Presence Of Endoplasmic Reticulum Stress Research Articles

An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

The Effects of Endoplasmic Reticulum Stress via Intratracheal Instillation of Water-Soluble Acrylic Acid Polymer on the Lungs of Rats.

Polyacrylic acid (PAA), an organic chemical, has been used as an intermediate in the manufacture of pharmaceuticals and cosmetics. It has been suggested recently that PAA has a high pulmonary inflammatory and fibrotic potential. Although endoplasmic reticulum stress is induced by various external and intracellular stimuli, there have been no reports examining the relationship between PAA-induced lung injury and endoplasmic reticulum stress. F344 rats were intratracheally instilled with dispersed PAA (molecular weight: 269,000) at low (0.5 mg/mL) and high (2.5 mg/mL) doses, and they were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure. PAA caused extensive inflammation and fibrotic changes in the lungs' histopathology over a month following instillation. Compared to the control group, the mRNA levels of endoplasmic reticulum stress markers Bip and Chop in BALF were significantly increased in the exposure group. In fluorescent immunostaining, both Bip and Chop exhibited co-localization with macrophages. Intratracheal instillation of PAA induced neutrophil inflammation and fibrosis in the rat lung, suggesting that PAA with molecular weight 269,000 may lead to pulmonary disorder. Furthermore, the presence of endoplasmic reticulum stress in macrophages was suggested to be involved in PAA-induced lung injury.

Effect on Endoplasmic Reticulum Stress of the Combined Oral Contraceptives in the Liver

Objective: We aimed to evaluate the effects of combined oral contraceptive active ingredients ethinylestradiol, drospirenone, and ethinylestradiol+drospirenone for histopathological changes, and endoplasmic reticulum stress levels in the liver.
 
 Methods: In the study, 37 to 8-week-old Balb/c female mice were used. Mice were randomly divided into the control, sham, ethinylestradiol, drospirenone, and ethinylestradiol+drospirenone groups. Experimental groups were administered ethinylestradiol, drospirenone, and ethinylestradiol+drospirenone with gavage for 35 days. In liver tissue sections, histopathological changes were detected with hematoxylin&eosin, orcein, Mallory's Azan, and periodic acid-Schiff, and the presence of endoplasmic reticulum stress was detected by Chop and Grp78 immunostaining.
 
 Results: The ethinylestradiol+drospirenone group showed significant histopathological changes compared to the control group. Some degenerative changes were noted such as swelling and size differences in hepatocytes in the ethinylestradiol+drospirenone group. When compared to the control group, an increased collagen and elastic fibers density around the vena centralis was observed in the ethinylestradiol+drospirenone group. The expression level of Grp78 protein in female mice given ethinylestradiol+drospirenone was statistically significantly increased compared to the control group. The expression level of Chop protein was significantly increased in the ethinylestradiol, drospirenone, and ethinylestradiol+drospirenone groups. 
 
 Conclusion: We concluded that the use of combined oral contraceptives increases endoplasmic reticulum stress in mouse liver tissue, and as a result, it may cause liver histopathological disorders by promoting cell death.

