQA were significantly decreased, the expression of GRP78 and CHOP protein and mRNA was significantly down-regulated(P 0.05). Conclusion Sevoflurane preconditioning can ameliorate lung injury induced by limb ischemia-reperfusion in the rats, and the mechanism is related to inhibition of endoplasmic reticulum stress. Key words: Anesthetics, inhalation; Ischemic preconditioning; Extremities; Reperfusion injury; Respiratory distress syndrome, adult; Endoplasmic reticulum; Stress

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

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

Ethanolic extract of Descurainia sophia seeds sensitizes A549 human lung cancer cells to TRAIL cytotoxicity by upregulating death receptors.

BackgroundOur previous genome-wide gene expression analysis revealed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors 4 (DR4) and 5 (DR5) are markedly upregulated by the ethanolic extract of D. sohia seeds (EEDS) in A549 TRAIL-refractory cancer cells. In the present study, we investigated whether the EEDS-mediated upregulation of TRAIL death receptors was associated with increased TRAIL-mediated toxicity in A549 cells in vitro.MethodsCell proliferation and viability were determined by an automatic cell counter. Gene silencing was performed by introducing small interfering RNA into cells. Expression changes of cellular proteins were determined by western blot analysis. Apoptotic cell death was monitored by western blot analysis. Analysis of variance followed by the post-hoc Dunnett’s test was used to compare the data.ResultsEEDS treatment increased both mRNA and protein levels of DR4 and DR5 in the TRAIL refractory A549 cells. Co-treatment of A549 cells with sub-lethal dose of EEDS and recombinant TRAIL increased the apoptotic cell death. Upregulation of DR5 by EEDS was mediated by an endoplasmic reticulum stress-induced transcription factor, CCAAT/enhancer-binding protein homologous protein (CHOP), and knockdown of CHOP expression inhibited EEDS-induced DR5 upregulation and abolished the EEDS-associated increase in TRAIL toxicity in A549 cells.ConclusionsEEDS can sensitize A549 cells to TRAIL cytotoxicity by upregulation of TRAIL death receptors. Our findings suggested that EEDS is a good initial herbal source for the development of an anticancer supplement for anticancer therapeutics associated with TRAIL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1094-0) contains supplementary material, which is available to authorized users.

Characterization of 2‐Chlorofatty Acid Signaling Pathways

Monocytes and macrophages are key inflammatory cell mediators in the response to infection and tissue injury. Monocytes and macrophages produce a respiratory burst, resulting in the release of reactive oxygen species. Upon activation of these leukocytes, myeloperoxidase (MPO) is released. MPO catalyzes the conversion of hydrogen peroxide into hypochlorous acid (HOCl). HOCl then is able to target the vinyl ether bond of plasmalogens, an abundant class of phospholipids. This results in the production of a novel class of chlorinated lipids, including 2‐chlorofatty aldehyde (2‐ClFALD) and its oxidation product, 2‐chlorofatty acid (2‐ClFA). 2‐Chlorofatty acids have been shown to accumulate in activated human neutrophils and monocytes, as well as the plasma of rats subjected to lipopolysaccharide treatment or the cecal ligation and puncture sepsis model. The present study is designed to identify signaling mechanisms mediated by 2‐ClFA. Initial studies used dynamic mass redistribution (DMR) assays, which is a label‐free functional cell‐based technology that measures whole cell response to ligands. DMR analyses demonstrated that the 2‐ClFA, 2‐chloropalmitic acid, selectively stimulates THP‐1 cells and not HEK293 cells. Furthermore, the dose‐response for 2‐chloropalmitic acid was striking compared to the lack of response to equimolar concentrations of the non‐chlorinated fatty acid, palmitic acid. CCAAT/enhancer‐binding protein homologous protein (CHOP) mRNA increases selectively in response to 2‐chloropalmitic acid compared to palmitic acid in both RAW 264.7 and THP‐1 cells. CHOP mRNA expression in RAW 264.7 cells is inhibited by Bisindolylmaleimide, SB203580, and U0126 indicating 2‐ClFA elicited CHOP expression is PKC, p38 and MEK1/2 dependent. Thus, THP‐1 and RAW 264.7 cells are ideal cells to use for expression analysis and DMR to further elucidate the mechanisms and receptor by which 2‐ClFA elicits cell responses.Support or Funding InformationNational Institutes of Health Grant GM115553

Niclosamide induced cell apoptosis via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells.