The Hsp90 Chaperone Regulates p38 Activation During ER Stress

Diseases such as diabetes, Alzheimer’s disease, and Parkinson’s disease can be characterized by the presence of endoplasmic reticulum stress. The endoplasmic reticulum is an organelle that plays a major role in protein synthesis, but an accumulation of misfolded proteins within the ER lumen causes the organelle to release a stress signal through the unfolded protein response (UPR). Despite being present in many disorders, there are alternative signaling routes within the UPR branches that are not fully understood. Perk dimerization initiates one of three main signaling cascades in the UPR by phosphorylating eIF2α and causing a cell‐wide pause in mRNA translation; however, a study has shown that outside of the established pathway, activation of Perk results in p38 activation, which induces apoptosis during prolonged ER stress. The mechanism responsible for p38 activation is still unknown. One route for elucidating the regulation of p38 activation during ER stress is the Hsp90 chaperone. The Hsp90 chaperone complex sequesters p38 in the cytoplasm and prevents its auto‐activation under normal conditions, so we hypothesized that Perk activation may mediate post‐translational modification of the Hsp90 complex in a manner that would release p38, allowing it to activate. In this study, Tunicamycin‐induced ER stress and Hsp90 inhibition were applied to BHK21 fibroblasts, a cell line in which ER stress has been well characterized, and dissociation of the p38‐Hsp90 complex was measured through immunoblot and co‐immunoprecipitation analysis. Initial immunoblot results from 50nM and 200nM of tunicamycin reconfirm that moderate doses of tunicamycin induced p38 activation in a dose dependent manner. Co‐immunoprecipitation of Hsp90 reconfirmed that tunicamycin‐induced ER stress causes p38 to dissociate from Hsp90. Previous experiments with co‐immunoprecipitation of p38 by Jingyi Chen revealed that treatment with 50nM of tunicamycin resulted in 0.3 decrease in fold induction after 15 minutes. Unexpectedly, we found that inhibition of Hsp90 causes it to exhibit increased binding of p38 with an peak average fold increase of 7 after treatment with 5uM of Hsp90‐I (17‐AAG) for 30 minutes, which suggests Hsp90 as a potential regulator for p38. Because p38 plays an important role in the induction of many intracellular signaling pathways, learning ways to regulate its actions within the context of ER stress may be beneficial in mediating symptoms of disease.

733 The association of endoplasmic reticulum (ER) stress with labor and evidence of placental vascular malperfusion

To determine if the degree of ER stress, as measured by staining intensity via glucose regulatory protein 78(GRP78), correlates with clinical outcomes of pregnancy; if there is a unique pattern of maternal (MVM) and fetal vascular malperfusion (FVM) lesions and if there is a correlation with duration of labor Women planning a pregnancy, were recruited between 2010-2014 (IRB approved). Chronic medical illnesses were excluded except chronic hypertension. Clinical or histopathologic evidence of chorioamnionitis(n=12) and missing data(n=5) were excluded resulting in 51 placentas(hypertensive n=14, normotensive n=37).Immunofluorescence was performed using GRP78 marker(CST ®) and staining was analyzed in the trophoblastic cytoplasm. Placentas were histologically classified and graded for individual MVM and FVM lesions, by a board-certified perinatal pathologist (blinded to clinical outcome), using the Amsterdam Placental Workshop Consensus Statement. Spearman’s correlation coefficients were used between GRP78 staining and labor duration, gestational age, birth weight, placental weight, MVM and FVM lesions. T-tests were used for dichotomous variables. Data is presented as mean ± SD with p<0.05 accepted for statistical significance. A positive correlation was noted between duration of second stage of labor and GRP78 staining(p=0.03) independent of hypertensive outcome. No significant associations were noted of GRP78 with total duration of labor, length of ruptured membranes, term or preterm preeclampsia or gestational hypertension. A statistically significant increase in GRP78 staining was noted with delayed villous maturation (1226+/-774 without vs w=1718+/-780 with; p=0.038).However, there were no associations between GRP78 staining and other individual MVM or FVM lesions, or their grades. The presence of ER stress in normal labor may confound the ability to elucidate its role in hypertensive disorders of pregnancy. The association between GRP78 and DVM is unexpected, since it is associated with macrosomia and gestational diabetes. Larger studies are needed to investigate this.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Regulation of lipid metabolism by the unfolded protein response.

The endoplasmic reticulum (ER) is the site of protein folding and secretion, Ca2+ storage and lipid synthesis in eukaryotic cells. Disruption to protein folding or Ca2+ homeostasis in the ER leads to the accumulation of unfolded proteins, a condition known as ER stress. This leads to activation of the unfolded protein response (UPR) pathway in order to restore protein homeostasis. Three ER membrane proteins, namely inositol‐requiring enzyme 1 (IRE1), protein kinase RNA‐like ER kinase (PERK) and activating transcription factor 6 (ATF6), sense the accumulation of unfolded/misfolded proteins and are activated, initiating an integrated transcriptional programme. Recent literature demonstrates that activation of these sensors can alter lipid enzymes, thus implicating the UPR in the regulation of lipid metabolism. Given the presence of ER stress and UPR activation in several diseases including cancer and neurodegenerative diseases, as well as the growing recognition of altered lipid metabolism in disease, it is timely to consider the role of the UPR in the regulation of lipid metabolism. This review provides an overview of the current knowledge on the impact of the three arms of the UPR on the synthesis, function and regulation of fatty acids, triglycerides, phospholipids and cholesterol.