BackgroundHepatocellular carcinoma (HCC) is one of most common and aggressive human malignancies in the world, especially, in eastern Asia, and its mortality is very high at any phase. We want to investigate mechanism of niclosamide inducing cell apoptosis in HCC.MethodsTwo hepatoma cell lines were used to evaluate activity of niclosamide inducing cell apoptosis and study its mechanism. Quantitative real-time PCR and western blotting were used in analysis of genes expression or protein active regulated by niclosamide.ResultsNiclosamide remarkably induced cell apoptosis in hepatoma cells. Furthermore, our study revealed that RNA-dependent protein kinase-like kinase (PERK) is activated and its expression is up-regulated in HCC cells which are exposed to niclosamide. niclosamide also significantly increase activating transcription factor 3 (ATF3), activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-homologous protein (CHOP) expression in HCC cells. It’s suggested that the function of niclosamide was abrogated by PERK inhibitor or absent ATF3. Expression of PERK and CHOP is correlated with ATF3 level in the cells.ConclusionTaken together, our results indicate that ATF3 plays an integral role in ER stress activated and cell apoptosis induced by niclosamide in HCC cells. In this study, the new mechanism of niclosamide as anti-cancer we investigated, too.

Tunicamycin-induced Endoplasmic Reticulum Stress Upregulates the Expression of Pentraxin 3 in Human Retinal Pigment Epithelial Cells.

PurposeTo investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19.MethodsPTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells.ResultsThe protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells.ConclusionsThese results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.

7,8-DHF Treatment Induces Cyr61 Expression to Suppress Hypoxia Induced ER Stress in HK-2 Cells.

Acute kidney injury (AKI) is a common syndrome which is strongly linked to high morbidity and mortality. Hypoxia is the leading cause of AKI and the proximal renal tubular cells are the most damaged part in the kidney during this period. It has been observed that 7,8-dihydroxyflavone (7,8-DHF) plays a protective role by acting on antiapoptosis and antioxidative stress. In this study we explored functions of 7,8-DHF in protecting human proximal tubular cell line HK-2 from hypoxia insults. We observed that treatment of 7,8-DHF could improve the viability of ischemic cell. Mechanistically, we found that 7,8-DHF could elevate the expression of cysteine-rich protein 61 (Cyr61), a protective immediate early gene in AKI. In addition, treatment of 7,8-DHF decreased CCAAT/enhancer-binding protein homologous protein (CHOP) expression, which is a marker protein during endoplasmic reticulum (ER) stress activation. Intriguingly, overexpression of Cyr61 significantly reduced CHOP expression. Taken together, our results provide novel insights into the possible protective role of 7,8-DHF by activating Cyr61 signaling and suppressing ER stress in hypoxic HK-2 cells which have potential clinical implications for the treatment of AKI.

RU486 Induces Pro-Apoptotic Endoplasmic Reticulum Stress Through the Induction of CHOP Expression by Enhancing C/EBPδ Expression in Human Renal Carcinoma Caki Cells.

RU486 (Mifepristone) is known as an antagonist of the progesterone receptor and glucocorticoid receptor. Here, we investigated the mechanism underlying anti-tumor activity of RU486 in renal carcinoma Caki cells. Treatment of Caki cells with RU486 was found to induce several signature ER stress markers; including ER stress-specific XBP1 splicing, and the up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. RU486-induced expression of CHOP involves the putative C/EBPδ site within the CHOP promoter region. Using a combination of C/EBPδ cDNA transfection, the luciferase assay with a mutated C/EBPδ binding site and siRNA-mediated C/EBPδ knockdown, we found that the C/EBPδ site is required for RU486-mediated activation of the CHOP promoter. In addition, RU486-induced CHOP expression is down-regulated by inhibition of the p38 MAPK and JNK signaling pathways at the post-translational levels. RU486 dose-dependently induced apoptotic cell death in renal carcinoma cells. Suppression of CHOP expression by CHOP siRNA attenuated RU486-induced apoptosis. Taken together, RU486 induces pro-apoptotic ER stress through the induction of CHOP expression.

Effect of fluvastatin on the expressions of caspase-12, CHOP and JNK in ischemia-reperfusion brain injury in rats

Objective To investigate the effect of fluvastatin on the expressions of caspase-12, CCAAT/enhancer-binding protein homologous protein(CHOP), and c-Jun N-terminal kinases (JNK) in ischemia-reperfusion brain injury in rats. Methods Forty two rats were randomly divided into sham operation group (6 rats), ischemia- reperfusion (I/R) group (18 rats), and fluvastatin (Flu) group (18 rats). The rats of I/R and Flu groups were molded by modified Longa intraluminal thread, then put to death at 2 h occlusion and 24 h reperfusion point. Expressions of caspase-12, CHOP, and JNK were detected with immunohistochemistry and Western blot. Results Immunohistochemistry and Western blot showed that the expressions of caspase-12, CHOP, and JNK were increased at 24 h reperfusion. Compared to I/R group, the expressions of caspase-12 and CHOP in Flu group were decreased significantly (all P 0.05). Conclusions The increased expression of caspase-12, CHOP, and JNK showed that endoplasmic reticulum stress was involved in the pathological process of ischemia-reperfusion brain injury. Fluvastatin could inhibit the expression of caspase-12 and CHOP, and could delete endoplasmic reticulum stress (ERS) in ischemia-reperfusion brain injury. Key words: Pravastatin/PD; Brain ischemia/DT/ME; Reperfusion injury/DT/ME; Caspase 12/ME; Transcription factor CHOP/ME; JNK mitogen-activated protein kinases/ME

The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway.

Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway.

Endoplasmic reticulum stress is involved in apoptosis of detrusor muscle in streptozocin-induced diabetic rats.

Endoplasmic reticulum stress (ERS) has been proven to be associated with apoptosis and plays a critical role in the development of many diabetic complications. In the pathogenesis of diabetic cystopathy (DCP), the role of ERS is still unclear. Our study is aimed at the investigation of the involvement of ERS-associated detrusor muscle apoptosis in streptozocin (STZ)-induced diabetic rats. At different timepoints (4, 8, 12, and 16 weeks after induction of type 1 diabetic rat models), hematoxylin & eosin (H&E) staining was performed to assess the histological changes of the diabetic detrusor; the sub-cellular ultrastructure, especially the zone of endoplasmic reticulum (ER), was observed by transmission electron microscopy (TEM), and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) staining was used to identify the enhanced apoptosis. Moreover, the expression of three hallmarks of ERS-associated apoptosis, including glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and caspase12, was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Light microscopic impairments of histology, including progressive loosely packed muscle bundles and increased fibrous tissue, can be seen; the ultrastructural changes featuring the swollen and fused cisternaes in ER zone and deformed nucleus were also observed in the detrusor smooth muscle (DSM). Increased apoptosis and elevated expression of GRP78, CHOP, and caspase12 at both protein and mRNA levels in a time-dependent fashion were detected. The occurrence of ERS-associated apoptosis may be involved in the development of DCP and may contribute to the diabetic detrusor impairment. Neurourol. Urodynam. 36:65-72, 2017. © 2015 Wiley Periodicals, Inc.