PKR inhibitors suppress endoplasmic reticulum stress and subdue glucolipotoxicity-mediated impairment of insulin secretion in pancreatic beta cells.

Type 2 diabetes mellitus is characterized by insulin resistance and hypersecretion of insulin from the pancreas to compensate for decreased insulin sensitivity in the peripheral tissues. In later stages of the disease insulin-secreting beta cell degeneration commences and patients require insulin replacement therapy in order to accomplish proper regulation of their blood glucose. Endoplasmic reticulum (ER) stress in the beta cells is one of the factors contributing to this detrimental effect. Protein kinase R (PKR) is a cellular stress kinase activated by ER stress and contributing to degeneration of pancreatic islets. In order to determine whether inhibition of PKR activation by specific small molecule inhibitors of PKR ameliorates pancreatic insulin secretion capacity, we treated beta cells with two imidazole/oxindole-derived inhibitors of PKR kinase, imoxin (C16) and 2-aminopurine (2-AP), in the presence of ER stress. Our results demonstrate that PKR inhibition suppresses tunicamycin-mediated ER stress without altering the insulin production capacity of the cells. Palmitic acid-mediated suppression of insulin secretion, however, was subdued significantly by PKR inhibitor treatment through an ER stress-related mechanism. We suggest that PKR inhibitor treatment may be used to increase the insulin secretion capacity of the pancreas in later stages of diabetes.

Endoplasmic reticulum stress attenuation promotes bovine oocyte maturation in vitro

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

Type I interferons and endoplasmic reticulum stress in health and disease.

Type I interferons (IFNs) comprise of pro-inflammatory cytokines created, as well as sensed, by all nucleated cells with the main objective of blocking pathogens-driven infections. Owing to this broad range of influence, type I IFNs also exhibit critical functions in many sterile inflammatory diseases and immunopathologies, especially those associated with endoplasmic reticulum (ER) stress-driven signaling pathways. Indeed, over the years accumulating evidence has indicated that the presence of ER stress can influence the production, or sensing of, type I IFNs induced by perturbations like pattern recognition receptor (PRR) agonists, infections (bacterial, viral or parasitic) or autoimmunity. In this article we discuss the link between type I IFNs and ER stress in various diseased contexts. We describe how ER stress regulates type I IFNs production or sensing, or how type I IFNs may induce ER stress, in various circumstances like microbial infections, autoimmunity, diabetes, cancer and other ER stress-related contexts.

Proteomics Highlights Common and Distinct Pathophysiological Processes Associated with Ileal and Colonic Ulcers in Crohn's Disease.

Based on genetics and natural history, Crohn's disease can be separated into two entities, an ileal and a colonic disease. Protein-based approaches are needed to elucidate whether such subphenotypes are related to distinct pathophysiological processes. The proteome of ulcer edges was compared with that of paired control tissue samples [n = 32 biopsies] by differential proteomics in the ileum and the colon of Crohn's disease patients [n = 16]. The results were analysed using a hypothesis-driven approach [based on the literature] and a hypothesis-free approach [pathway enrichment analyses] to determine common and segment-specific pathophysiological processes associated with ileal and colonic CD ulcer edges. To confirm the involvement of a key pathway highlighted by proteomics, two proteins were also studied by immunochemistry. In the ileum and the colon, 4428 and 5204 proteins, respectively, were identified and quantified. Ileal and colonic ulcer edges differed in having a distinct distribution of proteins associated with epithelial-mesenchymal transition, neutrophil degranulation, and ribosomes. Ileal and colonic ulcer edges were similarly characterized by an increase in the proteins implicated in the endoplasmic reticulum protein-processing pathway and a decrease in mitochondrial proteins. Immunochemistry confirmed the presence of endoplasmic reticulum stress in the mucosa of ileal and colonic ulcer edges. This study provides protein-based evidence for partially distinct pathophysiological processes being associated with ileal and colonic ulcer edges in Crohn's disease patients. This could constitute a first step toward the development of gut segment-specific diagnostic markers and therapeutics.

Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis

Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)-the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.

Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells

Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a β-subunit transmembrane protein 30 A (TMEM30A). Disruption of PS flippases led to several human diseases. However, the roles of TMEM30A in the central nervous system remain elusive. To investigate the role of Tmem30a in the cerebellum, we developed a Tmem30a Purkinje cell (PC)-specific knockout (KO) mouse model. The Tmem30a KO mice displayed early-onset ataxia and progressive PC death. Deficiency in Tmem30a led to an increased expression of Glial fibrillary acidic protein and astrogliosis in regions with PC loss. Elevated C/EBP homologous protein and BiP expression levels indicated the presence of endoplasmic reticulum stress in the PCs prior to visible cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis suggested that apoptotic cell death occurred in the cerebellum. Our data demonstrate that loss of Tmem30a in PCs results in protein folding and transport defects, a substantial decrease in dendritic spine density, increased astrogliosis and PC death. Taken together, our data demonstrate an essential role of Tmem30a in the cerebellum PCs.

Lunapark, a Novel Target to miR-126—Its Potential Role in Maintenance of ER and Glucose Homeostasis

Strong evidence suggests that an adverse in utero and/or early postnatal environment impacts on long-term risk of developing type 2 diabetes. We showed previously that miR-126 is increased in adipose tissue of mouse offspring born to obese mothers which leads to impaired insulin signaling pathway by silencing IRS-1 at the translational level. However, the full spectrum of targets of miR-126 and consequently the consequences of its overexpression for adipose tissue function are unknown. The aim of the current study was therefore to identify novel targets of miR-126 using the proteomic technique, known as Pulsed Stable Isotope Labeling by Amino Acids (pSILAC). 3T3-L1-cells were transfected with miR-126 and their proteome compared to those transfected with a scrambled sequence. We detected 4567 proteins that were translated in adipocytes and of these 401 demonstrated a &amp;gt;1.3 fold decrease following over-expression of miR-126. Bioinformatic analysis revealed that 43 of these contained a miR-126 seed sequence in their 3’un-translated region. This included known miR-126 targets such as IRS-1 and VCAM-1 as well as novel targets. One of the largest changes in expression was observed for Lunapark and through the use of luciferase assays and western blotting we independently confirmed this was a direct target of miR-126. Lunapark is a key component for stabilization of nascent three-way junctions in the endoplasmic reticulum (ER). Consistent with this role we observed altered levels of mTOR and XBP1 in cells treated with miR-126, reflecting the presence of ER stress. Together, the results suggest that overexpression of miR-126 can lead to both ER stress and Insulin Resistance and therefore represent a novel link between two pathways that contribute to development and progression to T2DM. Disclosure J.A. Faria: None. D. Duque Guimaraes: None. L. Pantaleao: None. T.P. Ong: None. A. Ozanne: None.

Non-canonical role of cancer-associated mutant SEC23B in the ribosome biogenesis pathway.

SEC23B is a component of coat protein complex II (COPII) vesicles that transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Loss-of-function SEC23B mutations cause a rare form of anemia, resulting from decreased SEC23B levels. We recently identified germline heterozygous SEC23B variants as potentially cancer-predisposing. Mutant SEC23B associated with ER stress-mediated tumorigenesis, without decreased SEC23B expression. However, our understanding of the processes behind these observations remain limited. Here, we show mutant SEC23B exists within nucleoli, in addition to classical distribution at the ER/Golgi. This occurs independent of other COPII proteins and does not compromise secretory function. Mutant cells have increased ribosomal protein and translation-related gene expression, and enhanced translational capacity, in the presence of ER stress. We show that mutant SEC23B binds to UBF transcription factor, with increased UBF transcription factor binding at the ribosomal DNA promoter. Our data indicate SEC23B has potential non-canonical COPII-independent function, particularly within the ribosome biogenesis pathway, and that may contribute to the pathogenesis of cancer-predisposition.

Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress

Retinoic acid (RA) in association with chemotherapy or with arsenic trioxide (ATO) results in high cure rates of acute promyelocytic leukemia (APL). We show that RA-induced differentiation of human leukemic cell lines and primary blasts dramatically increases their sensitivity to endoplasmic reticulum (ER) stress-inducing drugs at doses that are not toxic in the absence of RA. In addition, we demonstrate that the PERK pathway, triggered in response to ER stress, has a major protective role. Moreover, low amounts of pharmacologically induced ER stress are sufficient to strongly increase ATO toxicity. Indeed, in the presence of ER stress, ATO efficiently induced apoptosis in RA-sensitive and RA-resistant APL cell lines, at doses ineffective in the absence of ER stress. Our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-β (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

92 - Decreased Hepatic Polyunsaturated Fatty Acids and Inflammatory Response in a Rat Model of Endoplasmic Reticulum Stress

In this study, we sought to evaluate the effect of endoplasmic reticulum (ER) stress on hepatic polyunsaturated fatty acids (PUFAs) and inflammatory response through cyclooxygenase (COX)-mediated pathway. Male Wistar rats which were allowed free access to standard rat chow were randomly divided into control, tunicamycin (TM) treated and TM + tauroursodeoxycholic acid (TUDCA) treated groups. Hepatic ER stress was induced by intraperitoneal injection of tunicamycin and the ER stress inhibitor TUDCA was injected 30 minutes before hepatic induction of ER stress. The presence of ER stress was confirmed by increased intracellular levels of C/EBP-homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). Necroinflammation was evaluated in liver sections stained with hematoxylin-eosin using the Ishak-modified hepatic activity index. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fast-liquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), COX and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the inflammatory pathways. Tunicamycin treatment significantly decreased all measured PUFAs and increased AA/EPA ratio in liver tissue compared to controls. Tissue activity of PLA2, COX and PGE2 levels were significantly increased in liver tissue of TM treated rats compared to controls. Tauroursodeoxycholic acid lead to a partial restoration of liver PUFA levels and significantly decreased PLA2, COX and PGE2 levels compared to TM treated rats. The results of this study reveal the presence of a proinflammatory state in hepatic ER stress as shown by significantly increased AA/EPA ratio. To our best knowledge, this is the first study reporting altered PUFA levels in ER stress and supports the use of omega-3 fatty acids as adjuvant treatment in liver diseases demonstrating ER stress. Acknowledgement This study was supported by a grant from TUBITAK; # 214S223.

Liver metabolic disruption induced after a single exposure to PCB126 in rats.

Polychlorinated biphenyls (PCBs) have been recognized as metabolic disruptors. The liver plays a pivotal role in detoxification of an organism. Fatty liver results from altered intra-, and extra-hepatic mediators and is associated with increased glucose-related protein 78 (GRP78), commonly used as a marker for endoplasmic reticulum (ER) stress signaling. This pilot study aimed to study the effects of a single exposure on fatty liver metabolic parameters. The objective of the study is to characterize the effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) on ER stress protein chaperon GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP) and intra-hepatic mediators such as microsomal triglyceride transfer protein (MTP), sterol regulatory element-binding protein 1c (SREBP1c), and peroxisome proliferator-activated receptor alpha (PPARα), as well as extra-hepatic factors such as non-esterified fatty acid (NEFA) and tumor necrosis factor alpha (TNFα). Hepatic GRP78 mRNA and protein levels, indicating the presence of ER stress, were significantly increased following a single PCB126 exposure in rats. Intra-hepatic mechanisms such as lipoprotein secretion pathway (i.e., MTP), lipogenesis de novo (i.e., SREBP1c), and oxidation (i.e., PPARα) were altered in PCB126-treated rats. In addition, a state of inflammation measured by higher TNFα plasma levels was present in contaminated rats. These data indicate that a single injection of PCB126-modulated expression of GRP78 associated with hepatic ER stress and systemic inflammation in rats.

Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN) 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP) ribose polymerase (PARP) cleavage, and Bcl-associated X protein (Bax)/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP) and immunoglobulin heavy chain-binding protein (BiP), while cyclooxygenase (COX)-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.

Acetylcholine inhibits tumor necrosis factor α activated endoplasmic reticulum apoptotic pathway via EGFR-PI3K signaling in cardiomyocytes.

Previous findings have shown that acetylcholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh.